Advertisement

Evidence for Cooperative and Domain-specific Binding of the Signal Transducing Adaptor Molecule 2 (STAM2) to Lys63-linked Diubiquitin*

  • Anja Lange
    Footnotes
    Affiliations
    Université de Lyon, CNRS, UMR 5280 Institut des Sciences Analytiques, 69622 Villeurbanne, France
    Search for articles by this author
  • Carlos Castañeda
    Affiliations
    Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Organization, University of Maryland, College Park, Maryland 20910
    Search for articles by this author
  • Daniela Hoeller
    Affiliations
    Division of Medical Biochemistry, Innsbruck Medical University, Biocenter, Fritz-Pregl-Strasse 3, 6020 Innsbruck, Austria
    Search for articles by this author
  • Jean-Marc Lancelin
    Affiliations
    Université de Lyon, CNRS, UMR 5280 Institut des Sciences Analytiques, 69622 Villeurbanne, France
    Search for articles by this author
  • David Fushman
    Affiliations
    Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Organization, University of Maryland, College Park, Maryland 20910
    Search for articles by this author
  • Olivier Walker
    Correspondence
    To whom correspondence should be addressed: 43 boulevard du 11 Novembre 1918, 69622 Villeurbanne, France. Tel.: 33-4-72-43-18-27; Fax: 33-4-72-43-13-95
    Affiliations
    Université de Lyon, CNRS, UMR 5280 Institut des Sciences Analytiques, 69622 Villeurbanne, France
    Search for articles by this author
  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grant GM065334 (to D. F.) and National Science Foundation Postdoctoral Fellowship DBI-0905967 (to C. A. C.).
    This article contains supplemental Figs. S1–S14, Table S1, and Materials.
    1 Recipient of fellowships from the French MENRT and a French Rhone Alpes region for the ExploraDoc.
Open AccessPublished:April 04, 2012DOI:https://doi.org/10.1074/jbc.M111.324954
      As the upstream component of the ESCRT (endosomal sorting complexes required for transport) machinery, the ESCRT-0 complex is responsible for directing ubiquitinated membrane proteins to the multivesicular body pathway. ESCRT-0 is formed by two subunits known as Hrs (hepatocyte growth factor-regulated substrate) and STAM (signal transducing adaptor molecule), both of which harbor multiple ubiquitin-binding domains (UBDs). In particular, STAM2 possesses two UBDs, the VHS (Vps27/Hrs/Stam) and UIM (ubiquitin interacting motif) domains, connected by a 20-amino acid flexible linker. In the present study, we report the interactions of the UIM domain and VHS-UIM construct of STAM2 with monoubiquitin (Ub), Lys48- and Lys63-linked diubiquitins. Our results demonstrate that the UIM domain alone binds monoubiquitin, Lys48- and Lys63-linked diubiquitins with the same affinity and in the same binding mode. Interestingly, binding of VHS-UIM to Lys63-linked diubiquitin is not only avid, but also cooperative. We also show that the distal domain of Lys63-linked diubiquitin stabilizes the helical structure of the UIM domain and that the corresponding complex adopts a specific structural organization responsible for its greater affinity. In contrast, binding of VHS-UIM to Lys48-linked diubiquitin and monoubiquitin is not cooperative and does not show any avidity. These results may explain the better sorting efficiency of some cargoes polyubiquitinated with Lys63-linked chains over monoubiquitinated cargoes or those tagged with Lys48-linked chains.

      Introduction

      The turnover of many membrane proteins is determined through the endocytic pathway, which can either result in their recycling or lysosomal degradation (
      • Platta H.W.
      • Stenmark H.
      Cell structure and dynamics.
      ,
      • Sorkin A.
      • von Zastrow M.
      Endocytosis and signaling. Intertwining molecular networks.
      ,
      • Scita G.
      • Di Fiore P.P.
      The endocytic matrix.
      ). In the case of lysosomal degradation, membrane proteins are sorted into distinctive endosomes known as multivesicular bodies (MVBs).
      The abbreviations used are:
      MVB
      multivesicular body
      ESCRT
      endosomal sorting complexes required for transport
      Ub
      monoubiquitin
      UIM
      ubiquitin-interacting motif
      Ub2
      diubiquitin
      UBD
      ubiquitin-binding domain
      Hrs
      hepatocyte growth factor-regulated substrate
      STAM
      signal transducing adaptor molecule
      Lys63-Ub2
      Lys63-linked diubiquitin
      Lys48-Ub2
      Lys48-linked diubiquitin
      VHS
      Vps27/Hrs/STAM
      HSQC
      heteronuclear single quantum coherence
      CSP
      chemical shift perturbation
      MTSL
      1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl)methanesulfonate
      PRE
      paramagnetic relaxation enhancement
      tUIM
      tandem UIM.
      These MVBs fuse with lysosomes, resulting in the degradation of their cargoes. Prior to entering the MVB pathway, cargoes have to be properly tagged by a process called ubiquitination. Ubiquitination occurs through the attachment of a single ubiquitin (Ub), a 76-amino acid protein, to a lysine (monoubiquitination) (
      • Hicke L.
