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αα-Hub domains and intrinsically disordered proteins: A decisive combo

  • Katrine Bugge
    Affiliations
    REPIN and The Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Copenhagen, Denmark

    Structural Biology and NMR Laboratory, Department of Biology, University of Copenhagen, Copenhagen, Denmark
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  • Lasse Staby
    Affiliations
    REPIN and The Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Copenhagen, Denmark

    Structural Biology and NMR Laboratory, Department of Biology, University of Copenhagen, Copenhagen, Denmark
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  • Edoardo Salladini
    Affiliations
    REPIN and The Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Copenhagen, Denmark
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  • Rasmus G. Falbe-Hansen
    Affiliations
    REPIN and The Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Copenhagen, Denmark
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  • Birthe B. Kragelund
    Correspondence
    For correspondence: Birthe B. Kragelund; Karen Skriver
    Affiliations
    REPIN and The Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Copenhagen, Denmark

    Structural Biology and NMR Laboratory, Department of Biology, University of Copenhagen, Copenhagen, Denmark
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  • Karen Skriver
    Correspondence
    For correspondence: Birthe B. Kragelund; Karen Skriver
    Affiliations
    REPIN and The Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Copenhagen, Denmark
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Open AccessPublished:December 29, 2020DOI:https://doi.org/10.1074/jbc.REV120.012928
      Hub proteins are central nodes in protein–protein interaction networks with critical importance to all living organisms. Recently, a new group of folded hub domains, the αα-hubs, was defined based on a shared αα-hairpin supersecondary structural foundation. The members PAH, RST, TAFH, NCBD, and HHD are found in large proteins such as Sin3, RCD1, TAF4, CBP, and harmonin, which organize disordered transcriptional regulators and membrane scaffolds in interactomes of importance to human diseases and plant quality. In this review, studies of structures, functions, and complexes across the αα-hubs are described and compared to provide a unified description of the group. This analysis expands the associated molecular concepts of “one domain–one binding site”, motif-based ligand binding, and coupled folding and binding of intrinsically disordered ligands to additional concepts of importance to signal fidelity. These include context, motif reversibility, multivalency, complex heterogeneity, synergistic αα-hub:ligand folding, accessory binding sites, and supramodules. We propose that these multifaceted protein–protein interaction properties are made possible by the characteristics of the αα-hub fold, including supersite properties, dynamics, variable topologies, accessory helices, and malleability and abetted by adaptability of the disordered ligands. Critically, these features provide additional filters for specificity. With the presentations of new concepts, this review opens for new research questions addressing properties across the group, which are driven from concepts discovered in studies of the individual members. Combined, the members of the αα-hubs are ideal models for deconvoluting signal fidelity maintained by folded hubs and their interactions with intrinsically disordered ligands.

      Keywords

      Abbreviations:

      AD (activation domain), CBP (CREB binding protein), CCM2 (cerebral cavernous malformation 2), ETO (eight-twenty-one), GO (gene ontology), HDAC (histone deacetylase), HF (histone-fold), HHD (harmonin homology domain), HID (HDAC interacting domain), ID (intrinsic structural disorder), IDR (intrinsically disordered region), ITC (isothermal titration calorimetry), MD (molecular dynamics), NCBD (nuclear coactivator binding domain), NHR1 (nervy homology region 1), PAH (paired amphipathic helix), PARP (poly(ADP-ribose)polymerase), RCD1 (Radical-Induced Cell Death1), RST (RCD1, SRO, and TAF4), RTEL1 (regulator of telomere elongation helicase 1), SLiM (short linear motif), TAF4 (transcription initiation factor TFIID-subunit 4), TAFH (TATA-box associated factor homology), TF (transcription factor), TRD (TF regulatory domain)
      Fast and efficient regulation of complex cellular signaling pathways is mediated by highly connected protein nodes called hubs (
      • Oldfield C.J.
      • Meng J.
      • Yang J.Y.
      • Yang M.Q.
      • Uversky V.N.
      • Dunker A.K.
      Flexible nets: disorder and induced fit in the associations of p53 and 14-3-3 with their partners.
      ,
      • Cumberworth A.
      • Lamour G.
      • Babu M.M.
      • Gsponer J.
      Promiscuity as a functional trait: intrinsically disordered regions as central players of interactomes.
      ,
      • Patil A.
      • Kinoshita K.
      • Nakamura H.
      Hub promiscuity in protein-protein interaction networks.
      ,
      • Han J.J.
      • Bertin N.
      • Hao T.
      • Goldberg D.S.
      • Berriz G.F.
      • Zhang L.V.
      • Dupuy D.
      • Walhout A.J.M.
      • Cusick M.E.
      • Roth F.P.
      • Vidal M.
      Evidence for dynamically organized modularity in the yeast protein-protein interaction network.
      ,
      • Hu G.
      • Wu Z.
      • Uversky V.N.
      • Kurgan L.
      Functional analysis of human hub proteins and their interactors involved in the intrinsic disorder-enriched interactions.
      ). These hub proteins are intimately linked to intrinsic structural disorder (ID) (
      • Oldfield C.J.
      • Meng J.
      • Yang J.Y.
      • Yang M.Q.
      • Uversky V.N.
      • Dunker A.K.
      Flexible nets: disorder and induced fit in the associations of p53 and 14-3-3 with their partners.
      ,
      • Cumberworth A.
      • Lamour G.
      • Babu M.M.
      • Gsponer J.
      Promiscuity as a functional trait: intrinsically disordered regions as central players of interactomes.
      ,
      • Patil A.
      • Kinoshita K.
      • Nakamura H.
      Hub promiscuity in protein-protein interaction networks.
      ,
      • Han J.J.
      • Bertin N.
      • Hao T.
      • Goldberg D.S.
      • Berriz G.F.
      • Zhang L.V.
      • Dupuy D.
      • Walhout A.J.M.
      • Cusick M.E.
      • Roth F.P.
      • Vidal M.
      Evidence for dynamically organized modularity in the yeast protein-protein interaction network.
      ,
      • Hu G.
      • Wu Z.
      • Uversky V.N.
      • Kurgan L.
      Functional analysis of human hub proteins and their interactors involved in the intrinsic disorder-enriched interactions.
      ), either containing intrinsically disordered regions (IDRs) themselves (
      • Oldfield C.J.
      • Meng J.
      • Yang J.Y.
      • Yang M.Q.
      • Uversky V.N.
      • Dunker A.K.
      Flexible nets: disorder and induced fit in the associations of p53 and 14-3-3 with their partners.
      ) or being ordered proteins binding IDRs (
      • Moldovan G.-L.
      • Pfander B.
      • Jentsch S.
      PCNA, the maestro of the replication fork.
      ,
      • Prestel A.
      • Wichmann N.
      • Martins J.M.
      • Marabini R.
      • Kassem N.
      • Broendum S.S.
      • Otterlei M.
      • Nielsen O.
      • Willemoës M.
      • Ploug M.
      • Boomsma W.
      • Kragelund B.B.
      The PCNA interaction motifs revisited: thinking outside the PIP-box.
      ,
      • Bustos D.M.
      The role of protein disorder in the 14-3-3 interaction network.
      ). IDRs exist in ensembles of interconverting and dynamic states, endowing them with adaptability, multivalency, and high chemical modification potential (
      • Babu M.M.
      • van der Lee R.
      • de Groot N.S.
      • Gsponer J.
      Intrinsically disordered proteins: regulation and disease.
      ,
      • Dyson H.J.
      • Wright P.E.
      Coupling of folding and binding for unstructured proteins.
      ,
      • Huang J.-R.
      • Warner L.R.
      • Sanchez C.
      • Gabel F.
      • Madl T.
      • Mackereth C.D.
      • Sattler M.
      • Blackledge M.
      Transient electrostatic interactions dominate the conformational equilibrium sampled by multi-domain splicing factor U2AF65: a combined NMR and SAXS study.
      ). Indeed, ID appears to be a prerequisite for fidelity in signaling pathways through exploitation of the many different mechanisms encoded in these properties (
      • Dai W.
      • Wu A.
      • Ma L.
      • Li Y.-X.
      • Jiang T.
      • Li Y.-Y.
      A novel index of protein-protein interface propensity improves interface residue recognition.
      ,
      • Wright P.E.
      • Dyson H.J.
      Intrinsically disordered proteins in cellular signalling and regulation.
      ).
      Recognition of IDRs in and by hubs depends on short linear motifs (SLiMs), which are stretches of 2 to 12 residues with only a few highly conserved positions (
      • Neduva V.
      • Linding R.
      • Su-Angrand I.
      • Stark A.
      • de Masi F.
      • Gibson T.J.
      • Lewis J.
      • Serrano L.
      • Russell R.B.
      Systematic discovery of new recognition peptides mediating protein interaction networks.
      ,
      • Jespersen N.
      • Barbar E.
      Emerging features of linear motif-binding hub proteins.
      ). It has been proposed that the eukaryotic SLiMome consists of up to 1 million different SLiMs (
      • Tompa P.
      • Davey N.E.
      • Gibson T.J.
      • Babu M.M.
      A million peptide motifs for the molecular biologist.
      ), but SLiMs active in hub interactions have very similar features (
      • Plevin M.J.
      • Mills M.M.
      • Ikura M.
      The LxxLL motif: a multifunctional binding sequence in transcriptional regulation.
      ). Thus, it remains enigmatic how signal fidelity is orchestrated by hubs. Several folded domains present in large scaffolding proteins act as hubs, binding IDRs, typically transcription factor (TF) regulatory domains (TRDs), via SLiMs. These include the TAZ (
      • Berlow R.B.
      • Dyson H.J.
      • Wright P.E.
      Hypersensitive termination of the hypoxic response by a disordered protein switch.
      ,
      • Dyson H.J.
      • Wright P.E.
      Role of intrinsic protein disorder in the function and interactions of the transcriptional coactivators CREB-binding protein (CBP) and p300.
      ), the KIX (
      • Dyson H.J.
      • Wright P.E.
      Role of intrinsic protein disorder in the function and interactions of the transcriptional coactivators CREB-binding protein (CBP) and p300.
      ), the GACKIX (
      • Novatchkova M.
      • Eisenhaber F.
      Linking transcriptional mediators via the GACKIX domain super family.
      ), and the αα-hub domains (
      • Bugge K.
      • Staby L.
      • Kemplen K.R.
      • O’Shea C.
      • Bendsen S.K.
      • Jensen M.K.
      • Olsen J.G.
      • Skriver K.
      • Kragelund B.B.
      Structure of radical-induced cell Death1 hub domain reveals a common αα-scaffold for disorder in transcriptional networks.
      ). Despite belonging to different families, the domains share structural traits such as being a relatively short chain of <100 residues that folds into topologies constructed solely by α-helices (
      • Dyson H.J.
      • Wright P.E.
      Role of intrinsic protein disorder in the function and interactions of the transcriptional coactivators CREB-binding protein (CBP) and p300.
      ,
      • Bugge K.
      • Staby L.
      • Kemplen K.R.
      • O’Shea C.
      • Bendsen S.K.
      • Jensen M.K.
      • Olsen J.G.
      • Skriver K.
      • Kragelund B.B.
      Structure of radical-induced cell Death1 hub domain reveals a common αα-scaffold for disorder in transcriptional networks.
      ). All the domains are also part of multidomain hub proteins carrying both order and disorder (
      • Dyson H.J.
      • Wright P.E.
      Role of intrinsic protein disorder in the function and interactions of the transcriptional coactivators CREB-binding protein (CBP) and p300.
      ,
      • Bugge K.
      • Staby L.
      • Kemplen K.R.
      • O’Shea C.
      • Bendsen S.K.
      • Jensen M.K.
      • Olsen J.G.
      • Skriver K.
      • Kragelund B.B.
      Structure of radical-induced cell Death1 hub domain reveals a common αα-scaffold for disorder in transcriptional networks.
      ), increasing the valency of their interactions. As an additional layer of regulation, protein cofactors bring the transcriptional machinery to target genes through interactions with TFs (
      • Poss Z.C.
      • Ebmeier C.C.
      • Taatjes D.J.
      The mediator complex and transcription regulation.
      ) and aid in scaffolding of the transcriptional machinery (
      • Allen B.L.
      • Taatjes D.J.
      The mediator complex: a central integrator of transcription.
      ). How specificity and regulation within the associated multicomponent complexes are controlled is far from understood.
      The αα-hub domains have only recently emerged as a group of folded hub proteins (
      • Bugge K.
      • Staby L.
      • Kemplen K.R.
      • O’Shea C.
      • Bendsen S.K.
      • Jensen M.K.
      • Olsen J.G.
      • Skriver K.
      • Kragelund B.B.
      Structure of radical-induced cell Death1 hub domain reveals a common αα-scaffold for disorder in transcriptional networks.
      ,
      • Staby L.
      • Bugge K.
      • Falbe-Hansen R.G.
      • Saladini E.
      • Skriver K.
      • Kragelund B.B.
      Connecting the αα-hubs: same fold, disordered ligands, new functions.
      ), and hence, the full potential for understanding hub proteins from studies across these similar domains has yet to be unfolded. Furthermore, understanding their role in organizing disordered transcriptional regulators and membrane protein scaffolds in interactomes of importance to human diseases and plant quality is of broad interest. This review focuses on the αα-hub domains and brings an overview and comparative analysis of their structures, functions, complexes, and mechanisms. The αα-hub domains have low sequence identity (4%–15%) (
      • Bugge K.
      • Staby L.
      • Kemplen K.R.
      • O’Shea C.
      • Bendsen S.K.
      • Jensen M.K.
      • Olsen J.G.
      • Skriver K.
      • Kragelund B.B.
      Structure of radical-induced cell Death1 hub domain reveals a common αα-scaffold for disorder in transcriptional networks.
      ) and are diversely involved in distinct biological functions, while still binding ligands of similar structural and chemical properties. Thus, this group of hubs constitutes a suitable model for addressing how selectivity and specificity in interactomes are controlled and how fidelity is encoded. Through specific examples, we ask how the αα-hub domains maintain fidelity and highlight concepts and open questions related to the αα-hub modus operandi.

      The αα-hubs

      The αα-hub domains share key fold features

      The αα-hub domains were recently defined based on a common structural foundation (
      • Bugge K.
      • Staby L.
      • Kemplen K.R.
      • O’Shea C.
      • Bendsen S.K.
      • Jensen M.K.
      • Olsen J.G.
      • Skriver K.
      • Kragelund B.B.
      Structure of radical-induced cell Death1 hub domain reveals a common αα-scaffold for disorder in transcriptional networks.
      ) and originally included the following domains: RCD1, SRO, and TAF4 (RST) from Radical-Induced Cell Death1 (RCD1) (
      • Bugge K.
      • Staby L.
      • Kemplen K.R.
      • O’Shea C.
      • Bendsen S.K.
      • Jensen M.K.
      • Olsen J.G.
      • Skriver K.
      • Kragelund B.B.
      Structure of radical-induced cell Death1 hub domain reveals a common αα-scaffold for disorder in transcriptional networks.
      ); paired amphipathic helix (PAH)1, PAH2, and PAH3 from Sin3 of the Sin3/histone deacetylase corepressor complex (
      • Spronk C.A.E.M.
      • Tessari M.
      • Kaan A.M.
      • Jansen J.F.A.
      • Vermeulen M.
      • Stunnenberg H.G.
      • Vuister G.W.
      The Mad1-Sin3B interaction involves a novel helical fold.
      ); TATA-box associated factor homology (TAFH) (or nervy homology region 1 [NHR1]) from transcription initiation factor TFIID-subunit 4 (TAF4) and eight-twenty-one (ETO) (or MTG8/CBFA2T1) (
      • Wang X.
      • Truckses D.M.
      • Takada S.
      • Matsumura T.
      • Tanese N.
      • Jacobson R.H.
      Conserved region I of human coactivator TAF4 binds to a short hydrophobic motif present in transcriptional regulators.
      ); and nuclear coactivator binding domain (NCBD) (or IRF-binding domain) from CREB binding protein (CBP) (
      • Lin C.H.
      • Hare B.J.
      • Wagner G.
      • Harrison S.C.
      • Maniatis T.
      • Fraenkel E.
      A small domain of CBP/p300 binds diverse proteins: solution structure and functional studies.
      ) (Figure 1, Figure 2, Figure 3, AD). In addition, the harmonin homology domain (HHD) (or harmonin-N-terminal domain) from whirlin (
      • Delhommel F.
      • Cordier F.
      • Saul F.
      • Chataigner L.
      • Haouz A.
      • Wolff N.
      Structural plasticity of the HHD2 domain of whirlin.
      ), harmonin (
      • Bahloul A.
      • Pepermans E.
      • Raynal B.
      • Wolff N.
      • Cordier F.
      • England P.
      • Nouaille S.
      • Baron B.
      • El-Amraoui A.
      • Hardelin J.P.
      • Durand D.
      • Petit C.
      Conformational switch of harmonin, a submembrane scaffold protein of the hair cell mechanoelectrical transduction machinery.
      ), cerebral cavernous malformation 2 (CCM2) (
      • Fisher O.S.
      • Zhang R.
      • Li X.
      • Murphy J.W.
      • Demeler B.
      • Boggon T.J.
      Structural studies of cerebral cavernous malformations 2 (CCM2) reveal a folded helical domain at its C-terminus.
      ), and regulator of telomere elongation helicase 1 (RTEL1) (
      • Faure G.
      • Revy P.
      • Schertzer M.
      • Londono-Vallejo A.
      • Callebaut I.
      The C-terminal extension of human RTEL1, mutated in Hoyeraal-Hreidarsson syndrome, contains harmonin-N-like domains.
      ) was assigned to the αα-hub domain group (
      • Staby L.
      • Bugge K.
      • Falbe-Hansen R.G.
      • Saladini E.
      • Skriver K.
      • Kragelund B.B.
      Connecting the αα-hubs: same fold, disordered ligands, new functions.
      ) (Figs. 1E and 2E).
      Figure thumbnail gr1
      Figure 1Structures and evolution of αα-hub domains. AE, representative structures of the current αα-hub subgroups PAH (2rmr), RST (5n9q), TAFH (2pp4), NCBD (2kkj), and HHD (4fqn), respectively. For each domain, the helices are color coded with H1 (orange), H2 (blue), H3 (green), H4 (red), and H5 (pink). For the domains containing the αL4 loop, the hydrophobic β3-position is shown as gray sticks. Sequence logos below each domain illustrate the conservation of the H2-H3 loop region across phylogenetically representative species with each position named according to the αL4 loop nomenclature. In the structures of HHD the β2-residue (marked with an asterisk) is located in the site normally occupied by the β3-residue in the αL4 (see also C). Empty positions indicate either lack of conservation (for RST) or the presence of a gap in the alignment (HHD). F, compositional features of the prototypical αα-hub. Side and front views illustrate the different surfaces, helices, and loops as defined in this review. Zoom shows the configuration of the αL4 loop with the hydrophobic β3-position forming stabilizing interactions with side chains from H2 and H3. G, evolutionary proliferation of αα-hubs and relationships between major eukaryotic groups (
      • Burki F.
      • Roger A.J.
      • Brown M.W.
      • Simpson A.G.B.
      The new tree of eukaryotes.
      ). Branch lengths are arbitrary. Blue, PAH; green, RST; red, TAFH; orange, NCBD; purple, HHD.
      Figure thumbnail gr2
      Figure 2αα-Hub protein domain structures. αα-hub domain structures present in proteins with known functions or appearing more than 10 times in InterPro. The αα-hub domains included are: A, PAH, B, RST, C, TAF4, D, NCBD, E, HHD. The GO terms associated with the different αα-hub proteins are shown to the right. The schematics are not drawn to scale, the relative distance between the domains vary, and some of the proteins have more than one copy of the domains shown. GO, gene ontology.
      Figure thumbnail gr3
      Figure 3αα-Hub protein interactions. Examples of protein complexes and interactions of αα-hub proteins, focusing mainly on the interactions of the αα-hub domains. A, Sin3 as part of a coregulator complex. TFs, coregulators, adapters, and enzymes are shown bound to or as part of the binding pool of their target domains in Sin3. The classic functional role of Sin3 as a scaffold for chromatin remodeling is also shown. B, RCD1 in regulation of biotic and abiotic stress responses. The interactions mediate suppression of plant immunity (through HaRxL) and coordination of communication between ROS signals emitted from mitochondria and chloroplasts (through ANAC013 and ANAC017). C, TAF4 as part of the RNA Pol II preinitiation complex. TFs binding to the TAFH domain and implicated in embryonic pattering, and programmed cell death are shown. D, CBP/p300 is a central node in eukaryotic regulatory networks regulating TFs and chromatin via their histone acetyl transferase activity. The TAZ, KIX, and NCBD domains are scaffolds for interactions with IDRs of proteins shown with names for only NCBD ligands. E, Harmonin anchored interactions in regulation of hearing and vision. Cad23 assembles the upper part of the tip link, and its cytoplasmic tail is anchored to the actin filament of stereocilia via binding to harmonin.
      The αα-hub domains consist of 3 to 5 α-helices of ∼10 to 20 residues. Their core defining feature is an αα-hairpin supersecondary structure motif (
      • Efimov A.V.
      Structure of α-α-hairpins with short connections.
      ) constituted by two consecutive antiparallel α-helices with a crossing angle close to 180°, connected by an inflexible loop (L2) (Fig. 1F). For all members, except NCBD, these two helices are helix 2 (H2) and 3 (H3) from the N terminus, and for clarity, helices are for all members numbered relative to these. In the prototypical member, L2 is folded into the five-residue link motif αL4 (
      • Engel D.E.
      • DeGrado W.F.
      α-α linking motifs and interhelical orientations.
      ), with the β3-position carrying a well-sized (>100 Å3) hydrophobic side chain that anchors between H2 and H3 (Fig. 1F). For the PAH and TAFH domains β3 is either Ile, Leu, or Val, and for the RST domain either Ile or Met (Fig. 1, AC). Connected to the H2-H3 core are typically two, but sometimes one or three, additional α-helices organized on the same side of the hairpin (Fig. 1F) (here referred to as the front). This leaves the other side of the hairpin (the back) accessible (Fig. 1F). The antiparallel organization of H2 and H3 orient the short, but flexible loops connecting to the preceding and proceeding α-helices at the same end of the fold, resulting in the formation of an “open” and “closed” end (Fig. 1F). Together, the helices support a hydrophobic binding cleft at the open end. The prototypical αα-hub is thus a domain in a modular protein consisting of four α-helices (H1–H4), of which H2 and H3 make up the αα-hairpin supersecondary structure stabilized by the hydrophobic β3-loop anchoring residue. The organization of H1 and H4 is the distinctive feature of each αα-hub subgroup, resulting in different angles to H2-H3 (Fig. 1, AE). Based on this difference in topology, the five different subgroups of the αα-hubs can be defined: PAH1/2/3, RST, TAFH, NCBD, and HHD.

      Phylogenetic proliferation of the αα-hub domains

      According to the literature and InterPro (
      • Mitchell A.L.
      • Attwood T.K.
      • Babbitt P.C.
      • Blum M.
      • Bork P.
      • Bridge A.
      • Brown S.D.
      • Chang H.Y.
      • El-Gebali S.
      • Fraser M.I.
      • Gough J.
      • Haft D.R.
      • Huang H.
      • Letunic I.
      • Lopez R.
      • et al.
      InterPro in 2019: improving coverage, classification and access to protein sequence annotations.
      ) searches, the αα-hubs are exclusive to eukaryotes (Fig. 1G). The PAH domain is present in most of the major clades of eukaryotes (
      • Bowen A.J.
      • Gonzalez D.
      • Mullins J.G.L.
      • Bhatt A.M.
      • Martinez A.
      • Conlan R.S.
      PAH-domain-specific interactions of the arabidopsis transcription coregulator SIN3-LIKE1 (SNL1) with telomere-binding protein 1 and ALWAYS EARLY2 Myb-DNA binding factors.
      ), whereas the RST domain has been reported in land plants including mosses and liverworts (
      • Christensen L.F.
      • Staby L.
      • Bugge K.
      • O’Shea C.
      • Kragelund B.B.
      • Skriver K.
      Evolutionary conservation of the intrinsic disorder-based radical-induced cell Death1 hub interactome.
      ). HHD is present in animals and plants (
      • Delhommel F.
      • Cordier F.
      • Saul F.
      • Chataigner L.
      • Haouz A.
      • Wolff N.
      Structural plasticity of the HHD2 domain of whirlin.
      ), whereas TAFH and NCBD have been found only in animals (
      • Kazantseva J.
      • Palm K.
      Diversity in TAF proteomics: consequences for cellular differentiation and migration.
      ,
      • Hultqvist G.
      • Åberg E.
      • Camilloni C.
      • Sundell G.N.
      • Andersson E.
      • Dogan J.
      • Chi C.N.
      • Vendruscolo M.
      • Jemth P.
      Emergence and evolution of an interaction between intrinsically disordered proteins.
      ). As orthologous genes have a higher degree of intron position conservation than nonorthologous genes (
      • Henricson A.
      • Forslund K.
      • Sonnhammer E.L.L.
      Orthology confers intron position conservation.
      ), the structure of a gene may provide information about phylogenetic relationships. According to the RefSeq database at NCBI (
      • O’Leary N.A.
      • Wright M.W.
      • Brister J.R.
      • Ciufo S.
      • Haddad D.
      • McVeigh R.
      • Rajput B.
      • Robbertse B.
      • Smith-White B.
      • Ako-Adjei D.
      • Astashyn A.
      • Badretdin A.
      • Bao Y.
      • Blinkova O.
      • Brover V.
      • et al.
      Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion, and functional annotation.
      ), plant RST genes and animal TAFH genes have a conserved intron position right before the αL4 link motif, which is missing in the remaining αα-hubs. Since RST is unique to land plants and TAFH is unique to animals, but both are present in TAF4 proteins (Figs. 2, BC and 3C) (
      • Christensen L.F.
      • Staby L.
      • Bugge K.
      • O’Shea C.
      • Kragelund B.B.
      • Skriver K.
      Evolutionary conservation of the intrinsic disorder-based radical-induced cell Death1 hub interactome.
      ,
      • Kazantseva J.
      • Palm K.
      Diversity in TAF proteomics: consequences for cellular differentiation and migration.
      ) and they have a conserved intron structure, an early evolutionary relationship between these domains is likely. Thus, αα-hub domain proteins are dominant in higher eukaryotes with a likely evolutionary link between TAFH and RST dividing them into two different kingdoms of life. The remaining αα-hubs have no obvious evolutionary links, except for their structural similarities.

      The αα-hub subgroups have distinctive features and bear characteristics of analogous folds