      • Dunn R.
      Regulation of membrane protein transport by ubiquitin and ubiquitin-binding proteins.
      ) or several lysines (multi-monoubiquitination) (
      • Haglund K.
      • Di Fiore P.P.
      • Dikic I.
      Distinct monoubiquitin signals in receptor endocytosis.
      ) of a target protein, as well as by the attachment of polyubiquitin (poly-Ub) chains (
      • Pickart C.M.
      • Fushman D.
      Polyubiquitin chains. Polymeric protein signals.
      ). Interestingly, depending on the type of ubiquitin chain linkages, tagged proteins are committed to different pathways (
      • Clague M.J.
      • Urbé S.
      Ubiquitin, same molecule, different degradation pathways.
      ). Although monoubiquitination is a sufficient signal to direct cargoes through the MVB pathway (
      • Hicke L.
      Protein regulation by monoubiquitin.
      ,
      • Acconcia F.
      • Sigismund S.
      • Polo S.
      Ubiquitin in trafficking. The network at work.
      ), in many cases, polyubiquitination by Lys63-linked chains is a more efficient signal for cargo sorting (
      • Duncan L.M.
      • Piper S.
      • Dodd R.B.
      • Saville M.K.
      • Sanderson C.M.
      • Luzio J.P.
      • Lehner P.J.
      Lysine 63-linked ubiquitination is required for endolysosomal degradation of class I molecules.
      ,
      • Lauwers E.
      • Erpapazoglou Z.
      • Haguenauer-Tsapis R.
      • André B.
      The ubiquitin code of yeast permease trafficking.
      ,
      • Lauwers E.
      • Jacob C.
      • André B.
      Lys63-linked ubiquitin chains as a specific signal for protein sorting into the multivesicular body pathway.
      ). Most proteins that are not ubiquitinated follow other pathways and are recycled from the endosome to other cellular compartments (
      • Raiborg C.
      • Malerød L.
      • Pedersen N.M.
      • Stenmark H.
      Differential functions of Hrs and ESCRT proteins in endocytic membrane trafficking.
      ).
      The machinery responsible for committing ubiquitinated cargoes to the MVB pathway is the ESCRT machinery (
      • Henne W.M.
      • Buchkovich N.J.
      • Emr S.D.
      The ESCRT pathway.
      ), whose most upstream component is ESCRT-0 that is composed of the STAM/Hrs complex (
      • Mizuno E.
      • Kawahata K.
      • Okamoto A.
      • Kitamura N.
      • Komada M.
      Association with Hrs is required for the early endosomal localization, stability, and function of STAM.
      ) in mammalian cells (Vps27/Hse1 complex in yeast) (
      • Hurley J.H.
      The ESCRT complexes.
      ,
      • Hurley J.H.
      ESCRT complexes and the biogenesis of multivesicular bodies.
      ). Recognition of the ubiquitin signal is achieved by modular motifs called ubiquitin-binding domains (UBDs) (
      • Dikic I.
      • Wakatsuki S.
      • Walters K.J.
      Ubiquitin-binding domains. From structures to functions.
      ). Both STAM and Hrs harbor two UBDs. The VHS (
      • Mizuno E.
      • Kawahata K.
      • Kato M.
      • Kitamura N.
      • Komada M.
      STAM proteins bind ubiquitinated proteins on the early endosome via the VHS domain and ubiquitin-interacting motif.
      ) and UIM (ubiquitin-interacting motif) (
      • Fisher R.D.
      • Wang B.
      • Alam S.L.
      • Higginson D.S.
      • Robinson H.
      • Sundquist W.I.
      • Hill C.P.
      Structure and ubiquitin binding of the ubiquitin-interacting motif.
      ) domains of STAM, which are connected by a 20-amino acid linker, are key players in ubiquitin recognition and cargo sorting. Deletion of either the VHS or UIM domains of STAM gives rise to a partial loss of function of ESCRT-0 (
      • Mizuno E.
      • Kawahata K.
      • Kato M.
      • Kitamura N.
      • Komada M.
      STAM proteins bind ubiquitinated proteins on the early endosome via the VHS domain and ubiquitin-interacting motif.
      ,
      • Ren X.
      • Hurley J.H.
      VHS domains of ESCRT-0 cooperate in high-avidity binding to polyubiquitinated cargo.
      ). These results highlight the importance of both the VHS and UIM domains, and it has recently been proposed that ESCRT-0 as well as the VHS-UIM domains of STAM1 bind avidly to long polyubiquitin chains and show a moderate degree of selectivity for Lys63- over Lys48-linked chains (
      • Ren X.
      • Hurley J.H.
      VHS domains of ESCRT-0 cooperate in high-avidity binding to polyubiquitinated cargo.
      ). However, the molecular details of the recognition of Lys63-linked diubiquitin (Lys63-Ub2) chains by the ESCRT machinery remain poorly understood, and structural studies are required to decipher the underlying mechanisms. Because ESCRT-0 appears to be primarily responsible for cargo clustering (
      • Wollert T.