      Proteins with similar folds, such as the αα-hub domains, can be divided into three general categories: homologs (derived from a common ancestor), remote homologs (less obvious sequential similarity because of distant ancestor), and analogs (converged to similar advantageous fold independently) (
      • Russell R.B.
      • Saqi M.A.S.
      • Sayle R.A.
      • Bates P.A.
      • Sternberg M.J.E.
      Recognition of analogous and homologous protein folds: analysis of sequence and structure conservation.
      ,
      • Krishna S.S.
      • Grishin N.V.
      Structurally analogous proteins do exist!.
      ). Since the number of ways nature can arrange a few secondary structural elements in a stable manner is limited, analogous folds commonly occur for small and relatively simple protein structures (
      • Krishna S.S.
      • Grishin N.V.
      Structurally analogous proteins do exist!.
      ). Furthermore, analogous structures are typically similar but with distinct features and key binding site residues (
      • Russell R.B.
      • Saqi M.A.S.
      • Sayle R.A.
      • Bates P.A.
      • Sternberg M.J.E.
      Recognition of analogous and homologous protein folds: analysis of sequence and structure conservation.
      ). As alluded to above, various deviations from the prototypical features are found among the αα-hub subgroups. Some PAH2 domains differ by having extended H2s and L2s (Fig. 4B) (
      • Spronk C.A.E.M.
      • Tessari M.
      • Kaan A.M.
      • Jansen J.F.A.
      • Vermeulen M.
      • Stunnenberg H.G.
      • Vuister G.W.
      The Mad1-Sin3B interaction involves a novel helical fold.
      ,
      • Van Ingen H.
      • Lasonder E.
      • Jansen J.F.A.
      • Kaan A.M.
      • Spronk C.A.E.M.
      • Stunnenberg H.G.
      • Vuister G.W.
      Extension of the binding motif of the Sin3 interacting domain of the mad family proteins.
      ,
      • Swanson K.A.
      • Knoepfler P.S.
      • Huang K.
      • Kang R.S.
      • Cowley S.M.
      • Laherty C.D.
      • Eisenman R.N.
      • Radhakrishnan I.
      HBP1 and Mad1 repressors bind the Sin3 corepressor PAH2 domain with opposite helical orientations.
      ,
      • Brubaker K.
      • Cowley S.M.
      • Huang K.
      • Loo L.
      • Yochum G.S.
      • Ayer D.E.
      • Eisenman R.N.
      • Radhakrishnan I.
      Solution structure of the interacting domains of the mad-sin3 complex: implications for recruitment of a chromatin-modifying complex.
      ,
      • Kumar G.S.
      • Xie T.
      • Zhang Y.
      • Radhakrishnan I.
      Solution structure of the mSin3A PAH2-Pf1 SID1 complex: a Mad1/Mxd1-like interaction disrupted by MRG15 in the Rpd3S/Sin3S complex.
      ,
      • van Ingen H.
      • Baltussen M.A.H.
      • Aelen J.
      • Vuister G.W.
      Role of structural and dynamical plasticity in Sin3: the free PAH2 domain is a folded module in mSin3B.
      ), but with persistent β3-anchoring (Fig. 1A) (
      • Bugge K.
      • Staby L.
      • Kemplen K.R.
      • O’Shea C.
      • Bendsen S.K.
      • Jensen M.K.
      • Olsen J.G.
      • Skriver K.
      • Kragelund B.B.
      Structure of radical-induced cell Death1 hub domain reveals a common αα-scaffold for disorder in transcriptional networks.
      ). HHD differs by generally having an atypical L2 loop lacking β3-anchoring, but having a H5. In CCM2-HHD, the β2-residue is located in the site normally occupied by the β3-residue and is typically a small side chain residue (Fig. 1E and Fig. S1C), while in structures of harmonin-HHD (Protein Data Bank [PDB] codes 2kbq, 2lsr, 2kbr) (
      • Yan J.
      • Pan L.
      • Chen X.
      • Wu L.
      • Zhang M.
      The structure of the harmonin/sans complex reveals an unexpected interaction mode of the two Usher syndrome proteins.
      ,
      • Wu L.
      • Pan L.
      • Zhang C.
      • Zhang M.
      Large protein assemblies formed by multivalent interactions between cadherin23 and harmonin suggest a stable anchorage structure at the tip link of stereocilia.
      ), β3 is a Met but does not anchor between H2 and H3. For both, H5 packs between H3 and H4, possibly rescuing any lost stability from lack of β3-anchoring. Indeed, in harmonin-HHD the β3 Met interacts with side chains of H5. NCBD also lacks β3-anchoring, but here this coincides with a lack of H1. Of note, NCBD stands out by existing in a molten globule-like state when free (
      • Kjaergaard M.
      • Andersen L.
      • Nielsen L.D.
      • Teilum K.
      A folded excited state of ligand-free nuclear coactivator binding domain (NCBD) underlies plasticity in ligand recognition.
      ,
      • Papaleo E.
      • Camilloni C.
      • Teilum K.
      • Vendruscolo M.
      • Lindorff-Larsen K.
      Molecular dynamics ensemble refinement of the heterogeneous native state of NCBD using chemical shifts and NOEs.
      ). Hence, the absence of prototypical features appears to be counteracted by helices outside the αα-hairpin, either intrinsically present in the hub or from binding partners (
      • Bugge K.
      • Staby L.
      • Kemplen K.R.
      • O’Shea C.
      • Bendsen S.K.
      • Jensen M.K.
      • Olsen J.G.
      • Skriver K.
      • Kragelund B.B.
      Structure of radical-induced cell Death1 hub domain reveals a common αα-scaffold for disorder in transcriptional networks.
      ). This way, the hubs may maintain stability, while exposing a hydrophobic and solvent accessible binding pocket. Taken together, the αα-hub domains have with their similar, but small, simplistic folds with distinct differences between subgroups, the typical characteristics of analogous folds.
      Figure thumbnail gr4
      Figure 4Alignments of sequences of PAH1, PAH2, and RST, respectively, from phylogenetically representative species and comparison with 3D structures. Sequences were aligned with Clustal Omega and visualized in Jalview. Available 3D structures of each subgroup were manually inspected and compared with the conservation alignment, and residues with identity >50% that could not be readily explained by fold-conservation (no tertiary side chain contacts) were highlighted in red (alignments and structures). The fold-defining positions (identity above 50% and tertiary side chain contacts) were colored blue in accordance with percentage identity (darker is higher identity, alignments, and structures). Above each alignment, the β3-position is highlighted with “∗,” and the gray boxes indicate the helix boundaries in the free (light gray) and complexed (darker gray, variations are different structures) αα-hubs. Species are given as four-letter abbreviations, with full names given in . A, PAH1. Protein Data Bank (PDB) codes 2czy, 2rms. The peptides of the ligands REST (2czy) and SAP25 (2rms) are shown semitransparent in orange variations. B, PAH2. PDB codes 1s5r, 1e91, 1g1e. The peptides of the ligands HBP1 (1s5r) and Mad1 (1e91, 1g1e) are shown semitransparent in orange variations. C, RST. PDB codes 5oao, 5oap. The ligand peptide of DREB2a (5oap) is shown semitransparent in yellow as an ensemble of 10 lowest-energy structures.
      To address conservation of the hub topology in terms of fold-defining positions across subgroups, we compared the sequences for each subgroup across phylogenetically representative species (Fig. 4, Figs. S1, and S2). 3D structures of each subgroup were manually inspected and compared with the sequence alignment to identify fold-defining positions (identity >50% and with tertiary side chain contacts). Within subgroups, many residues making up the hydrophobic core, and hence defining the fold, are highly conserved (Fig. 4, Figs. S1, and S2). Most distinctively, PAH1 and PAH2 have highly conserved cores, sharing many of the conserved core residues across all four helices, whereas PAH3 is the least conserved of all the αα-hubs (Fig. 4, AB, and Fig. S1A). Across the subgroups, however, no clear conservation pattern of even H2-H3 core residues is evident, consistent with their low sequence identity (
      • Bugge K.
      • Staby L.
      • Kemplen K.R.
      • O’Shea C.
      • Bendsen S.K.
      • Jensen M.K.
      • Olsen J.G.
      • Skriver K.
      • Kragelund B.B.
      Structure of radical-induced cell Death1 hub domain reveals a common αα-scaffold for disorder in transcriptional networks.
      ), and despite a high degree of core residues within each subgroup. However, despite the identified evolutionary relationship described in the previous section, a sequence-relationship between RST and TAFH could not be established through this analysis (Fig. 4C and Fig. S1B). Hence, the structural similarity between the αα-hubs cannot be traced from any recognizable sequential relatedness, and besides the conserved intron structure between RST and TAFH, we found no evidence to support emergence from a common ancestor. Rather, the αα-hub folds should be considered analogous folds (
      • Russell R.B.
      • Sasieni P.D.
      • Sternberg M.J.E.
      Supersites within superfolds. Binding site similarity in the absence of homology.
      ), although more extensive analysis would be required to rule out remote homology. As a consequence, the possibilities for identification of new αα-hubs directly from sequence alone is currently limited. An alternative will be searches through 3D-structure alignment using, e.g., PDBeFold (
      • Krissinel E.
      • Henrick K.
      Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions.
      ), as done in the defining work on the αα-hub group (
      • Bugge K.
      • Staby L.
      • Kemplen K.R.
      • O’Shea C.
      • Bendsen S.K.
      • Jensen M.K.
      • Olsen J.G.
      • Skriver K.
      • Kragelund B.B.
      Structure of radical-induced cell Death1 hub domain reveals a common αα-scaffold for disorder in transcriptional networks.
      ). However, this approach is naturally limited to targets with described 3D structures, and the identification of potential additional αα-hub group members is therefore a challenge.

      The functions of αα-hub domains

      The αα-hub domains are linked to different domains of diverse functions

      To obtain an overview of the domain compositions of the αα-hub domain proteins, we searched the literature and InterPro (
      • Mitchell A.L.
      • Attwood T.K.
      • Babbitt P.C.
      • Blum M.
      • Bork P.
      • Bridge A.
      • Brown S.D.
      • Chang H.Y.
      • El-Gebali S.
      • Fraser M.I.
      • Gough J.
      • Haft D.R.
      • Huang H.
      • Letunic I.
      • Lopez R.
      • et al.
      InterPro in 2019: improving coverage, classification and access to protein sequence annotations.
      ). Most PAH-domain proteins, including Sin3, also contain a histone deacetylase (HDAC) interacting domain (HID) and a Sin3 C-terminal domain (Fig. 2A) (
      • Adams G.E.
      • Chandru A.
      • Cowley S.M.
      Co-repressor, co-activator and general transcription factor: the many faces of the Sin3 histone deacetylase (HDAC) complex.
      ), but numerous PAH-domain proteins contain only some of these domains. The PAH domain is also present in the plant protein WRKY19, which additionally contains a WRKY DNA-binding domain, a kinase domain, and a central TIR-NB-ARC-LRR module implicated in plant immunity (
      • Warmerdam S.
      • Sterken M.G.
      • Sukarta O.C.A.
      • van Schaik C.C.
      • Oortwijn M.E.P.
      • Lozano-Torres J.L.
      • Bakker J.
      • Smant G.
      • Goverse A.
      The TIR-NB-LRR pair DSC1 and WRKY19 contributes to basal immunity of Arabidopsis to the root-knot nematode Meloidogyne incognita.
      ). The gene ontology (GO) terms for the PAH domain proteins suggest a function in transcriptional regulation. The RST domain is found in RCD1 and is responsible for most RCD1 interactions (
      • Jaspers P.
      • Blomster T.
      • Brosché M.
      • Salojärvi J.
      • Ahlfors R.
      • Vainonen J.P.
      • Reddy R.A.
      • Immink R.
      • Angenent G.
      • Turck F.
      • Overmyer K.
      • Kangasjärvi J.
      Unequally redundant RCD1 and SRO1 mediate stress and developmental responses and interact with transcription factors.
      ,
      • O’Shea C.
      • Kryger M.
      • Stender E.G.P.
      • Kragelund B.B.
      • Willemoës M.
      • Skriver K.
      Protein intrinsic disorder in Arabidopsis NAC transcription factors: transcriptional activation by ANAC013 and ANAC046 and their interactions with RCD1.
      ,
      • O’Shea C.
      • Staby L.
      • Bendsen S.K.
      • Tidemand F.G.
      • Redsted A.
      • Willemoës M.
      • Kragelund B.B.
      • Skriver K.
      Structures and short linear motif of disordered transcription factor regions provide clues to the interactome of the cellular hub protein radical-induced cell death 1.
      ). In addition, RCD1 contains an N-terminal WWE domain followed by a poly(ADP-ribose)polymerase (PARP) domain (Fig. 2B). The RST domain is also present in proteins lacking either the WWE or the PARP domain, or both, and in combination with a histone-fold (HF) domain in plant TAF4, which is reflected in GO terms related to transcription. Human TAF4 consists of a TAFH domain followed by a HF domain and is crucial for structural integrity of the TFIID complex (
      • Kazantseva J.
      • Palm K.
      Diversity in TAF proteomics: consequences for cellular differentiation and migration.
      ,
      • Wright K.J.
      • Marr M.T.
      • Tjian R.
      TAF4 nucleates a core subcomplex of TFIID and mediates activated transcription from a TATA-less promoter.
      ) (Fig. 2C). TAFH-domains are also found in conjunction with NHR-like domains and in ETO proteins, in which a MYND zinc finger for corepressor recruitment is also found (
      • Lutterbach B.
      • Sun D.
      • Schuetz J.
      • Hiebert S.W.
      The MYND motif is required for repression of basal transcription from the multidrug resistance 1 promoter by the t(8;21) fusion protein.
      ). Overall, GO terms reveal a function of TAFH domain proteins in transcription. The multidomain proteins, CBP and its paralog p300 (
      • Dyson H.J.
      • Wright P.E.
      Role of intrinsic protein disorder in the function and interactions of the transcriptional coactivators CREB-binding protein (CBP) and p300.
      ), both have histone acetyltransferase activity (Fig. 2D), as reflected in the GO terms associated with the NCBD-containing proteins suggesting functions within transcription. HHD is present in proteins with several PDZ domains, as in the case of whirlin (
      • Delhommel F.
      • Cordier F.
      • Saul F.
      • Chataigner L.
      • Haouz A.
      • Wolff N.
      Structural plasticity of the HHD2 domain of whirlin.
      ) and harmonin (
      • Pan L.
      • Yan J.
      • Wu L.
      • Zhang M.
      Assembling stable hair cell tip link complex via multidentate interactions between harmonin and cadherin 23.
      ) (Fig. 2E). In addition, HHD is found in combination with DEAD-, phosphotyrosine binding-, and formin homology domains (
      • Delhommel F.
      • Cordier F.
      • Saul F.
      • Chataigner L.
      • Haouz A.
      • Wolff N.
      Structural plasticity of the HHD2 domain of whirlin.
      ). The GO terms for the HHD proteins suggest functions in sensory perception and teleomere maintenance. Thus, both similar and versatile functions and domains are linked to αα-hub domains.