      • Hurley J.H.
      Molecular mechanism of multivesicular body biogenesis by ESCRT complexes.
      ), we are seeking to understand if any particular structural organization forms the basis for the polyubiquitin chain selectivity of ESCRT-0 and, more specifically, of the VHS-UIM fragment of STAM2. To address this question, we compared interactions of the UIM domain and the VHS-UIM construct of STAM2 with monoubiquitin (mono-Ub), Lys48- and Lys63-Ub2. Here we show that the UIM domain binds mono-Ub, Lys48- and Lys63-Ub2 with the same affinity and binding mode, in striking contrast with our previous findings for the VHS domain (
      • Lange A.
      • Hoeller D.
      • Wienk H.
      • Marcillat O.
      • Lancelin J.M.
      • Walker O.
      NMR reveals a different mode of binding of the Stam2 VHS domain to ubiquitin and diubiquitin.
      ). However, the situation is somewhat different when the UIM and the VHS domains are connected. Our results indicate that interaction of the VHS-UIM construct with Lys63-Ub2, but not with mono-Ub or Lys48-Ub2, is cooperative. In addition, site-directed paramagnetic spin labeling data allow modeling of the VHS-UIM/Lys63-Ub2 complex and clearly demonstrate that each domain of STAM2 VHS-UIM interacts with a specific Ub unit in Lys63-Ub2.

      DISCUSSION

      The human STAM2 protein is involved in lysosomal degradation and associates with Hrs to form the ESCRT-0 component of the ESCRT machinery. STAM2 possesses two UBDs: the UIM and VHS domains. Deletion of one of these domains or mutation alters the transport of ubiquitinated cargoes (
      • Mizuno E.
      • Kawahata K.
      • Kato M.
      • Kitamura N.
      • Komada M.
      STAM proteins bind ubiquitinated proteins on the early endosome via the VHS domain and ubiquitin-interacting motif.
      ,
      • Ren X.
      • Hurley J.H.
      VHS domains of ESCRT-0 cooperate in high-avidity binding to polyubiquitinated cargo.
      ,
      • Bilodeau P.S.
      • Urbanowski J.L.
      • Winistorfer S.C.
      • Piper R.C.
      The Vps27p Hse1p complex binds ubiquitin and mediates endosomal protein sorting.
      ,
      • Shih S.C.
      • Katzmann D.J.
      • Schnell J.D.
      • Sutanto M.
      • Emr S.D.
      • Hicke L.
      Epsins and Vps27p/Hrs contain ubiquitin-binding domains that function in receptor endocytosis.
      ). Additionally, ESCRT-0 binds polyubiquitin chains with high avidity and seems to have a preference for Lys63-linked chains (
      • Ren X.
      • Hurley J.H.
      VHS domains of ESCRT-0 cooperate in high-avidity binding to polyubiquitinated cargo.
      ). For instance, Lys63-linked ubiquitination is required for MVB sorting of Gap1, CPS, and probably other cargoes (
      • Lauwers E.
      • Jacob C.
      • André B.
      Lys63-linked ubiquitin chains as a specific signal for protein sorting into the multivesicular body pathway.
      ). Because it has been reported that the binding of ESCRT-0 showed a modest increase in affinity for Lys63-Ub2 over Lys48-Ub2 and “linear,” head-to-tail linked chains (NC-Ub2) (
      • Ren X.
      • Hurley J.H.
      VHS domains of ESCRT-0 cooperate in high-avidity binding to polyubiquitinated cargo.
      ), it is likely that other factors are responsible for the specificity of Lys63-linked chains toward cargo sorting. We previously characterized the interaction of an isolated VHS domain with mono-Ub, Lys48- and Lys63-Ub2 and reported that Lys63- and Lys48-Ub2 chains bind VHS via different binding modes (
      • Lange A.
      • Hoeller D.
      • Wienk H.
      • Marcillat O.
      • Lancelin J.M.
      • Walker O.
      NMR reveals a different mode of binding of the Stam2 VHS domain to ubiquitin and diubiquitin.
      ).
      In the present study, we first examined whether the UIM domain of STAM2 also exhibits such structural features when binding to Lys48- and Lys63-Ub2 chains. A UIM was originally identified in the S5a subunit of the 26 S proteasome and is often found in proteins involved in the proteasomal and lysosomal degradation (
      • Hofmann K.
      • Falquet L.
      A ubiquitin-interacting motif conserved in components of the proteasomal and lysosomal protein degradation systems.
      ). UIMs fold into a single α-helix and are embedded as independent domains into various modular proteins. Our NMR mapping revealed that the Ub-binding surface is located on one face of the helical region of STAM2 UIM (supplemental Fig. S13B) and involves a set of conserved hydrophobic residues (supplemental Fig. S13A). These findings are in good agreement with the Vps27-UIM1/mono-Ub and STAM-UIM/mono-Ub structures (
      • Swanson K.A.
      • Kang R.S.
      • Stamenova S.D.
      • Hicke L.
      • Radhakrishnan I.
      Solution structure of Vps27 UIM-ubiquitin complex important for endosomal sorting and receptor down-regulation.
      ,
      • Lim J.
      • Son W.S.
      • Park J.K.
      • Kim E.E.
      • Lee B.J.
      • Ahn H.C.
      Solution structure of UIM and interaction of tandem ubiquitin binding domains in STAM1 with ubiquitin.
      ). Using NMR titration experiments, we determined the STAM2-UIM/mono-Ub dissociation constant of 287 ± 33 μm, which implies that the UIM domain of STAM2 binds to mono-Ub weaker than VHS (
      • Lange A.
      • Hoeller D.
      • Wienk H.
      • Marcillat O.
      • Lancelin J.M.
      • Walker O.
      NMR reveals a different mode of binding of the Stam2 VHS domain to ubiquitin and diubiquitin.
      ). Furthermore, our study revealed that Lys48- and Lys63-Ub2 show comparable affinities for UIM (Table 1) and can accommodate up to two STAM2-UIM molecules per chain. A similar structural organization has already been seen in the case of the UIM2 domain of S5a (
      • Haririnia A.
      • D'Onofrio M.
      • Fushman D.
      Mapping the interactions between Lys48- and Lys63-linked diubiquitins and a ubiquitin-interacting motif of S5a.
      ). According to our results, the UIM domain of STAM2 does not discriminate between mono-Ub, Lys48- and Lys63-Ub2.
      Our data suggest that the UIM adopts the same mode of binding to mono-Ub, Lys48- and Lys63-Ub2 chains. Several lines of evidence support this conclusion. First, the trajectories of the NMR signals in the course of titration with UIM are similar for mono-Ub and the distal and proximal Ubs of Lys48-Ub2 and Lys63-Ub2 chains (see supplemental Fig. S6). Second, the perturbation maps (i.e. residues showing strong CSPs and/or signal attenuations as a result of UIM binding) are similar for mono-Ub, Lys48- and Lys63-Ub2 chains. Based on the present results on UIM binding and previous work on VHS (
      • Lange A.
      • Hoeller D.
      • Wienk H.
      • Marcillat O.
      • Lancelin J.M.
      • Walker O.
      NMR reveals a different mode of binding of the Stam2 VHS domain to ubiquitin and diubiquitin.
      ), we can rule out the possibility that the individual UIM or VHS domains are solely responsible for the better affinity of STAM2 for Lys63-Ub2.
      To determine the impact of having two UBDs in tandem (as in STAM2) on binding to ubiquitin and polyubiquitin chains, we characterized the interaction of the VHS-UIM construct from STAM2 with mono-Ub, Lys48- and Lys63-Ub2 chains. Our titration experiments suggest that both UBDs in VHS-UIM bind mono-Ub independently and with affinities similar to those observed for the isolated VHS and UIM domains (see Lange et al. (
      • Lange A.
      • Hoeller D.
      • Wienk H.
      • Marcillat O.
      • Lancelin J.M.
      • Walker O.
      NMR reveals a different mode of binding of the Stam2 VHS domain to ubiquitin and diubiquitin.
      ) and Table 2). Note that VHS binds Ub stronger than UIM does. Switching from mono-Ub to Ub2, the VHS-UIM/Lys48-Ub2 interaction gives a somewhat more complicated picture of the equilibrium, where various intermolecular arrangements (and perhaps with different populations) can exist. The VHS-UIM construct binds Lys48-Ub2 with the same affinity as mono-Ub (average Kd = 115 ± 41 μm), and thus does not bind avidly to Lys48-Ub2 chains. As shown above, the isolated VHS and UIM exhibit different stoichiometry in binding to Lys48-Ub2. Then one might hypothesize that, due to the steric hindrance introduced by the VHS domain, it is unlikely that the UIM domain of VHS-UIM could bind another Ub unit of the same Lys48-Ub2 chain. An attempt to model the VHS-UIM/Lys48-Ub2 complex with a 1:1 stoichiometry supports this hypothesis. Indeed, the resulting structure is in disagreement with our CSP data and would induce a severe bending of the VHS-UIM linker (supplemental Fig. S14). In addition, it should be borne in mind that Lys48-Ub2 is in dynamic equilibrium between a closed conformation, in which the functionally important hydrophobic patch residues of each Ub unit are sequestered at the Ub/Ub interface, and one or more open conformations (
      • Varadan R.
      • Walker O.
      • Pickart C.
      • Fushman D.
      Structural properties of polyubiquitin chains in solution.
      ,
      • Ryabov Y.
      • Fushman D.
      Interdomain mobility in diubiquitin revealed by NMR.
      ). VHS-UIM binding would require opening of the interface to allow contacts between the hydrophobic residues of the two Ub units and VHS-UIM. In such a situation, the molecular recognition process would likely proceed through a conformational selection mechanism, namely selection of the right (open) conformation of Lys48-Ub2. At neutral pH the closed state is predominantly populated (∼85%), and the time of interconversion between the closed and open conformations, estimated by NMR to be ∼10 ns, is comparable with the overall tumbling time (
      • Ryabov Y.