      Orchestration of function from networks by αα-hubs

      As hubs, the αα-hub domains serve to organize larger networks and multicomponent complexes. Sin3 proteins are coregulators of gene expression and implicated in processes such as cell cycle regulation, energy metabolism, senescence, and organ development (for recent reviews see (
      • Adams G.E.
      • Chandru A.
      • Cowley S.M.
      Co-repressor, co-activator and general transcription factor: the many faces of the Sin3 histone deacetylase (HDAC) complex.
      ,
      • Chaubal A.
      • Pile L.A.
      Same agent, different messages: insight into transcriptional regulation by SIN3 isoforms.
      ,
      • Kadamb R.
      • Mittal S.
      • Bansal N.
      • Batra H.
      • Saluja D.
      Sin3: insight into its transcription regulatory functions.
      )). Early studies showed that Sin3 is associated with HDAC1 and HDAC2 in multiprotein complexes, with its central domains, PAH3 and HID (Fig. 3A), interacting with the core complex components HDAC1, HDAC2, Rbbp4/7, SAP30, SAP18, and SDS3 (
      • Laherty C.D.
      • Yang W.M.
      • Jian-Min S.
      • Davie J.R.
      • Seto E.
      • Eisenman R.N.
      Histone deacetylases associated with the mSin3 corepressor mediate Mad transcriptional repression.
      ,
      • Zhang Y.
      • Iratni R.
      • Erdjument-Bromage H.
      • Tempst P.
      • Reinberg D.
      Histone deacetylases and SAP18, a novel polypeptide, are components of a human Sin3 complex.
      ,
      • Xie T.
      • He Y.
      • Korkeamaki H.
      • Zhang Y.
      • Imhoff R.
      • Lohi O.
      • Radhakrishnan I.
      Structure of the 30-kDa Sin3-associated protein (SAP30) in complex with the mammalian Sin3A corepressor and its role in nucleic acid binding.
      ,
      • Clark M.D.
      • Marcum R.
      • Graveline R.
      • Chan C.W.
      • Xie T.
      • Chen Z.
      • Ding Y.
      • Zhang Y.
      • Mondragón A.
      • David G.
      • Radhakrishnan I.
      Structural insights into the assembly of the histone deacetylase-associated Sin3L/Rpd3L corepressor complex.
      ,
      • Streubel G.
      • Fitzpatrick D.J.
      • Oliviero G.
      • Scelfo A.
      • Moran B.
      • Das S.
      • Munawar N.
      • Watson A.
      • Wynne K.
      • Negri G.L.
      • Dillon E.T.
      • Jammula S.
      • Hokamp K.
      • O’Connor D.P.
      • Pasini D.
      • et al.
      Fam60a defines a variant Sin3a-Hdac complex in embryonic stem cells required for self-renewal.
      ). The Sin3–HDAC complex mediates histone deacetylation, which together with methylation, leads to gene repression (
      • van Oevelen C.
      • Wang J.
      • Asp P.
      • Yan Q.
      • Kaelin W.G.
      • Kluger Y.
      • Dynlacht B.D.
      A role for mammalian Sin3 in permanent gene silencing.
      ), but Sin3 also interacts with the DNA demethylase Tet1 to regulate transcription epigenetically (
      • Williams K.
      • Christensen J.
      • Pedersen M.T.
      • Johansen J.V.
      • Cloos P.A.C.
      • Rappsilber J.
      • Helin K.
      TET1 and hydroxymethylcytosine in transcription and DNA methylation fidelity.
      ). The PAH1 and PAH2 domains bind numerous TFs, as shown using various different methods including biochemical methods such as pulldown assays (
      • Swanson K.A.
      • Knoepfler P.S.
      • Huang K.
      • Kang R.S.
      • Cowley S.M.
      • Laherty C.D.
      • Eisenman R.N.
      • Radhakrishnan I.
      HBP1 and Mad1 repressors bind the Sin3 corepressor PAH2 domain with opposite helical orientations.
      ,
      • Nomura M.
      • Uda-Tochio H.
      • Murai K.
      • Mori N.
      • Nishimura Y.
      The neural repressor NRSF/REST binds the PAH1 domain of the Sin3 corepressor by using its distinct short hydrophobic helix.
      ,
      • David G.
      • Alland L.
      • Hong S.H.
      • Wong C.W.
      • DePinho R.A.
      • Dejean A.
      Histone deacetylase associated with mSin3A mediates repression by the acute promyelocytic leukemia-associated PLZF protein.
      ,
      • Hurlin P.J.
      • Quéva C.
      • Eisenman R.N.
      Mnt, a novel Max-interacting protein is coexpressed with Myc in proliferating cells and mediates repression at Myc binding sites.
      ,
      • Zhang J.-S.
      • Moncrieffe M.C.
      • Kaczynski J.
      • Ellenrieder V.
      • Prendergast F.G.
      • Urrutia R.
      A conserved α-helical motif mediates the interaction of sp1-like transcriptional repressors with the corepressor mSin3A.
      ,
      • Yang Q.
      • Kong Y.
      • Rothermel B.
      • Garry D.J.
      • Bassel-Duby R.
      • Williams R.S.
      The winged-helix/forkhead protein myocyte nuclear factor beta (MNF-beta) forms a co-repressor complex with mammalian sin3B.
      ) and fluorescence anisotropy (
      • Marcum R.D.
      • Radhakrishnan I.
      The neuronal transcription factor Myt1L interacts via a conserved motif with the PAH1 domain of Sin3 to recruit the Sin3L/Rpd3L histone deacetylase complex.
      ), genetic methods such as yeast two-hybrid assays (
      • Yang Q.
      • Kong Y.
      • Rothermel B.
      • Garry D.J.
      • Bassel-Duby R.
      • Williams R.S.
      The winged-helix/forkhead protein myocyte nuclear factor beta (MNF-beta) forms a co-repressor complex with mammalian sin3B.
      ,
      • Le Guezennec X.
      • Vriend G.
      • Stunnenberg H.G.
      Molecular determinants of the interaction of Mad with the PAH2 domain of mSin3.
      ) and biophysical methods such as NMR spectroscopy (
      • Swanson K.A.
      • Knoepfler P.S.
      • Huang K.
      • Kang R.S.
      • Cowley S.M.
      • Laherty C.D.
      • Eisenman R.N.
      • Radhakrishnan I.
      HBP1 and Mad1 repressors bind the Sin3 corepressor PAH2 domain with opposite helical orientations.
      ,
      • Marcum R.D.
      • Radhakrishnan I.
      The neuronal transcription factor Myt1L interacts via a conserved motif with the PAH1 domain of Sin3 to recruit the Sin3L/Rpd3L histone deacetylase complex.
      ,
      • Brubaker K.
      • Cowley S.M.
      • Huang K.
      • Loo L.
      • Yochum G.S.
      • Ayer D.E.
      • Eisenman R.N.
      • Radhakrishnan I.
      Solution structure of the interacting domains of the mad–sin3 complex.
      ). The TF ligands include REST/NRSF (
      • Nomura M.
      • Uda-Tochio H.
      • Murai K.
      • Mori N.
      • Nishimura Y.
      The neural repressor NRSF/REST binds the PAH1 domain of the Sin3 corepressor by using its distinct short hydrophobic helix.
      ), PLZF (
      • David G.
      • Alland L.
      • Hong S.H.
      • Wong C.W.
      • DePinho R.A.
      • Dejean A.
      Histone deacetylase associated with mSin3A mediates repression by the acute promyelocytic leukemia-associated PLZF protein.
      ), Mad1/Mdx1 (
      • Brubaker K.
      • Cowley S.M.
      • Huang K.
      • Loo L.
      • Yochum G.S.
      • Ayer D.E.
      • Eisenman R.N.
      • Radhakrishnan I.
      Solution structure of the interacting domains of the mad-sin3 complex: implications for recruitment of a chromatin-modifying complex.
      ), NRL (
      • Le Guezennec X.
      • Vermeulen M.
      • Stunnenberg H.G.
      Molecular characterization of Sin3 PAH-domain interactor specificity and identification of PAH partners.
      ), HBP1 (
      • Swanson K.A.
      • Knoepfler P.S.
      • Huang K.
      • Kang R.S.
      • Cowley S.M.
      • Laherty C.D.
      • Eisenman R.N.
      • Radhakrishnan I.
      HBP1 and Mad1 repressors bind the Sin3 corepressor PAH2 domain with opposite helical orientations.
      ), FoxK1/MNF-β (
      • Yang Q.
      • Kong Y.
      • Rothermel B.
      • Garry D.J.
      • Bassel-Duby R.
      • Williams R.S.
      The winged-helix/forkhead protein myocyte nuclear factor beta (MNF-beta) forms a co-repressor complex with mammalian sin3B.
      ), Mnt/Rox (
      • Hurlin P.J.
      • Quéva C.
      • Eisenman R.N.
      Mnt, a novel Max-interacting protein is coexpressed with Myc in proliferating cells and mediates repression at Myc binding sites.
      ), KLF11/TIEG2 (
      • Zhang J.-S.
      • Moncrieffe M.C.
      • Kaczynski J.
      • Ellenrieder V.
      • Prendergast F.G.
      • Urrutia R.
      A conserved α-helical motif mediates the interaction of sp1-like transcriptional repressors with the corepressor mSin3A.
      ), and Myt1L (
      • Marcum R.D.
      • Radhakrishnan I.
      The neuronal transcription factor Myt1L interacts via a conserved motif with the PAH1 domain of Sin3 to recruit the Sin3L/Rpd3L histone deacetylase complex.
      ), which recruit the Sin3 complex to target genes to regulate expression (
      • Adams G.E.
      • Chandru A.
      • Cowley S.M.
      Co-repressor, co-activator and general transcription factor: the many faces of the Sin3 histone deacetylase (HDAC) complex.
      ). The importance of the PAH domain:ligand interactions is apparent from several studies. For example, Tet1 depends on interactions with Sin3a-PAH1 for repression of transcription in cells (
      • Chandru A.
      • Bate N.
      • Vuister G.W.
      • Cowley S.M.
      Sin3A recruits Tet1 to the PAH1 domain via a highly conserved Sin3-interaction domain.
      ) and for PLZF, which interacts with Sin3a-PAH1, histone deacetylation inhibition interferes with its ability to mediate transcriptional repression (
      • David G.
      • Alland L.
      • Hong S.H.
      • Wong C.W.
      • DePinho R.A.
      • Dejean A.
      Histone deacetylase associated with mSin3A mediates repression by the acute promyelocytic leukemia-associated PLZF protein.
      ).
      In agreement with RCD1 being a hub (
      • Jaspers P.
      • Blomster T.
      • Brosché M.
      • Salojärvi J.
      • Ahlfors R.
      • Vainonen J.P.
      • Reddy R.A.
      • Immink R.
      • Angenent G.
      • Turck F.
      • Overmyer K.
      • Kangasjärvi J.
      Unequally redundant RCD1 and SRO1 mediate stress and developmental responses and interact with transcription factors.
      ,
      • Kragelund B.B.
      • Jensen M.K.
      • Skriver K.
      Order by disorder in plant signaling.
      ), rcd1 knockout mutants, which have premature stop codons in the region encoding the PARP domain (Fig. 2B), thus affecting the RST and PARP domains, display pleiotropic phenotypes in gene expression, stress responses, and developmental processes. More specifically, rcd1 shows increased ozone and salt sensitivities, changed leaf morphologies and early flowering times, as well as altered stomatal regulation (
      • Jaspers P.
      • Blomster T.
      • Brosché M.
      • Salojärvi J.
      • Ahlfors R.
      • Vainonen J.P.
      • Reddy R.A.
      • Immink R.
      • Angenent G.
      • Turck F.
      • Overmyer K.
      • Kangasjärvi J.
      Unequally redundant RCD1 and SRO1 mediate stress and developmental responses and interact with transcription factors.
      ,
      • Kragelund B.B.
      • Jensen M.K.
      • Skriver K.
      Order by disorder in plant signaling.
      ,
      • Overmyer K.
      • Tuominen H.
      • Kettunen R.
      • Betz C.
      • Langebartels C.
      • Sandermann H.,J.
      • Kangasjarvi J.
      Ozone-sensitive Arabidopsis rcd1 mutant reveals opposite roles for ethylene and jasmonate signaling pathways in regulating superoxide-dependent cell death.
      ,
      • Ahlfors R.
      • Lång S.
      • Overmyer K.
      • Jaspers P.
      • Brosché M.
      • Tauriainen A.
      • Kollist H.
      • Tuominen H.
      • Belles-Boix E.
      • Piippo M.
      • Inzé D.
      • Palva E.T.
      • Kangasjärvi J.
      Arabidopsis radical-induced cell Death1 belongs to the WWE protein–protein interaction domain protein family and modulates abscisic acid, ethylene, and methyl jasmonate responses.
      ). The WWE domain interacts with itself and the RCD1 paralog SRO1, and with the downy mildew effector HaRxL106 to suppress plant immunity (
      • Wirthmueller L.
      • Asai S.
      • Rallapalli G.
      • Sklenar J.
      • Fabro G.
      • Kim D.S.
      • Lintermann R.
      • Jaspers P.
      • Wrzaczek M.
      • Kangasjärvi J.
      • MacLean D.
      • Menke F.L.H.
      • Banfield M.J.
      • Jones J.D.G.
      Arabidopsis downy mildew effector HaRxL106 suppresses plant immunity by binding to radical-induced cell Death1.
      ) (Fig. 3B). The interactions of RCD1-RST with TFs, which have been studied using both yeast two-hybrid assays and biophysical techniques (
      • Bugge K.
      • Staby L.
      • Kemplen K.R.
      • O’Shea C.
      • Bendsen S.K.
      • Jensen M.K.
      • Olsen J.G.
      • Skriver K.
      • Kragelund B.B.
      Structure of radical-induced cell Death1 hub domain reveals a common αα-scaffold for disorder in transcriptional networks.
      ,
      • Jaspers P.
      • Blomster T.
      • Brosché M.
      • Salojärvi J.
      • Ahlfors R.
      • Vainonen J.P.
      • Reddy R.A.
      • Immink R.
      • Angenent G.
      • Turck F.
      • Overmyer K.
      • Kangasjärvi J.
      Unequally redundant RCD1 and SRO1 mediate stress and developmental responses and interact with transcription factors.
      ,
      • O’Shea C.
      • Staby L.
      • Bendsen S.K.
      • Tidemand F.G.
      • Redsted A.
      • Willemoës M.
      • Kragelund B.B.
      • Skriver K.
      Structures and short linear motif of disordered transcription factor regions provide clues to the interactome of the cellular hub protein radical-induced cell death 1.
      ), play important roles in plant biology. Thus, the RST domain of RCD1 affects stress responses via interactions with DREB2a (
      • Vainonen J.P.
      • Jaspers P.
      • Wrzaczek M.
      • Lamminmäki A.
      • Reddy R. a
      • Vaahtera L.
      • Brosché M.
      • Kangasjärvi J.
      RCD1-DREB2A interaction in leaf senescence and stress responses in Arabidopsis thaliana.
      ) and ANAC013 and ANAC017 (
      • Shapiguzov A.
      • Vainonen J.P.
      • Hunter K.
      • Tossavainen H.
      • Tiwari A.
      • Järvi S.
      • Hellman M.
      • Aarabi F.
      • Alseekh S.
      • Wybouw B.
      • Van Der Kelen K.
      • Nikkanen L.
      • Krasensky-Wrzaczek J.
      • Sipari N.
      • Keinänen M.
      • et al.
      Arabidopsis RCD1 coordinates chloroplast and mitochondrial functions through interaction with ANAC transcription factors.
      ,
      • De Clercq I.
      • Vermeirssen V.
      • Van Aken O.
      • Vandepoele K.
      • Murcha M.W.
      • Law S.R.
      • Inzé A.
      • Ng S.
      • Ivanova A.
      • Rombaut D.
      • van de Cotte B.
      • Jaspers P.
      • Van de Peer Y.
      • Kangasjärvi J.
      • Whelan J.
      • et al.
      The membrane-bound NAC transcription factor ANAC013 functions in mitochondrial retrograde regulation of the oxidative stress response in Arabidopsis.
      ), the latter two of which contribute to coordination of reactive oxygen species signals emitted from mitochondria and chloroplasts (
      • Shapiguzov A.
      • Vainonen J.P.
      • Hunter K.
      • Tossavainen H.
      • Tiwari A.
      • Järvi S.
      • Hellman M.
      • Aarabi F.
      • Alseekh S.
      • Wybouw B.
      • Van Der Kelen K.
      • Nikkanen L.
      • Krasensky-Wrzaczek J.
      • Sipari N.
      • Keinänen M.
      • et al.
      Arabidopsis RCD1 coordinates chloroplast and mitochondrial functions through interaction with ANAC transcription factors.
      ).
      The TAFH protein TAF4 is crucial for structural integrity of the TFIID complex (
      • Wright K.J.
      • Marr M.T.
      • Tjian R.
      TAF4 nucleates a core subcomplex of TFIID and mediates activated transcription from a TATA-less promoter.
      ), which contains 13 additional TAF subunits and TATA binding protein (TBP) that by binding to genes triggers formation of the transcriptional preinitiation complex (
      • Kazantseva J.
      • Palm K.
      Diversity in TAF proteomics: consequences for cellular differentiation and migration.
      ,
      • Papai G.
      • Weil P.A.
      • Schultz P.
      New insights into the function of transcription factor TFIID from recent structural studies.
      ,
      • Thuault S.
      • Gangloff Y.G.
      • Kirchner J.
      • Sanders S.
      • Werten S.
      • Romier C.
      • Weil P.A.
      • Davidson I.
      Functional analysis of the TFIID-specific yeast TAF4 (yTAFII48) reveals an unexpected organization of its histone-fold domain.
      ) (Fig. 3C). This, in addition, contains RNA polymerase II, general TFs, and the large Mediator complex. Through HF domains, TAF4 interacts with TAF12 to stabilize the TFIID complex (
      • Gazit K.
      • Moshonov S.
      • Elfakess R.
      • Sharon M.
      • Mengus G.
      • Davidson I.
      • Dikstein R.
      TAF4/4b·TAF12 displays a unique mode of DNA binding and is required for core promoter function of a subset of genes.
      ). The TAFH domain contributes to the regulation of the expression of approximately 400 genes (
      • Chen W.Y.
      • Zhang J.
      • Geng H.
      • Du Z.
      • Nakadai T.
      • Roeder R.G.
      A TAF4 coactivator function for E proteins that involves enhanced TFIID binding.
      ) and has been experimentally shown to interact directly with TFs such as ZF and LZIP (
      • Wang X.
      • Truckses D.M.
      • Takada S.
      • Matsumura T.
      • Tanese N.
      • Jacobson R.H.
      Conserved region I of human coactivator TAF4 binds to a short hydrophobic motif present in transcriptional regulators.
      ). TAFH binding of the E-protein TFs, HEB and E2A, implicated in embryonic pattering and programmed cell death (
      • Quong M.W.
      • Romanow W.J.
      • Murre C.
      E protein function in lymphocyte development.
      ), is critical to gene activation by enhancing TFIID promoter binding (
      • Chen W.Y.
      • Zhang J.
      • Geng H.
      • Du Z.
      • Nakadai T.
      • Roeder R.G.
      A TAF4 coactivator function for E proteins that involves enhanced TFIID binding.
      ).
      CBP is a central node in eukaryotic regulatory networks (
      • Dyson H.J.
      • Wright P.E.
      Role of intrinsic protein disorder in the function and interactions of the transcriptional coactivators CREB-binding protein (CBP) and p300.
      ) and regulates TFs and chromatin via its histone acetyl transferase activity (
      • Janknecht R.
      • Hunter T.
      A growing coactivator network.
      ). The TAZ, KIX, and NCBD domains form the scaffold for the interactions of CBP with IDRs of regulatory proteins (
      • Dyson H.J.
      • Wright P.E.
      Role of intrinsic protein disorder in the function and interactions of the transcriptional coactivators CREB-binding protein (CBP) and p300.
      ). NCBD alone has multiple experimentally identified interaction partners, including IRF-3 (
      • Lin C.H.
      • Hare B.J.
      • Wagner G.
      • Harrison S.C.
      • Maniatis T.
      • Fraenkel E.
      A small domain of CBP/p300 binds diverse proteins: solution structure and functional studies.
      ), p160 nuclear receptor coactivator 1 (NCOA1;Src1), NCOA2 (Tif2), and NCOA3 (ACTR) (
      • Lin C.H.
      • Hare B.J.
      • Wagner G.
      • Harrison S.C.
      • Maniatis T.
      • Fraenkel E.
      A small domain of CBP/p300 binds diverse proteins: solution structure and functional studies.
      ,
      • Demarest S.J.
      Packing, specificity, and mutability at the binding interface between the p160 coactivator and CREB-binding protein.
      ,
      • Kamei Y.
      • Xu L.
      • Heinzel T.
      • Torchia J.
      • Kurokawa R.
      • Gloss B.
      • Lin S.C.
      • Heyman R.A.
      • Rose D.W.
      • Glass C.K.
      • Rosenfeld M.G.
      A CBP integrator complex mediates transcriptional activation and AP-1 inhibition by nuclear receptors.
      ), tumor suppressor p53 (
      • Lee C.W.
      • Martinez-Yamout M.A.
      • Dyson H.J.
      • Wright P.E.
      Structure of the p53 transactivation domain in complex with the nuclear receptor coactivator binding domain of CREB binding protein.
      ), Ets-2 (
      • Lin C.H.
      • Hare B.J.
      • Wagner G.
      • Harrison S.C.
      • Maniatis T.
      • Fraenkel E.
      A small domain of CBP/p300 binds diverse proteins: solution structure and functional studies.
      ), Smad3 and 4 (
      • Janknecht R.
      • Wells N.J.
      • Hunter T.
      TGF-β-stimulated cooperation of Smad proteins with the coactivators CBP/p300.
      ), Stat6 (
      • Gingras S.
      • Simard J.
      • Groner B.
      • Pfitzner E.
      p300/CBP is required for transcriptional induction by interleukin-4 and interacts with Stat6.
      ), and the adenoviral protein E1A (
      • Lin C.H.
      • Hare B.J.
      • Wagner G.
      • Harrison S.C.
      • Maniatis T.
      • Fraenkel E.
      A small domain of CBP/p300 binds diverse proteins: solution structure and functional studies.
      ) (Fig. 3D). Also, in these cases, the ligands may depend on interactions with the αα-hub domain, both in vivo and in vitro, as in the transcriptional activation by IRF-3 (
      • Lin C.H.
      • Hare B.J.
      • Wagner G.
      • Harrison S.C.
      • Maniatis T.
      • Fraenkel E.
      A small domain of CBP/p300 binds diverse proteins: solution structure and functional studies.
      ).
      The HHD proteins whirlin and harmonin are implicated in Usher syndrome causing hearing-vision loss (
      • Saihan Z.
      • Webster A.R.
      • Luxon L.
      • Bitner-Glindzicz M.
      Update on Usher syndrome.
      ). Usher syndrome proteins are organized in interactomes with harmonin, whirlin, and sans as scaffolds and cadherin23 (Cad23), protocad15, sans, VlgR, and Ush2C binding to harmonin (Fig. 3E) (
      • Sorusch N.
      • Wunderlich K.
      • Bauss K.
      • Nagel-Wolfrum K.
      • Wolfrum U.
      Usher syndrome protein network functions in the retina and their relation to other retinal ciliopathies.
      ,
      • Siemens J.
      • Kazmierczak P.
      • Reynolds A.
      • Sticker M.
      • Littlewood-Evans A.
      • Müller U.
      The Usher syndrome proteins cadherin 23 and harmonin form a complex by means of PDZ-domain interactions.
      ). Structural and thermodynamic analyses have shown that HHD and PDZ1 of harmonin form a supramodule that binds sans with high affinity (
      • Yan J.
      • Pan L.
      • Chen X.
      • Wu L.
      • Zhang M.
      The structure of the harmonin/sans complex reveals an unexpected interaction mode of the two Usher syndrome proteins.
      ), and harmonin-HHD also binds Cad23 (
      • Pan L.
      • Yan J.
      • Wu L.
      • Zhang M.
      Assembling stable hair cell tip link complex via multidentate interactions between harmonin and cadherin 23.
      ). This interaction, together with the harmonin-PDZ2:Cad23 interaction, represents multidentate binding via supramodule exploitation (
      • Yan J.
      • Pan L.
      • Chen X.
      • Wu L.
      • Zhang M.
      The structure of the harmonin/sans complex reveals an unexpected interaction mode of the two Usher syndrome proteins.
      ), providing a structural platform for the tip link complex of stereocilia (
      • Pan L.
      • Yan J.
      • Wu L.
      • Zhang M.
      Assembling stable hair cell tip link complex via multidentate interactions between harmonin and cadherin 23.
      ). Furthermore, the tail of Cad23 promotes Cad23:harmonin polymer formation by binding to harmonin-HHD or by self-dimerization (
      • Wu L.
      • Pan L.
      • Zhang C.
      • Zhang M.
      Large protein assemblies formed by multivalent interactions between cadherin23 and harmonin suggest a stable anchorage structure at the tip link of stereocilia.
      ). Harmonin thus connects tip link complexes with the actin cytoskeleton (
      • Boëda B.
      • El-Amraoui A.
      • Bahloul A.
      • Goodyear R.
      • Daviet L.
      • Blanchard S.
      • Perfettini I.
      • Fath K.R.
      • Shorte S.
      • Reiners J.
      • Houdusse A.
      • Legrain P.
      • Wolfrum U.
      • Richardson G.
      • Petit C.
      Myosin VIIa, harmonin and cadherin 23, three Usher I gene products that cooperate to shape the sensory hair cell bundle.
      ). For an αα-hub protein, harmonin has an atypical biological function and sensory perception, but typical molecular function in scaffolding.