      • Fushman D.
      Interdomain mobility in diubiquitin revealed by NMR.
      ). Analysis of our relaxation data indicates that UIM and VHS tumble essentially independently from each other with respective rotational correlation times of 3.8 ± 0.5 and 15.0 ± 0.4 ns, respectively. Thus, slower tumbling of the VHS domain could be a limiting factor in the recognition and binding to the open state of Lys48-Ub2, possibly explaining the less efficient sorting of Lys48-polyubiquitinated cargoes by ESCRT-0.
      A strikingly different picture arises in the case of VHS-UIM interaction with Lys63-Ub2, where the VHS and the UIM domains preferentially bind to the proximal and the distal Ub of Lys63-Ub2, respectively. Lys63-Ub2 adopts an extended conformation (
      • Varadan R.
      • Assfalg M.
      • Haririnia A.
      • Raasi S.
      • Pickart C.
      • Fushman D.
      Solution conformation of Lys63-linked diubiquitin chain provides clues to functional diversity of polyubiquitin signaling.
      ,
      • Datta A.B.
      • Hura G.L.
      • Wolberger C.
      The structure and conformation of Lys63-linked tetraubiquitin.
      ) with the hydrophobic patches exposed to the solvent; this allows the interaction with the VHS and UIM domains without the need to compete with the Ub/Ub interaction as in Lys48-Ub2. Interestingly, the titration curves corresponding to the interaction of VHS-UIM with Lys63-Ub2 have a sigmoidal shape. This is likely to result from two binding events and a cooperative effect, thus demonstrating a clear difference in the binding of mono-Ub, Lys48-Ub2, and Lys63-Ub2 to VHS-UIM. We derived two dissociation constants (average Kd1 = 41 ± 11 μm Kd2 = 7 ± 4 μm) that reflect an increase in the affinity of VHS-UIM for Lys63-Ub2 compared with Lys48-Ub2. Assuming that VHS binds first and UIM second, it appears that the binding of VHS to Lys63-Ub2 is only slightly stronger than to Lys48-Ub2 or mono-Ub, whereas the UIM binds ∼40 times stronger. This agrees with the notion that the presence of multiple UBDs (in tandem) can greatly enhance the affinity for binding partners and/or induce a preference for a given polyubiquitin chain (
      • Sims J.J.
      • Cohen R.E.
      Linkage-specific avidity defines the lysine 63-linked polyubiquitin-binding preference of Rap80.
      ). From a mechanistic point of view, the binding can be envisioned to occur stepwise by first binding one of the UBDs to a Ub unit. Upon binding, the first UBD becomes part of the complex, and the binding of the second UBD becomes intramolecular. One can assume that one of the roles of the flexible linker is to keep the UBD in proximity, thus increasing its effective local concentration to ∼10 mm. This value is of the same order of magnitude as the one calculated for the RAP80-tUIM/Lys63-Ub2 (∼12 mm) or S5A/Lys48-Ub2 (∼3 mm) complexes (
      • Markin C.J.
      • Xiao W.
      • Spyracopoulos L.
      Mechanism for recognition of polyubiquitin chains. Balancing affinity through interplay between multivalent binding and dynamics.
      ) and reflects the dependence of the effective local concentration on the length of the inter-UBD linker. In addition to stepwise binding and increasing the local effective concentration, one has to take into account the cooperative nature of the VHS-UIM/Lys63-Ub2 interaction. A possible mechanism to explain the cooperativity is that the binding of VHS to the first Ub unit would position UIM favorably for interaction with a nearby Ub, leading to the cooperative stabilization of the UIMs helix. The difference in tumbling between the VHS and UIM in VHS-UIM could have important consequences regarding molecular events, as the initial binding of VHS to 1 Ub unit can be followed by a rapid reorientation (on the nanosecond time scale), binding, and subsequent stabilization of the UIM helix. Binding of the first UBD to a Ub unit will result in a loss of its translational and rotational degrees of freedom (
      • Finkelstein A.V.
      • Janin J.
      The price of lost freedom. Entropy of bimolecular complex formation.
      ), hence reducing the conformational entropy. However, the flexibility of the linker as well as the cooperative stabilization of the UIM helix can overcome the entropic penalty. One has to keep in mind that flexibility has an important influence on the thermodynamics of binding and may both favor and disfavor association (
      • Grünberg R.
      • Nilges M.
      • Leckner J.
      Flexibility and conformational entropy in protein-protein binding.
      ). In the light of our results, the differences between the binding of VHS-UIM to mono-Ub, Lys48-Ub2, and Lys63-Ub2 could be one explanation for the better sorting efficiency of Lys63- compared with Lys48-polyubiquitinated targets in the context of the full ESCRT-0.