      The αα-hub domains as protein–protein interaction hubs

      Disordered αα-hub ligands have SLiMs of similar characteristics that maintain specificity

      Many αα-hub ligands use IDRs for binding, but identification of most αα-hub ligands dates back before the general appreciation of ID. Still, ID has often been mentioned as a feature of the free state of the hub-binding regions (
      • Kumar G.S.
      • Xie T.
      • Zhang Y.
      • Radhakrishnan I.
      Solution structure of the mSin3A PAH2-Pf1 SID1 complex: a Mad1/Mxd1-like interaction disrupted by MRG15 in the Rpd3S/Sin3S complex.
      ,
      • Brubaker K.
      • Cowley S.M.
      • Huang K.
      • Loo L.
      • Yochum G.S.
      • Ayer D.E.
      • Eisenman R.N.
      • Radhakrishnan I.
      Solution structure of the interacting domains of the mad–sin3 complex.
      ,
      • Sahu S.C.
      • Swanson K.A.
      • Kang R.S.
      • Huang K.
      • Brubaker K.
      • Ratcliff K.
      • Radhakrishnan I.
      Conserved themes in target recognition by the PAH1 and PAH2 domains of the Sin3 transcriptional corepressor.
      ) or has been computationally predicted (
      • Staby L.
      • Bugge K.
      • Falbe-Hansen R.G.
      • Saladini E.
      • Skriver K.
      • Kragelund B.B.
      Connecting the αα-hubs: same fold, disordered ligands, new functions.
      ), whereas experimental characterization of the IDRs has mostly appeared in studies of RST (
      • Christensen L.F.
      • Staby L.
      • Bugge K.
      • O’Shea C.
      • Kragelund B.B.
      • Skriver K.
      Evolutionary conservation of the intrinsic disorder-based radical-induced cell Death1 hub interactome.
      ,
      • O’Shea C.
      • Kryger M.
      • Stender E.G.P.
      • Kragelund B.B.
      • Willemoës M.
      • Skriver K.
      Protein intrinsic disorder in Arabidopsis NAC transcription factors: transcriptional activation by ANAC013 and ANAC046 and their interactions with RCD1.
      ,
      • Kjaersgaard T.
      • Jensen M.K.
      • Christiansen M.W.
      • Gregersen P.
      • Kragelund B.B.
      • Skriver K.
      Senescence-associated barley NAC (NAM, ATAF1,2, CUC) transcription factor interacts with radical-induced cell death 1 through a disordered regulatory domain.
      ,
      • Staby L.
      • O’Shea C.
      • Willemoës M.
      • Theisen F.
      • Kragelund B.B.
      • Skriver K.
      Eukaryotic transcription factors: paradigms of protein intrinsic disorder.
      ) and NCBD ligands (
      • Demarest S.J.
      Packing, specificity, and mutability at the binding interface between the p160 coactivator and CREB-binding protein.
      ,
      • Lee C.W.
      • Martinez-Yamout M.A.
      • Dyson H.J.
      • Wright P.E.
      Structure of the p53 transactivation domain in complex with the nuclear receptor coactivator binding domain of CREB binding protein.
      ,
      • Haberz P.
      • Arai M.
      • Martinez-Yamout M.A.
      • Dyson H.J.
      • Wright P.E.
      Mapping the interactions of adenoviral E1A proteins with the p160 nuclear receptor coactivator binding domain of CBP.
      ). Molecular dynamics (MD) simulations have also been used for characterizing free αα-hub ligands. Thus, the Sin3b-PAH1-binding region of REST was suggested to fluctuate between hairpins, helices, and bent structures with population shifts and induced folding working cooperatively in coupled folding and binding (Fig. 5A) (
      • Higo J.
      • Nishimura Y.
      • Nakamura H.
      A free-energy landscape for coupled folding and binding of an intrinsically disordered protein in explicit solvent from detailed all-atom computations.
      ). ID-associated flexibility provides the structural adaptability needed for REST to function as a hub itself, and for the αα-hub ligands, ID is in general a prerequisite for adaptable SLiM-based interactions.
      Figure thumbnail gr5
      Figure 5The modus operandi of αα-hubs. A, the αα-hub-binding region of free protein ligand may fluctuate between hairpins, helices, and bent structures as in the case of the Sin3b-PAH1-binding SLiM of REST (
      • Higo J.
      • Nishimura Y.
      • Nakamura H.
      A free-energy landscape for coupled folding and binding of an intrinsically disordered protein in explicit solvent from detailed all-atom computations.
      ). B, protein ligand using a SLiM with hydrophobic and acidic residues for αα-hub binding as in the Sin3-PAH2-binding SLiM of Mad1 (
      • Cowley S.M.
      • Kang R.S.
      • Frangioni J.V.
      • Yada J.J.
      • DeGrand A.M.
      • Radhakrishnan I.
      • Eisenman R.N.
      Functional analysis of the Mad1-mSin3A repressor-corepressor interaction reveals determinants of specificity, affinity, and transcriptional response.
      ). The SLiM is often part of a larger intrinsically disordered context. C, protein ligands may use SLiM reversibility for governing specificity as in the case of Sap25 and REST binding to Sin3-PAH1 (Protein Data Bank [PDB] codes 1s5q and 1s5r) (
      • Swanson K.A.
      • Knoepfler P.S.
      • Huang K.
      • Kang R.S.
      • Cowley S.M.
      • Laherty C.D.
      • Eisenman R.N.
      • Radhakrishnan I.
      HBP1 and Mad1 repressors bind the Sin3 corepressor PAH2 domain with opposite helical orientations.
      ). D, ligands using coupled folding and binding, through conformational selection and/or induced fit, as in the case of ACTR association with NCBD (based on PDB codes 2kkj and 1kbh) (
      • Iešmantavičius V.
      • Dogan J.
      • Jemth P.
      • Teilum K.
      • Kjaergaard M.
      Helical propensity in an intrinsically disordered protein accelerates ligand binding.
      ). E, αα-Hub:ligand complexes may retain some disorder as in the case of the Sin3a-PAH1:SAP25 complex (PDB code 2rms) (
      • Sahu S.C.
      • Swanson K.A.
      • Kang R.S.
      • Huang K.
      • Brubaker K.
      • Ratcliff K.
      • Radhakrishnan I.
      Conserved themes in target recognition by the PAH1 and PAH2 domains of the Sin3 transcriptional corepressor.
      ). F, structural heterogeneity in an αα-hub:ligand complex as in the case of RCD1-RST complexes with NAC and DREB2a transcription factors (PDB codes 5oao and 5oap) (
      • Bugge K.
      • Staby L.
      • Kemplen K.R.
      • O’Shea C.
      • Bendsen S.K.
      • Jensen M.K.
      • Olsen J.G.
      • Skriver K.
      • Kragelund B.B.
      Structure of radical-induced cell Death1 hub domain reveals a common αα-scaffold for disorder in transcriptional networks.
      ,
      • Christensen L.F.
      • Staby L.
      • Bugge K.
      • O’Shea C.
      • Kragelund B.B.
      • Skriver K.
      Evolutionary conservation of the intrinsic disorder-based radical-induced cell Death1 hub interactome.
      ,
      • O’Shea C.
      • Staby L.
      • Bendsen S.K.
      • Tidemand F.G.
      • Redsted A.
      • Willemoës M.
      • Kragelund B.B.
      • Skriver K.
      Structures and short linear motif of disordered transcription factor regions provide clues to the interactome of the cellular hub protein radical-induced cell death 1.
      ). G, αα-hub domains may fold synergistically with a disordered protein ligand to form different bound ligand structures as in the case of NCBD complexes with Src1 (left) and ACTR (right), respectively (PDB codes 2c52 and 1kbh) (
      • Waters L.
      • Yue B.
      • Veverka V.
      • Renshaw P.
      • Bramham J.
      • Matsuda S.
      • Frenkiel T.
      • Kelly G.
      • Muskett F.
      • Carr M.
      • Heery D.M.
      Structural diversity in p160/CREB-binding protein coactivator complexes.
      ). H, allosteric effects of the SLiM context on ligand association with αα-hubs as in the case of RCD1-RST association with ANAC013 (
      • O’Shea C.
      • Staby L.
      • Bendsen S.K.
      • Tidemand F.G.
      • Redsted A.
      • Willemoës M.
      • Kragelund B.B.
      • Skriver K.
      Structures and short linear motif of disordered transcription factor regions provide clues to the interactome of the cellular hub protein radical-induced cell death 1.
      ). I, αα-hubs may be part of supramodules as in the case of the harmonin:sans complex (PDB code 3k1r) (
      • Yan J.
      • Pan L.
      • Chen X.
      • Wu L.
      • Zhang M.
      The structure of the harmonin/sans complex reveals an unexpected interaction mode of the two Usher syndrome proteins.
      ).
      Although different intrinsically disordered ligands use different SLiMs for αα-hub binding, most are simple and depend on hydrophobic residues for contacts with the hydrophobic αα-hub cleft (Fig. 5B). Initial work to identify a PAH2-binding SLiM based on screenings, sequence comparisons, ligand affinity measurements, as well as structural analysis revealed the motif φΖΖφφΧAAΧΧφnΧΧn (X, nonproline residue; φ, bulky hydrophobic residue; Z, aliphatic side chain; n, negatively charged) (
      • Spronk C.A.E.M.
      • Tessari M.
      • Kaan A.M.
      • Jansen J.F.A.
      • Vermeulen M.
      • Stunnenberg H.G.
      • Vuister G.W.
      The Mad1-Sin3B interaction involves a novel helical fold.
      ,
      • Van Ingen H.
      • Lasonder E.
      • Jansen J.F.A.
      • Kaan A.M.
      • Spronk C.A.E.M.
      • Stunnenberg H.G.
      • Vuister G.W.
      Extension of the binding motif of the Sin3 interacting domain of the mad family proteins.
      ,
      • Brubaker K.
      • Cowley S.M.
      • Huang K.
      • Loo L.
      • Yochum G.S.
      • Ayer D.E.
      • Eisenman R.N.
      • Radhakrishnan I.
      Solution structure of the interacting domains of the mad-sin3 complex: implications for recruitment of a chromatin-modifying complex.
      ,
      • Le Guezennec X.
      • Vermeulen M.
      • Stunnenberg H.G.
      Molecular characterization of Sin3 PAH-domain interactor specificity and identification of PAH partners.
      ). Later, structural work identified two orientations of PAH-bound SLiMs, types I and II, as exemplified by the PAH1-binding SLiMs from REST (φXφφSXφS) (
      • Nomura M.
      • Uda-Tochio H.
      • Murai K.
      • Mori N.
      • Nishimura Y.
      The neural repressor NRSF/REST binds the PAH1 domain of the Sin3 corepressor by using its distinct short hydrophobic helix.
      ,
      • Sahu S.C.
      • Swanson K.A.
      • Kang R.S.
      • Huang K.
      • Brubaker K.
      • Ratcliff K.
      • Radhakrishnan I.
      Conserved themes in target recognition by the PAH1 and PAH2 domains of the Sin3 transcriptional corepressor.
      ) and Sap25 (
      • Sahu S.C.
      • Swanson K.A.
      • Kang R.S.
      • Huang K.
      • Brubaker K.
      • Ratcliff K.
      • Radhakrishnan I.
      Conserved themes in target recognition by the PAH1 and PAH2 domains of the Sin3 transcriptional corepressor.
      ) (SφXSφφXφ) (S, short side chain) (Table 1), respectively. In PAH2-complexes, the SLiMs of Pf1 and Mad1 (φΖΖφφΧAAΧΧφn) and of HBP1 (A(A/V)XφφXXφ) also adapt different orientations (Fig. 5C) (
      • Van Ingen H.
      • Lasonder E.
      • Jansen J.F.A.
      • Kaan A.M.
      • Spronk C.A.E.M.
      • Stunnenberg H.G.
      • Vuister G.W.
      Extension of the binding motif of the Sin3 interacting domain of the mad family proteins.
      ,
      • Swanson K.A.
      • Knoepfler P.S.
      • Huang K.
      • Kang R.S.
      • Cowley S.M.
      • Laherty C.D.
      • Eisenman R.N.
      • Radhakrishnan I.
      HBP1 and Mad1 repressors bind the Sin3 corepressor PAH2 domain with opposite helical orientations.
      ,
      • Kumar G.S.
      • Xie T.
      • Zhang Y.
      • Radhakrishnan I.
      Solution structure of the mSin3A PAH2-Pf1 SID1 complex: a Mad1/Mxd1-like interaction disrupted by MRG15 in the Rpd3S/Sin3S complex.
      ). Despite fold similarities and SLiM simplicities, the αα-hubs show remarkable selectivity. The ∼40 times difference in affinities of Sin3-PAH2 for Mad1 (Kd ∼50 nM) (
      • Brubaker K.
      • Cowley S.M.
      • Huang K.
      • Loo L.
      • Yochum G.S.
      • Ayer D.E.
      • Eisenman R.N.
      • Radhakrishnan I.
      Solution structure of the interacting domains of the mad-sin3 complex: implications for recruitment of a chromatin-modifying complex.
      ,
      • Sahu S.C.
      • Swanson K.A.
      • Kang R.S.
      • Huang K.
      • Brubaker K.
      • Ratcliff K.
      • Radhakrishnan I.
      Conserved themes in target recognition by the PAH1 and PAH2 domains of the Sin3 transcriptional corepressor.
      ,
      • Cowley S.M.
      • Kang R.S.
      • Frangioni J.V.
      • Yada J.J.
      • DeGrand A.M.
      • Radhakrishnan I.
      • Eisenman R.N.
      Functional analysis of the Mad1-mSin3A repressor-corepressor interaction reveals determinants of specificity, affinity, and transcriptional response.
      ) and Pf1 (Kd ∼2 μM) (
      • Kumar G.S.
      • Xie T.
      • Zhang Y.
      • Radhakrishnan I.
      Solution structure of the mSin3A PAH2-Pf1 SID1 complex: a Mad1/Mxd1-like interaction disrupted by MRG15 in the Rpd3S/Sin3S complex.
      ) was explained by a phenylalanine in the first position of the Pf1-SLiM constituting a steric disadvantage (
      • Kumar G.S.
      • Xie T.
      • Zhang Y.
      • Radhakrishnan I.
      Solution structure of the mSin3A PAH2-Pf1 SID1 complex: a Mad1/Mxd1-like interaction disrupted by MRG15 in the Rpd3S/Sin3S complex.
      ). The minimal Mad1-SLiM consists of eight residues, with only three being essential for the interactions with Sin3-PAH2. One of these, L12, inserts into the hydrophobic cleft of PAH2 and is important for affinity, whereas the other two, A15 and A16, determine specificity for PAH2, owing to their proximity to bulky side chains of PAH2 in the complex. Thus, hydrophobic residues are implicated in both affinity and specificity of PAH:SLiM interactions (
      • Cowley S.M.
      • Kang R.S.
      • Frangioni J.V.
      • Yada J.J.
      • DeGrand A.M.
      • Radhakrishnan I.
      • Eisenman R.N.
      Functional analysis of the Mad1-mSin3A repressor-corepressor interaction reveals determinants of specificity, affinity, and transcriptional response.
      ).
      Table 1αα-Hub-interacting SLiMs
      Ligandαα-HubSLiMReference
      ACTRCBP-NCBDφφXXφ and φXXφφ
      X, nonproline residue; φ, bulky hydrophobic residues; Z, aliphatic component in the side chain; S, short side chain; n, negatively charged.
      (
      • Demarest S.J.
      • Martinez-Yamout M.
      • Chung J.
      • Chen H.
      • Xu W.
      • Dyson H.J.
      • Evans R.M.
      • Wright P.E.
      Mutual synergistic folding in recruitment of CBP/p300 by p160 nuclear receptor coactivators.
      )
      ANAC013/016/017/046, bZIP23, COL10, DREB2a/b/c, STORCD1-RST(D/E)X(1,2)(Y/F)X(1,4)(D/E)L(
      • O’Shea C.
      • Staby L.
      • Bendsen S.K.
      • Tidemand F.G.
      • Redsted A.
      • Willemoës M.
      • Kragelund B.B.
      • Skriver K.
      Structures and short linear motif of disordered transcription factor regions provide clues to the interactome of the cellular hub protein radical-induced cell death 1.
      )
      ANAC087RCD1-RST(Y/F)X(1,4)(D/E)(LI)(
      • Christensen L.F.
      • Staby L.
      • Bugge K.
      • O’Shea C.
      • Kragelund B.B.
      • Skriver K.
      Evolutionary conservation of the intrinsic disorder-based radical-induced cell Death1 hub interactome.
      )
      cMyb. HEB, N-Cor, STAT6ETO-TAFH(D/E)φXφφ(
      • Park S.
      • Chen W.
      • Cierpicki T.
      • Tonelli M.
      • Cai X.
      • Speck N.A.
      • Bushweller J.H.
      Structure of the AML1-ETO eTAFH domain-HEB peptide complex and its contribution to AML1-ETO activity.
      ,
      • Zhang J.
      • Kalkum M.
      • Yamamura S.
      • Chait B.T.
      • Roeder R.G.
      E protein silencing by the leukemogenic AML1-ETO fusion protein.
      )
      E2A, LZIP, ZFTAF4-TAFHDφφXXφφ(
      • Wang X.
      • Truckses D.M.
      • Takada S.
      • Matsumura T.
      • Tanese N.
      • Jacobson R.H.
      Conserved region I of human coactivator TAF4 binds to a short hydrophobic motif present in transcriptional regulators.
      )
      HBP1Sin3-PAH2A(A/V)XφφXXφ (type II)(
      • Swanson K.A.
      • Knoepfler P.S.
      • Huang K.
      • Kang R.S.
      • Cowley S.M.
      • Laherty C.D.
      • Eisenman R.N.
      • Radhakrishnan I.
      HBP1 and Mad1 repressors bind the Sin3 corepressor PAH2 domain with opposite helical orientations.
      )
      Mad1, Pf1Sin3-PAH2φΖΖφφΧAAΧΧφn (type I)(
      • Van Ingen H.
      • Lasonder E.
      • Jansen J.F.A.
      • Kaan A.M.
      • Spronk C.A.E.M.
      • Stunnenberg H.G.
      • Vuister G.W.
      Extension of the binding motif of the Sin3 interacting domain of the mad family proteins.
      ,
      • Kumar G.S.
      • Xie T.
      • Zhang Y.
      • Radhakrishnan I.
      Solution structure of the mSin3A PAH2-Pf1 SID1 complex: a Mad1/Mxd1-like interaction disrupted by MRG15 in the Rpd3S/Sin3S complex.
      ,
      • Cowley S.M.
      • Kang R.S.
      • Frangioni J.V.
      • Yada J.J.
      • DeGrand A.M.
      • Radhakrishnan I.
      • Eisenman R.N.
      Functional analysis of the Mad1-mSin3A repressor-corepressor interaction reveals determinants of specificity, affinity, and transcriptional response.
      )
      p53CBP-NCBDφφXXφ and φXXφφ(
      • Lee C.W.
      • Martinez-Yamout M.A.
      • Dyson H.J.
      • Wright P.E.
      Structure of the p53 transactivation domain in complex with the nuclear receptor coactivator binding domain of CREB binding protein.
      )
      RESTSin3-PAH1φXφφSXφS (type I)(
      • Nomura M.
      • Uda-Tochio H.
      • Murai K.
      • Mori N.
      • Nishimura Y.
      The neural repressor NRSF/REST binds the PAH1 domain of the Sin3 corepressor by using its distinct short hydrophobic helix.
      ,
      • Sahu S.C.
      • Swanson K.A.
      • Kang R.S.
      • Huang K.
      • Brubaker K.
      • Ratcliff K.
      • Radhakrishnan I.
      Conserved themes in target recognition by the PAH1 and PAH2 domains of the Sin3 transcriptional corepressor.
      )
      SAP25Sin3-PAH1SφXSφφXφ (type II)(
      • Sahu S.C.
      • Swanson K.A.
      • Kang R.S.
      • Huang K.
      • Brubaker K.
      • Ratcliff K.
      • Radhakrishnan I.
      Conserved themes in target recognition by the PAH1 and PAH2 domains of the Sin3 transcriptional corepressor.
      )
      a X, nonproline residue; φ, bulky hydrophobic residues; Z, aliphatic component in the side chain; S, short side chain; n, negatively charged.
      A combined bioinformatics and experimental approach, including substitution analysis, was used to identify the RST-binding SLiM (D/E)X(1,2)(Y/F)X(1,4)(D/E)L (where X(1,2) denotes 1 or 2 Xs) (Table 1), which has essential binding contributions from aromatic, acidic, and leucine residues (
      • O’Shea C.
      • Kryger M.
      • Stender E.G.P.
      • Kragelund B.B.
      • Willemoës M.
      • Skriver K.
      Protein intrinsic disorder in Arabidopsis NAC transcription factors: transcriptional activation by ANAC013 and ANAC046 and their interactions with RCD1.
      ,
      • O’Shea C.
      • Staby L.
      • Bendsen S.K.
      • Tidemand F.G.
      • Redsted A.
      • Willemoës M.
      • Kragelund B.B.
      • Skriver K.
      Structures and short linear motif of disordered transcription factor regions provide clues to the interactome of the cellular hub protein radical-induced cell death 1.
      ). The RST domain and the RST-binding SLiM were traced back 480 million years to the emergence of land plants, and SLiM variants, identified from the evolutionary analysis, suggested numerous additional RCD1-interactome members (
      • Christensen L.F.
      • Staby L.
      • Bugge K.
      • O’Shea C.
      • Kragelund B.B.
      • Skriver K.
      Evolutionary conservation of the intrinsic disorder-based radical-induced cell Death1 hub interactome.
      ). Among the few known TAFH ligands (Fig. 3D), the TFs HEB, cMyb, and STAT6 and the corepressor N-Cor use the SLiM (D/E)φXφφ for binding ETO-TAFH (
      • Park S.
      • Chen W.
      • Cierpicki T.
      • Tonelli M.
      • Cai X.
      • Speck N.A.
      • Bushweller J.H.
      Structure of the AML1-ETO eTAFH domain-HEB peptide complex and its contribution to AML1-ETO activity.
      ,
      • Zhang J.
      • Kalkum M.
      • Yamamura S.
      • Chait B.T.
      • Roeder R.G.
      E protein silencing by the leukemogenic AML1-ETO fusion protein.
      ). Using phage display, DφφXXφφ was identified as the TAF4-TAFH-binding SLiM present in ZF, LZIP, and E2A (
      • Wang X.
      • Truckses D.M.
      • Takada S.
      • Matsumura T.
      • Tanese N.
      • Jacobson R.H.
      Conserved region I of human coactivator TAF4 binds to a short hydrophobic motif present in transcriptional regulators.
      ). The lack of a common NCBD-binding SLiM likely reflects partner-templated modulation of the NCBD structures. However, similar SLiMs, φφXXφ or φXXφφ, mediate the interactions between NCBD and ACTR, and the TRD regions activation domain (AD)1 and AD2 of p53 (
      • Lee C.W.
      • Martinez-Yamout M.A.
      • Dyson H.J.
      • Wright P.E.
      Structure of the p53 transactivation domain in complex with the nuclear receptor coactivator binding domain of CREB binding protein.
      ,
      • Demarest S.J.
      • Martinez-Yamout M.
      • Chung J.
      • Chen H.
      • Xu W.
      • Dyson H.J.
      • Evans R.M.
      • Wright P.E.
      Mutual synergistic folding in recruitment of CBP/p300 by p160 nuclear receptor coactivators.
      ). HHDs have only a few identified ligands and no known SLiMs. Still, similar to other αα-hub ligands, hydrophobic residues are prominent in the HHD-binding ligand region (
      • Wu L.
      • Pan L.
      • Zhang C.
      • Zhang M.
      Large protein assemblies formed by multivalent interactions between cadherin23 and harmonin suggest a stable anchorage structure at the tip link of stereocilia.
      ,
      • Pan L.
      • Yan J.
      • Wu L.
      • Zhang M.
      Assembling stable hair cell tip link complex via multidentate interactions between harmonin and cadherin 23.
      ).
      In summary, the simple generic φXXφφ, recurring in TF:coregulator interactions (
      • Plevin M.J.
      • Mills M.M.
      • Ikura M.
      The LxxLL motif: a multifunctional binding sequence in transcriptional regulation.
      ), is also dominant among the αα-hub-interacting SLiMs, which use both hydrophobic and charged residues for securing binding affinity and specificity. Furthermore, PAH1 and PAH2 may use SLiM reversibility for governing specificity.