      A polypeptide chain linking two successive UBDs as well as the nature of the UBDs could favor a specific ubiquitin chain linkage. Among the few existing structures reporting the interaction of multiple UBDs with polyubiquitin chains, S5a, which is involved in proteasomal degradation, contains two UIMs and binds preferentially to Lys48-Ub2. Moreover, the UIMs of S5a bind the two Ub units simultaneously and with UIM1 showing a 3:1 preference for the distal Ub of Lys48-Ub2 possibly reflecting specific interactions with the Ub-Ub linker (
      • Zhang N.
      • Wang Q.
      • Ehlinger A.
      • Randles L.
      • Lary J.W.
      • Kang Y.
      • Haririnia A.
      • Storaska A.J.
      • Cole J.L.
      • Fushman D.
      • Walters K.J.
      Structure of the S5a:K48-linked diubiquitin complex and its interactions with Rpn13.
      ). Rap80 is another protein that harbors two UIM domains linked in tandem (tUIM). The tUIM fragment specifically recognizes Lys63-Ub2 chains where the length of the linker is critical for binding with high affinity. By engineering different linker lengths, Sims and Cohen (
      • Sims J.J.
      • Cohen R.E.
      Linkage-specific avidity defines the lysine 63-linked polyubiquitin-binding preference of Rap80.
      ) demonstrated that the linker length and composition modulated the affinity of RAP80-tUIM for Lys63-Ub2 in a periodic fashion. Changes in the length of the helical linker not only modify the distance between the two UIMs, but also the relative orientation of their Ub-binding surfaces. Thus, the Rap80 linker defines selectivity through domain positioning rather than specific contact with the Ub-Ub linkage. Another example is given by NEMO (NF-κ essential modulator) that forms a heterodimer, which preferentially binds to NC-Ub2 via its UBAN domain (ubiquitin binding in ABIN and NEMO) (
      • Rahighi S.
      • Ikeda F.
      • Kawasaki M.
      • Akutsu M.
      • Suzuki N.
      • Kato R.
      • Kensche T.
      • Uejima T.
      • Bloor S.
      • Komander D.
      • Randow F.
      • Wakatsuki S.
      • Dikic I.
      Specific recognition of linear ubiquitin chains by NEMO is important for NF-κB activation.
      ,
      • Lo Y.C.
      • Lin S.C.
      • Rospigliosi C.C.
      • Conze D.B.
      • Wu C.J.
      • Ashwell J.D.
      • Eliezer D.
      • Wu H.
      Structural basis for recognition of diubiquitins by NEMO.
      ). The NEMO dimer accommodates two Ub2s, whereas both NEMO protomers contribute to the binding to each Ub2. In contrast to Rap80, NEMO specifically recognizes the linker region of head to tail chains and can distinguish between NC- and Lys63-linked chains. These examples demonstrate that an array of homologous UBDs can define chain linkage selectivity. Therefore, not only the combination of UBDs but also the length of the intervening linker and the arrangement of the UBDs could form the basis for linkage-specific polyubiquitin recognition. Additionally, the combination of heterologous UBDs of different sizes, like in STAM2, could influence the discrimination between different polyubiquitin chain linkages. Other factors like oligomerization, compartmentalization, and concentration of UBD-containing proteins could contribute to and possibly enhance/complicate the discrimination between different Ub chains.
      In the context of the full ESCRT machinery, STAM2 and Hrs form a heterodimer/tetramer (
      • Hurley J.H.
      The ESCRT complexes.
      ,
      • Mayers J.R.
      • Fyfe I.
      • Schuh A.L.
      • Chapman E.R.
      • Edwardson J.M.
      • Audhya A.
      ESCRT-0 assembles as a heterotetrameric complex on membranes and binds multiple ubiquitinylated cargoes simultaneously.
      ) through their GAT domain to form the ESCRT-0 component. The fact that Lys63-linked polyubiquitin chains now emerge as a specific signal for protein sorting into the MVB pathway (
      • Lauwers E.
      • Jacob C.
      • André B.
      Lys63-linked ubiquitin chains as a specific signal for protein sorting into the multivesicular body pathway.
      ,
      • Jencks W.P.
      On the attribution and additivity of binding energies.
      ,
      • Barriere H.
      • Nemes C.
      • Du K.
      • Lukacs G.L.
      Plasticity of polyubiquitin recognition as lysosomal targeting signals by the endosomal sorting machinery.
      ) could be linked to different factors, one of them being the higher affinity and cooperativity of the STAM2 VHS-UIM domains for Lys63-Ub2 chains. From the general view of the ESCRT-0 complex, two different models have been proposed. In one of them, cargoes are passed sequentially from ESCRT-0 to ESCRT-I, -II, and -III. If one considers that ESCRT-0 originally possesses four UBDs being part of STAM (VHS and UIM) and Hrs (VHS and DUIM), one can wonder how cargoes carrying Lys63-Ub2 chains can be transferred to the ESCRT-I complex, with a UEV domain of Tsg101 that binds to mono-Ub with a Kd as high as 510 μm (
      • Garrus J.E.
      • von Schwedler U.K.
      • Pornillos O.W.
      • Morham S.G.
      • Zavitz K.H.
      • Wang H.E.