      The affinities and thermodynamic profiles of αα-hub interactions vary

      The affinities of the αα-hub:ligand interactions have been determined using a number of different methods including stopped-flow fluorescence spectroscopy, fluorescence titration, NMR spectroscopy, surface plasmon resonance, and isothermal titration calorimetry (ITC), with ITC being the most frequently used (
      • Van Ingen H.
      • Lasonder E.
      • Jansen J.F.A.
      • Kaan A.M.
      • Spronk C.A.E.M.
      • Stunnenberg H.G.
      • Vuister G.W.
      Extension of the binding motif of the Sin3 interacting domain of the mad family proteins.
      ,
      • O’Shea C.
      • Staby L.
      • Bendsen S.K.
      • Tidemand F.G.
      • Redsted A.
      • Willemoës M.
      • Kragelund B.B.
      • Skriver K.
      Structures and short linear motif of disordered transcription factor regions provide clues to the interactome of the cellular hub protein radical-induced cell death 1.
      ,
      • Pan L.
      • Yan J.
      • Wu L.
      • Zhang M.
      Assembling stable hair cell tip link complex via multidentate interactions between harmonin and cadherin 23.
      ,
      • Lee C.W.
      • Martinez-Yamout M.A.
      • Dyson H.J.
      • Wright P.E.
      Structure of the p53 transactivation domain in complex with the nuclear receptor coactivator binding domain of CREB binding protein.
      ,
      • Haberz P.
      • Arai M.
      • Martinez-Yamout M.A.
      • Dyson H.J.
      • Wright P.E.
      Mapping the interactions of adenoviral E1A proteins with the p160 nuclear receptor coactivator binding domain of CBP.
      ,
      • Demarest S.J.
      • Martinez-Yamout M.
      • Chung J.
      • Chen H.
      • Xu W.
      • Dyson H.J.
      • Evans R.M.
      • Wright P.E.
      Mutual synergistic folding in recruitment of CBP/p300 by p160 nuclear receptor coactivators.
      ,
      • Dogan J.
      • Jonasson J.
      • Andersson E.
      • Jemth P.
      Binding rate constants reveal distinct features of disordered protein domains.
      ) (Table S1). In addition to providing information about affinities, ITC also allows determination of changes in binding enthalpy and entropy. It is generally assumed that IDRs pay an entropic cost upon binding owing to conformational restrictions (
      • Flock T.
      • Weatheritt R.J.
      • Latysheva N.S.
      • Babu M.M.
      Controlling entropy to tune the functions of intrinsically disordered regions.
      ,
      • Teilum K.
      • Olsen J.G.
      • Kragelund B.B.
      Globular and disordered—the non-identical twins in protein-protein interactions.
      ). However, IDRs may also use entropy for binding through counter-ion release (
      • Borgia A.
      • Borgia M.B.
      • Bugge K.
      • Kissling V.M.
      • Heidarsson P.O.
      • Fernandes C.B.
      • Sottini A.
      • Soranno A.
      • Buholzer K.J.
      • Nettels D.
      • Kragelund B.B.
      • Best R.B.
      • Schuler B.
      Extreme disorder in an ultrahigh-affinity protein complex.
      ), increased conformational flexibility (
      • Heller G.T.
      • Sormanni P.
      • Vendruscolo M.
      Targeting disordered proteins with small molecules using entropy.
      ), or expansion of the surrounding IDRs (
      • Das R.K.
      • Huang Y.
      • Phillips A.H.
      • Kriwacki R.W.
      • Pappu R.V.
      Cryptic sequence features within the disordered protein p27 Kip1 regulate cell cycle signaling.
      ). For the αα-hubs, complexes form with Kds ranging from low nanomolar to mid micromolar, with most affinities in the low micromolar range (Table S1). In the high-affinity end, the Sin3a-PAH3:Sap30 complex has a Kd of 9 nM, resulting from cooperative recognition of two discrete Sin3a-PAH3 surfaces by the tripartite binding region in SAP30 (
      • Xie T.
      • He Y.
      • Korkeamaki H.
      • Zhang Y.
      • Imhoff R.
      • Lohi O.
      • Radhakrishnan I.
      Structure of the 30-kDa Sin3-associated protein (SAP30) in complex with the mammalian Sin3A corepressor and its role in nucleic acid binding.
      ). The high affinity may reflect constitutive Sin3:SAP30 association (
      • Streubel G.
      • Fitzpatrick D.J.
      • Oliviero G.
      • Scelfo A.
      • Moran B.
      • Das S.
      • Munawar N.
      • Watson A.
      • Wynne K.
      • Negri G.L.
      • Dillon E.T.
      • Jammula S.
      • Hokamp K.
      • O’Connor D.P.
      • Pasini D.
      • et al.
      Fam60a defines a variant Sin3a-Hdac complex in embryonic stem cells required for self-renewal.
      ). A similar high affinity (Kd 9 nM) was measured for the RCD1-RST:ANAC013 complex, notwithstanding the lack of demonstrated induced structure in ANAC013 upon binding (
      • O’Shea C.
      • Staby L.
      • Bendsen S.K.
      • Tidemand F.G.
      • Redsted A.
      • Willemoës M.
      • Kragelund B.B.
      • Skriver K.
      Structures and short linear motif of disordered transcription factor regions provide clues to the interactome of the cellular hub protein radical-induced cell death 1.
      ). The Sin3a-PAH2:HBP1 complex has a Kd ∼2 orders of magnitude larger than that of the Sin3a-PAH2:Mad1 complex (
      • Swanson K.A.
      • Knoepfler P.S.
      • Huang K.
      • Kang R.S.
      • Cowley S.M.
      • Laherty C.D.
      • Eisenman R.N.
      • Radhakrishnan I.
      HBP1 and Mad1 repressors bind the Sin3 corepressor PAH2 domain with opposite helical orientations.
      ,
      • Brubaker K.
      • Cowley S.M.
      • Huang K.
      • Loo L.
      • Yochum G.S.
      • Ayer D.E.
      • Eisenman R.N.
      • Radhakrishnan I.
      Solution structure of the interacting domains of the mad-sin3 complex: implications for recruitment of a chromatin-modifying complex.
      ,
      • Sahu S.C.
      • Swanson K.A.
      • Kang R.S.
      • Huang K.
      • Brubaker K.
      • Ratcliff K.
      • Radhakrishnan I.
      Conserved themes in target recognition by the PAH1 and PAH2 domains of the Sin3 transcriptional corepressor.
      ,
      • Cowley S.M.
      • Kang R.S.
      • Frangioni J.V.
      • Yada J.J.
      • DeGrand A.M.
      • Radhakrishnan I.
      • Eisenman R.N.
      Functional analysis of the Mad1-mSin3A repressor-corepressor interaction reveals determinants of specificity, affinity, and transcriptional response.
      ), possibly reflecting the biological functions of the two ligands with Mad1 replacing HBP1 in Sin3a complexes during differentiation (
      • Swanson K.A.
      • Knoepfler P.S.
      • Huang K.
      • Kang R.S.
      • Cowley S.M.
      • Laherty C.D.
      • Eisenman R.N.
      • Radhakrishnan I.
      HBP1 and Mad1 repressors bind the Sin3 corepressor PAH2 domain with opposite helical orientations.
      ). Large differences in affinities (40-fold) have also been detected for Sin3a-PAH1 interactions with SAP25 and Myt1L, explained by the Myt1L-SLiM diverting from the canonical SLiM (
      • Marcum R.D.
      • Radhakrishnan I.
      The neuronal transcription factor Myt1L interacts via a conserved motif with the PAH1 domain of Sin3 to recruit the Sin3L/Rpd3L histone deacetylase complex.
      ).
      The thermodynamic profiles for ligand binding vary among the hubs, even for the same αα-hub under the same experimental conditions. Some complexes are entropy driven, as exemplified by RCD1-RST complexes with Col10 (ΔH −9.2 kJ mol−1, −TΔS −27.2 kJ mol−1; Kd 418 nM), STO (ΔH −3.8 kJ mol−1, −TΔS −36.3 kJ mol−1, Kd 90 nM), and ANAC087 (ΔH −15.9 kJ mol−1, −TΔS −16.9 kJ mol−1, Kd 1.8 μM) (
      • Krishna S.S.
      • Grishin N.V.
      Structurally analogous proteins do exist!.
      ). Other, such as the RCD1-RST:DREB2a (ΔH −63.3 kJ mol−1, −TΔS 18.7 kJ mol−1, Kd 16.0 nM) and the CBP-NCBD:ACTR (ΔH −132.6 kJ mol−1, −TΔS 89.1 kJ mol−1, Kd 34 nM) complexes are driven by enthalpy (
      • Christensen L.F.
      • Staby L.
      • Bugge K.
      • O’Shea C.
      • Kragelund B.B.
      • Skriver K.
      Evolutionary conservation of the intrinsic disorder-based radical-induced cell Death1 hub interactome.
      ,
      • Demarest S.J.
      • Martinez-Yamout M.
      • Chung J.
      • Chen H.
      • Xu W.
      • Dyson H.J.
      • Evans R.M.
      • Wright P.E.
      Mutual synergistic folding in recruitment of CBP/p300 by p160 nuclear receptor coactivators.
      ). In a study addressing binding of Sin3 isoforms, Sin3a-PAH2 and Sin3b-PAH2 bound Pf1 with comparable affinities but apparently different thermodynamic profiles. This likely reflects that apo-Sin3a-PAH2 samples both folded and partially folded conformations and forms a monomer–dimer equilibrium and that apo-Sin3b-PAH2 is monomeric and mostly folded (see below) (
      • Van Ingen H.
      • Lasonder E.
      • Jansen J.F.A.
      • Kaan A.M.
      • Spronk C.A.E.M.
      • Stunnenberg H.G.
      • Vuister G.W.
      Extension of the binding motif of the Sin3 interacting domain of the mad family proteins.
      ,
      • Kumar G.S.
      • Xie T.
      • Zhang Y.
      • Radhakrishnan I.
      Solution structure of the mSin3A PAH2-Pf1 SID1 complex: a Mad1/Mxd1-like interaction disrupted by MRG15 in the Rpd3S/Sin3S complex.
      ,
      • Zhang Y.
      • Zhang Z.
      • Demeler B.
      • Radhakrishnan I.
      Coupled unfolding and dimerization by the PAH2 domain of the mammalian Sin3A corepressor.
      ,
      • He Y.
      • Radhakrishnan I.
      Solution NMR studies of apo-mSin3A and -mSin3B reveal that the PAH1 and PAH2 domains are structurally independent.
      ). Accordingly, Sin3a-PAH2 and HBP1 undergo mutual coupled conformational transitions upon association (
      • Swanson K.A.
      • Knoepfler P.S.
      • Huang K.
      • Kang R.S.
      • Cowley S.M.
      • Laherty C.D.
      • Eisenman R.N.
      • Radhakrishnan I.
      HBP1 and Mad1 repressors bind the Sin3 corepressor PAH2 domain with opposite helical orientations.
      ). The thermodynamics of the αα-hub interactions with ligands thus appears diverse, ranging from highly entropically to highly enthalpically driven. However, since the different ligands can be folding to different degrees upon binding, and since both ΔH and ΔS vary with temperature, a comparison of the profiles across the different hubs is complex. Thus, it would be relevant to include more in-depth analyses under varying temperatures, which will allow determination of ΔCp, and through that infer on differences in binding-induced folding.

      Properties of αα-hub–ligand complexes

      The αα-hubs share a common supersite with topological variations

      The majority of αα-hub complex structures have been solved with ligand peptide fragments, entailing an amphipathic α-helix bound through coupled folding and binding (Fig. 5D) in the hydrophobic cleft (
      • Bugge K.
      • Staby L.
      • Kemplen K.R.
      • O’Shea C.
      • Bendsen S.K.
      • Jensen M.K.
      • Olsen J.G.
      • Skriver K.
      • Kragelund B.B.
      Structure of radical-induced cell Death1 hub domain reveals a common αα-scaffold for disorder in transcriptional networks.
      ,
      • Spronk C.A.E.M.
      • Tessari M.
      • Kaan A.M.
      • Jansen J.F.A.
      • Vermeulen M.
      • Stunnenberg H.G.
      • Vuister G.W.
      The Mad1-Sin3B interaction involves a novel helical fold.
      ,
      • Van Ingen H.
      • Lasonder E.
      • Jansen J.F.A.
      • Kaan A.M.
      • Spronk C.A.E.M.
      • Stunnenberg H.G.
      • Vuister G.W.
      Extension of the binding motif of the Sin3 interacting domain of the mad family proteins.
      ,
      • Swanson K.A.
      • Knoepfler P.S.
      • Huang K.
      • Kang R.S.
      • Cowley S.M.
      • Laherty C.D.
      • Eisenman R.N.
      • Radhakrishnan I.
      HBP1 and Mad1 repressors bind the Sin3 corepressor PAH2 domain with opposite helical orientations.
      ,
      • Brubaker K.
      • Cowley S.M.
      • Huang K.
      • Loo L.
      • Yochum G.S.
      • Ayer D.E.
      • Eisenman R.N.
      • Radhakrishnan I.
      Solution structure of the interacting domains of the mad-sin3 complex: implications for recruitment of a chromatin-modifying complex.
      ,
      • Kumar G.S.
      • Xie T.
      • Zhang Y.
      • Radhakrishnan I.
      Solution structure of the mSin3A PAH2-Pf1 SID1 complex: a Mad1/Mxd1-like interaction disrupted by MRG15 in the Rpd3S/Sin3S complex.
      ,
      • Nomura M.
      • Uda-Tochio H.
      • Murai K.
      • Mori N.
      • Nishimura Y.
      The neural repressor NRSF/REST binds the PAH1 domain of the Sin3 corepressor by using its distinct short hydrophobic helix.
      ,
      • Chandru A.
      • Bate N.
      • Vuister G.W.
      • Cowley S.M.
      Sin3A recruits Tet1 to the PAH1 domain via a highly conserved Sin3-interaction domain.
      ,
      • Sahu S.C.
      • Swanson K.A.
      • Kang R.S.
      • Huang K.
      • Brubaker K.
      • Ratcliff K.
      • Radhakrishnan I.
      Conserved themes in target recognition by the PAH1 and PAH2 domains of the Sin3 transcriptional corepressor.
      ,
      • Staby L.
      • O’Shea C.
      • Willemoës M.
      • Theisen F.
      • Kragelund B.B.
      • Skriver K.
      Eukaryotic transcription factors: paradigms of protein intrinsic disorder.
      ,
      • Shammas S.L.
      • Crabtree M.D.
      • Dahal L.
      • Wicky B.I.M.
      • Clarke J.
      Insights into coupled folding and binding mechanisms from kinetic studies.
      ). For all the αα-hubs, this occurs without substantial changes to the backbone structure of the αα-hub, and thus while maintaining the relative helix orientations. For PAH1/2/3, RST, and HHD, binding of these ligands occurs in a shared supersite (
      • Russell R.B.
      • Sasieni P.D.
      • Sternberg M.J.E.
      Supersites within superfolds. Binding site similarity in the absence of homology.
      ), consisting of the hydrophobic cleft formed at the open end of the fold (Figs. 1, AB, EF and 4, AC and Fig. S1, A and C). The shared location of a binding site within apparent analogous domains suggests that it has arisen because it is a particularly advantageous structural motif (
      • Russell R.B.
      • Sasieni P.D.
      • Sternberg M.J.E.
      Supersites within superfolds. Binding site similarity in the absence of homology.
      ). In this case, the open-end hydrophobic cleft seems particularly well suited for versatile binding of IDRs forming amphipathic α-helices upon binding. NCBD only fully populates the αα-hub fold upon complex formation with some ligands (
      • Demarest S.J.
      • Martinez-Yamout M.
      • Chung J.
      • Chen H.
      • Xu W.
      • Dyson H.J.
      • Evans R.M.
      • Wright P.E.
      Mutual synergistic folding in recruitment of CBP/p300 by p160 nuclear receptor coactivators.
      ,
      • Waters L.
      • Yue B.
      • Veverka V.
      • Renshaw P.
      • Bramham J.
      • Matsuda S.
      • Frenkiel T.
      • Kelly G.
      • Muskett F.
      • Carr M.
      • Heery D.M.
      Structural diversity in p160/CREB-binding protein coactivator complexes.
      ) and hence does not have the supersite in a traditional sense. For PAH1/2/3 and HHD, the cleft is primarily located between H1 and H2. Here α-helices engage in a mostly hydrophobic contact surface of 650 to 750 Å2 (
      • Wu L.
      • Pan L.
      • Zhang C.
      • Zhang M.
      Large protein assemblies formed by multivalent interactions between cadherin23 and harmonin suggest a stable anchorage structure at the tip link of stereocilia.
      ,
      • Pan L.
      • Yan J.
      • Wu L.
      • Zhang M.
      Assembling stable hair cell tip link complex via multidentate interactions between harmonin and cadherin 23.
      ,
      • Sahu S.C.
      • Swanson K.A.
      • Kang R.S.
      • Huang K.
      • Brubaker K.
      • Ratcliff K.
      • Radhakrishnan I.
      Conserved themes in target recognition by the PAH1 and PAH2 domains of the Sin3 transcriptional corepressor.
      ), which is close to the average protein–protein interface size of 800 Å2 (
      • Dai W.
      • Wu A.
      • Ma L.
      • Li Y.-X.
      • Jiang T.
      • Li Y.-Y.
      A novel index of protein-protein interface propensity improves interface residue recognition.
      ). For RST, the cleft opening is primarily located between H3 and H4 (
      • Bugge K.
      • Staby L.
      • Kemplen K.R.
      • O’Shea C.
      • Bendsen S.K.
      • Jensen M.K.
      • Olsen J.G.
      • Skriver K.
      • Kragelund B.B.
      Structure of radical-induced cell Death1 hub domain reveals a common αα-scaffold for disorder in transcriptional networks.
      ). TAFH deviates from the prototypical αα-hub traits by its hydrophobic open-end cleft (i.e., the supersite) being occupied by a repositioned H4 (Fig. 1, C and F and Fig. S1B). Structures of TAFH complexes revealed binding of ligands in the interfaces between H1 and H4 (Fig. S1B), resulting in a mostly hydrophobic contact surface of 700 Å2 (
      • Park S.
      • Chen W.
      • Cierpicki T.
      • Tonelli M.
      • Cai X.
      • Speck N.A.
      • Bushweller J.H.
      Structure of the AML1-ETO eTAFH domain-HEB peptide complex and its contribution to AML1-ETO activity.
      ). This different relative orientation of H1, H4, and H5 in the αα-hubs, resulting in different positioning of side chains and geometry of the binding site, may be an additional filter for specificity tuning (
      • Bugge K.
      • Staby L.
      • Kemplen K.R.
      • O’Shea C.
      • Bendsen S.K.
      • Jensen M.K.
      • Olsen J.G.
      • Skriver K.
      • Kragelund B.B.
      Structure of radical-induced cell Death1 hub domain reveals a common αα-scaffold for disorder in transcriptional networks.
      ).

      The positions of binding residues are not always conserved across subgroups

      The sequence alignments of the individual αα-hub subgroups presented above together with manual inspection of 3D structures allowed identification of fold-defining residues (Fig. 4, Figs. S1 and S2). However, each subgroup also revealed between 4 to 13 conserved residues that cannot be explained by apparent fold-conservation (>50% identity, lack of tertiary contacts) (Fig. 4, Figs. S1 and S2). These are likely conserved because they are crucial components of interaction sites. In, e.g., PAH2, TAFH, and NCBD, 7 of 10, 6 of 7, and 10 of 13, respectively, of the suggested binding residues are in known complex structures indeed in contact with ligands (
      • Spronk C.A.E.M.
      • Tessari M.
      • Kaan A.M.
      • Jansen J.F.A.
      • Vermeulen M.
      • Stunnenberg H.G.
      • Vuister G.W.
      The Mad1-Sin3B interaction involves a novel helical fold.
      ,
      • Van Ingen H.
      • Lasonder E.
      • Jansen J.F.A.
      • Kaan A.M.
      • Spronk C.A.E.M.
      • Stunnenberg H.G.
      • Vuister G.W.
      Extension of the binding motif of the Sin3 interacting domain of the mad family proteins.
      ,
      • Swanson K.A.
      • Knoepfler P.S.
      • Huang K.
      • Kang R.S.
      • Cowley S.M.
      • Laherty C.D.
      • Eisenman R.N.
      • Radhakrishnan I.
      HBP1 and Mad1 repressors bind the Sin3 corepressor PAH2 domain with opposite helical orientations.
      ,
      • Brubaker K.
      • Cowley S.M.
      • Huang K.
      • Loo L.
      • Yochum G.S.
      • Ayer D.E.
      • Eisenman R.N.
      • Radhakrishnan I.
      Solution structure of the interacting domains of the mad-sin3 complex: implications for recruitment of a chromatin-modifying complex.
      ,
      • Kumar G.S.
      • Xie T.
      • Zhang Y.
      • Radhakrishnan I.
      Solution structure of the mSin3A PAH2-Pf1 SID1 complex: a Mad1/Mxd1-like interaction disrupted by MRG15 in the Rpd3S/Sin3S complex.
      ,
      • Lee C.W.
      • Martinez-Yamout M.A.
      • Dyson H.J.
      • Wright P.E.
      Structure of the p53 transactivation domain in complex with the nuclear receptor coactivator binding domain of CREB binding protein.
      ,
      • Park S.
      • Chen W.
      • Cierpicki T.
      • Tonelli M.
      • Cai X.
      • Speck N.A.
      • Bushweller J.H.
      Structure of the AML1-ETO eTAFH domain-HEB peptide complex and its contribution to AML1-ETO activity.
      ,
      • Demarest S.J.
      • Martinez-Yamout M.
      • Chung J.
      • Chen H.
      • Xu W.
      • Dyson H.J.
      • Evans R.M.
      • Wright P.E.
      Mutual synergistic folding in recruitment of CBP/p300 by p160 nuclear receptor coactivators.
      ,
      • Nady N.
      • Gupta A.
      • Ma Z.
      • Swigut T.
      • Koide A.
      • Koide S.
      • Wysocka J.
      ETO family protein Mtgr1 mediates Prdm14 functions in stem cell maintenance and primordial germ cell formation.
      ).
      In the αα-hub complex structures, the majority of ligand contacts are through the open-end hydrophobic supersite (
      • Bugge K.
      • Staby L.
      • Kemplen K.R.
      • O’Shea C.
      • Bendsen S.K.
      • Jensen M.K.
      • Olsen J.G.
      • Skriver K.
      • Kragelund B.B.
      Structure of radical-induced cell Death1 hub domain reveals a common αα-scaffold for disorder in transcriptional networks.
      • Spronk C.A.E.M.
      • Tessari M.
      • Kaan A.M.
      • Jansen J.F.A.
      • Vermeulen M.
      • Stunnenberg H.G.
      • Vuister G.W.
      The Mad1-Sin3B interaction involves a novel helical fold.
      ,
      • Van Ingen H.
      • Lasonder E.
      • Jansen J.F.A.
      • Kaan A.M.
      • Spronk C.A.E.M.
      • Stunnenberg H.G.
      • Vuister G.W.
      Extension of the binding motif of the Sin3 interacting domain of the mad family proteins.
      ,
      • Swanson K.A.
      • Knoepfler P.S.
      • Huang K.
      • Kang R.S.
      • Cowley S.M.
      • Laherty C.D.
      • Eisenman R.N.
      • Radhakrishnan I.
      HBP1 and Mad1 repressors bind the Sin3 corepressor PAH2 domain with opposite helical orientations.
      ,
      • Brubaker K.
      • Cowley S.M.
      • Huang K.
      • Loo L.
      • Yochum G.S.
      • Ayer D.E.
      • Eisenman R.N.
      • Radhakrishnan I.
      Solution structure of the interacting domains of the mad-sin3 complex: implications for recruitment of a chromatin-modifying complex.
      ,
      • Kumar G.S.
      • Xie T.
      • Zhang Y.
      • Radhakrishnan I.
      Solution structure of the mSin3A PAH2-Pf1 SID1 complex: a Mad1/Mxd1-like interaction disrupted by MRG15 in the Rpd3S/Sin3S complex.
      ,
      • Yan J.
      • Pan L.
      • Chen X.
      • Wu L.
      • Zhang M.
      The structure of the harmonin/sans complex reveals an unexpected interaction mode of the two Usher syndrome proteins.
      ,
      • Wu L.
      • Pan L.
      • Zhang C.
      • Zhang M.
      Large protein assemblies formed by multivalent interactions between cadherin23 and harmonin suggest a stable anchorage structure at the tip link of stereocilia.
      ,
      • Nomura M.
      • Uda-Tochio H.
      • Murai K.
      • Mori N.
      • Nishimura Y.
      The neural repressor NRSF/REST binds the PAH1 domain of the Sin3 corepressor by using its distinct short hydrophobic helix.
      ,
      • Lee C.W.
      • Martinez-Yamout M.A.
      • Dyson H.J.
      • Wright P.E.
      Structure of the p53 transactivation domain in complex with the nuclear receptor coactivator binding domain of CREB binding protein.
      ,
      • Sahu S.C.
      • Swanson K.A.
      • Kang R.S.
      • Huang K.
      • Brubaker K.
      • Ratcliff K.
      • Radhakrishnan I.
      Conserved themes in target recognition by the PAH1 and PAH2 domains of the Sin3 transcriptional corepressor.
      ,
      • Park S.
      • Chen W.
      • Cierpicki T.
      • Tonelli M.
      • Cai X.
      • Speck N.A.
      • Bushweller J.H.
      Structure of the AML1-ETO eTAFH domain-HEB peptide complex and its contribution to AML1-ETO activity.
      ,
      • Demarest S.J.
      • Martinez-Yamout M.
      • Chung J.
      • Chen H.
      • Xu W.
      • Dyson H.J.
      • Evans R.M.
      • Wright P.E.
      Mutual synergistic folding in recruitment of CBP/p300 by p160 nuclear receptor coactivators.
      ,
      • Nady N.
      • Gupta A.
      • Ma Z.
      • Swigut T.
      • Koide A.
      • Koide S.
      • Wysocka J.
      ETO family protein Mtgr1 mediates Prdm14 functions in stem cell maintenance and primordial germ cell formation.
      ,
      • Wang X.
      • Hou Y.
      • Deng K.
      • Zhang Y.
      • Wang D.C.
      • Ding J.
      Structural insights into the molecular recognition between cerebral cavernous malformation 2 and mitogen-activated protein kinase kinase kinase 3.
      ,
      • Fisher O.S.
      • Deng H.
      • Liu D.
      • Zhang Y.
      • Wei R.
      • Deng Y.
      • Zhang F.
      • Louvi A.
      • Turk B.E.
      • Boggon T.J.
      • Su B.
      Structure and vascular function of MEKK3-cerebral cavernous malformations 2 complex.
      ). Since the analysis does not pick up residues of the hydrophobic supersite that are also part of the core fold, the identified potential binding residues are primarily solvent exposed and, hence, the majority are hydrophilic and charged (Fig. 4, Figs. S1 and S2). All the αα-hub domains have conserved binding residues in both H1 and H2 (except for H1 of RST), whereas their presence in H3, H4, and H5 varies between subgroups. The relative position of the conserved binding residues is, however, not consistently conserved throughout the domains, supporting that binding discrimination may be partially encoded in the position of key residues. Even for PAH1 and PAH2, which as described above have many common conserved core residues, the pattern of conserved binding residues is entirely different (Fig. 4, AB). For PAH1, the 9 identified residues are distributed throughout the domain, whereas for PAH2, 6 of 10 residues are in H1 and none is in H3 and H4. Nonetheless, when inspecting their positions in available structures (Fig. 4, AB), it is clear that they cluster around the open-end binding pocket between H1 and H2 in both PAH1 and PAH2. This difference is consistent with previous studies showing that conservative replacements of PAH2 residues with equivalent PAH1 residues were sufficient to alter affinity as well as specificity. Thus, substitution of Sin3-PAH2-Leu332, positioned in H2 of the ligand-binding cleft of PAH1/2, with Met, present in the corresponding position in PAH1, resulted in a 7-fold decrease in the affinity for Mad1 (
      • Sahu S.C.
      • Swanson K.A.
      • Kang R.S.
      • Huang K.
      • Brubaker K.
      • Ratcliff K.
      • Radhakrishnan I.
      Conserved themes in target recognition by the PAH1 and PAH2 domains of the Sin3 transcriptional corepressor.
      ). For all the αα-hubs, particularly the last or second-to-last turn of H2 situated between the core and solvent-exposed side of H2 almost always has a conserved binding residue, which is in contact with ligands in known structures (Fig. 4 and Fig. S1).
      NCBD is an outlier, only substantially populating the αα-hub fold with certain ligands. Here, the ligand takes the position of H1 in the complex, resulting in many conserved residues engaging in intermolecular interactions. For this reason, it is omitted from the cross comparisons. From the sequences and αα-hub-like structures of NCBD (
      • Demarest S.J.
      • Martinez-Yamout M.
      • Chung J.
      • Chen H.
      • Xu W.
      • Dyson H.J.
      • Evans R.M.
      • Wright P.E.
      Mutual synergistic folding in recruitment of CBP/p300 by p160 nuclear receptor coactivators.
      ,
      • Lee C.W.
      • Ferreon J.C.
      • Ferreon A.C.M.
      • Arai M.
      • Wright P.E.
      Graded enhancement of p53 binding to CREB-binding protein (CBP) by multisite phosphorylation.
      ), 13 conserved binding residues were identified, and 10 of these can be recognized as partaking in complexes (Fig. S2, (
      • Demarest S.J.
      • Martinez-Yamout M.
      • Chung J.
      • Chen H.
      • Xu W.
      • Dyson H.J.
      • Evans R.M.
      • Wright P.E.
      Mutual synergistic folding in recruitment of CBP/p300 by p160 nuclear receptor coactivators.
      ,
      • Lee C.W.
      • Ferreon J.C.
      • Ferreon A.C.M.
      • Arai M.
      • Wright P.E.
      Graded enhancement of p53 binding to CREB-binding protein (CBP) by multisite phosphorylation.
      )). The remainder are on the backside of H2-H3 (two residues) or in H2 (one residue) and may engage in complexes with other ligands.

      Conserved binding residues across subgroups suggest expanded binding

      Many of the identified binding-conserved residues are at the rim of the hydrophobic supersite. However, a subset has geometrically distant locations. Especially noteworthy is that all αα-hubs have conserved residues positioned at the backside of H2-H3, and a few also on the solvent-exposed side of H1 (PAH2, CCM2-HHD, RST), H4 (PAH1, RST), or H5 (CCM2-HHD). This suggests these to constitute one or more accessory binding (super)sites. A few complex structures solved with relatively large intrinsically disordered ligand fragments of ∼60 to 90 residues (Sin3-PAH1: PDB 2rms (
      • Sahu S.C.
      • Swanson K.A.
      • Kang R.S.
      • Huang K.
      • Brubaker K.
      • Ratcliff K.
      • Radhakrishnan I.
      Conserved themes in target recognition by the PAH1 and PAH2 domains of the Sin3 transcriptional corepressor.
      ), Sin3-PAH3: PDB 2ld7 (
      • Xie T.
      • He Y.
      • Korkeamaki H.
      • Zhang Y.
      • Imhoff R.
      • Lohi O.
      • Radhakrishnan I.
      Structure of the 30-kDa Sin3-associated protein (SAP30) in complex with the mammalian Sin3A corepressor and its role in nucleic acid binding.
      )) or folded partners (CCM2-HHD: PDB 4y5o (
      • Fisher O.S.
      • Deng H.
      • Liu D.
      • Zhang Y.
      • Wei R.
      • Deng Y.
      • Zhang F.
      • Louvi A.
      • Turk B.E.
      • Boggon T.J.
      • Su B.
      Structure and vascular function of MEKK3-cerebral cavernous malformations 2 complex.
      ), Mtgr1-TAFH: PDB 5ecj (
      • Nady N.
      • Gupta A.
      • Ma Z.
      • Swigut T.
      • Koide A.
      • Koide S.
      • Wysocka J.
      ETO family protein Mtgr1 mediates Prdm14 functions in stem cell maintenance and primordial germ cell formation.
      )) are available. In the Sin3-PAH1 complex, the additional ∼25 disordered residues do not engage with the αα-hub (Fig. 5E), whereas in the Sin3-PAH3 complex, the additional ∼60 residues, intrinsically disordered in the free ligand, form two α-helices engaging with conserved contact residues on the backside of H2-H3 (Fig. S1A). A similar pattern is observed in the CCM2–HHD complex with a folded partner (PDB 4y5o (
      • Fisher O.S.
      • Deng H.
      • Liu D.
      • Zhang Y.
      • Wei R.
      • Deng Y.
      • Zhang F.
      • Louvi A.
      • Turk B.E.
      • Boggon T.J.
      • Su B.
      Structure and vascular function of MEKK3-cerebral cavernous malformations 2 complex.