      • Wettstein D.A.
      • Stray K.M.
      • Côté M.
      • Rich R.L.
      • Myszka D.G.
      • Sundquist W.I.
      Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding.
      ). In contrast, our data tend to support a second model where UBDs within the different ESCRT complexes cooperate to increase the binding affinity for ubiquitinated cargoes and work as a “supercomplex” (
      • Shields S.B.
      • Oestreich A.J.
      • Winistorfer S.
      • Nguyen D.
      • Payne J.A.
      • Katzmann D.J.
      • Piper R.
      ESCRT ubiquitin-binding domains function cooperatively during MVB cargo sorting.
      ), with ESCRT-0 being the main complex to cluster ubiquitinated cargoes (
      • Slagsvold T.
      • Pattni K.
      • Malerød L.
      • Stenmark H.
      Endosomal and nonendosomal functions of ESCRT proteins.
      ).

      Acknowledgments

      The eNMR project (European FP7 e-Infrastructure grant, contract no. 213010), supported by the national GRID Initiatives of Italy, Germany, and the Dutch BiG Grid project (Netherlands Organization for Scientific Research) are acknowledged for the use of web portals, computing, and storage facilities.

      References

        • Platta H.W.
        • Stenmark H.
        Cell structure and dynamics.
        Curr. Opin. Cell Biol. 2011; 23: 393-403
        • Sorkin A.
        • von Zastrow M.
        Endocytosis and signaling. Intertwining molecular networks.
        Nat. Rev. Mol. Cell Biol. 2009; 10: 609-622
        • Scita G.
        • Di Fiore P.P.
        The endocytic matrix.
        Nature. 2010; 463: 464-473
        • Hicke L.
        • Dunn R.
        Regulation of membrane protein transport by ubiquitin and ubiquitin-binding proteins.
        Annu. Rev. Cell Dev. Biol. 2003; 19: 141-172
        • Haglund K.
        • Di Fiore P.P.
        • Dikic I.
        Distinct monoubiquitin signals in receptor endocytosis.
        Trends Biochem. Sci. 2003; 28: 598-603
        • Pickart C.M.
        • Fushman D.
        Polyubiquitin chains. Polymeric protein signals.
        Curr. Opin. Chem. Biol. 2004; 8: 610-616
        • Clague M.J.
        • Urbé S.
        Ubiquitin, same molecule, different degradation pathways.
        Cell. 2010; 143: 682-685
        • Hicke L.
        Protein regulation by monoubiquitin.
        Nat. Rev. Mol. Cell Biol. 2001; 2: 195-201
        • Acconcia F.
        • Sigismund S.
        • Polo S.
        Ubiquitin in trafficking. The network at work.
        Exp. Cell Res. 2009; 315: 1610-1618
        • Duncan L.M.
        • Piper S.
        • Dodd R.B.
        • Saville M.K.
        • Sanderson C.M.
        • Luzio J.P.
        • Lehner P.J.
        Lysine 63-linked ubiquitination is required for endolysosomal degradation of class I molecules.
        EMBO J. 2006; 25: 1635-1645
        • Lauwers E.
        • Erpapazoglou Z.
        • Haguenauer-Tsapis R.
        • André B.
        The ubiquitin code of yeast permease trafficking.
        Trends Cell Biol. 2010; 20: 196-204
        • Lauwers E.
        • Jacob C.
        • André B.
        Lys63-linked ubiquitin chains as a specific signal for protein sorting into the multivesicular body pathway.
        J. Cell Biol. 2009; 185: 493-502
        • Raiborg C.
        • Malerød L.
        • Pedersen N.M.
        • Stenmark H.
        Differential functions of Hrs and ESCRT proteins in endocytic membrane trafficking.
        Exp. Cell Res. 2008; 314: 801-813
        • Henne W.M.
        • Buchkovich N.J.
        • Emr S.D.
        The ESCRT pathway.
        Dev. Cell. 2011; 21: 77-91
        • Mizuno E.
        • Kawahata K.
        • Okamoto A.
        • Kitamura N.
        • Komada M.
        Association with Hrs is required for the early endosomal localization, stability, and function of STAM.
        J. Biochem. 2004; 135: 385-396
        • Hurley J.H.
        The ESCRT complexes.
        Crit. Rev. Biochem. Mol. Biol. 2010; 45: 463-487
        • Hurley J.H.
        ESCRT complexes and the biogenesis of multivesicular bodies.
        Curr. Opin. Cell Biol. 2008; 20: 4-11
        • Dikic I.
        • Wakatsuki S.
        • Walters K.J.
        Ubiquitin-binding domains. From structures to functions.
        Nat. Rev. Mol. Cell Biol. 2009; 10: 659-671
        • Mizuno E.
        • Kawahata K.
        • Kato M.
        • Kitamura N.
        • Komada M.
        STAM proteins bind ubiquitinated proteins on the early endosome via the VHS domain and ubiquitin-interacting motif.
        Mol. Biol. Cell. 2003; 14: 3675-3689
        • Fisher R.D.
        • Wang B.
        • Alam S.L.
        • Higginson D.S.
        • Robinson H.
        • Sundquist W.I.
        • Hill C.P.
        Structure and ubiquitin binding of the ubiquitin-interacting motif.
        J. Biol. Chem. 2003; 278: 28976-28984
        • Ren X.
        • Hurley J.H.
        VHS domains of ESCRT-0 cooperate in high-avidity binding to polyubiquitinated cargo.
        EMBO J. 2010; 29: 1045-1054
        • Wollert T.
        • Hurley J.H.
        Molecular mechanism of multivesicular body biogenesis by ESCRT complexes.
        Nature. 2010; 464: 864-869
        • Lange A.
        • Hoeller D.
        • Wienk H.
        • Marcillat O.
        • Lancelin J.M.
        • Walker O.
        NMR reveals a different mode of binding of the Stam2 VHS domain to ubiquitin and diubiquitin.
        Biochemistry. 2011; 50: 48-62
        • Varadan R.
        • Walker O.
        • Pickart C.
        • Fushman D.
        Structural properties of polyubiquitin chains in solution.
        J. Mol. Biol. 2002; 324: 637-647
        • Pickart C.M.
        • Raasi S.
        Controlled synthesis of polyubiquitin chains.
        Methods Enzymol. 2005; 399: 21-36
        • Zhang D.
        • Raasi S.
        • Fushman D.
        Affinity makes the difference. Nonselective interaction of the UBA domain of ubiquilin-1 with monomeric ubiquitin and polyubiquitin chains.
        J. Mol. Biol. 2008; 377: 162-180
        • Varadan R.
        • Assfalg M.
        • Haririnia A.
        • Raasi S.
        • Pickart C.
        • Fushman D.
        Solution conformation of Lys63-linked diubiquitin chain provides clues to functional diversity of polyubiquitin signaling.
        J. Biol. Chem. 2004; 279: 7055-7063
        • Varadan R.
        • Assfalg M.
        • Fushman D.
        Using NMR spectroscopy to monitor ubiquitin chain conformation and interactions with ubiquitin-binding domains.
        Methods Enzymol. 2005; 399: 177-192
        • Delaglio F.
        • Grzesiek S.
        • Vuister G.W.
        • Zhu G.
        • Pfeifer J.
        • Bax A.
        NMRPipe, a multidimensional spectral processing system based on UNIX pipes.
        J. Biomol. NMR. 1995; 6: 277-293
        • Keller R.
        The Computer-aided Resonance Assignment Tutorial. 2004; (CARA NMR)
        • Pervushin K.
        • Riek R.
        • Wider G.
        • Wüthrich K.
        Attenuated T2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution.
        Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 12366-12371
        • Fushman D.
        • Cahill S.
        • Cowburn D.
        The main-chain dynamics of the dynamin pleckstrin homology (PH) domain in solution. Analysis of 15N relaxation with monomer/dimer equilibration.
        J. Mol. Biol. 1997; 266: 173-194
        • Hall J.B.
        • Fushman D.
        Characterization of the overall and local dynamics of a protein with intermediate rotational anisotropy. Differentiating between conformational exchange and anisotropic diffusion in the B3 domain of protein G.
        J. Biomol. NMR. 2003; 27: 261-275
        • Kay L.E.
        • Torchia D.A.
        • Bax A.
        Backbone dynamics of proteins as studied by 15N inverse detected heteronuclear NMR spectroscopy. Application to staphylococcal nuclease.
        Biochemistry. 1989; 28: 8972-8979
        • Grzesiek S.
        • Bax A.
        The importance of not saturating water in protein NMR. Application to sensitivity enhancement and NOE measurements.
        J. Am. Chem. Soc. 1993; 115: 12593-12594
        • Goddard T.D.
        • Kneller D.G.
        SPARKY 3. University of California, San Francisco, CA2001
        • Ryabov Y.
        • Fushman D.
        Interdomain mobility in diubiquitin revealed by NMR.
        Proteins. 2006; 63: 787-796
        • Swanson K.A.
        • Kang R.S.
        • Stamenova S.D.
        • Hicke L.
        • Radhakrishnan I.
        Solution structure of Vps27 UIM-ubiquitin complex important for endosomal sorting and receptor down-regulation.
        EMBO J. 2003; 22: 4597-4606
        • Sali A.
        • Blundell T.L.
        Comparative protein modelling by satisfaction of spatial restraints.
        J. Mol. Biol. 1993; 234: 779-815
        • Wishart D.S.
        • Sykes B.D.
        The 13C chemical-shift index. A simple method for the identification of protein secondary structure using 13C chemical shift data.
        J. Biomol. NMR. 1994; 4: 171-180
        • Lim J.
        • Son W.S.
        • Park J.K.
        • Kim E.E.
        • Lee B.J.
        • Ahn H.C.
        Solution structure of UIM and interaction of tandem ubiquitin binding domains in STAM1 with ubiquitin.
        Biochem. Biophys. Res. Commun. 2011; 405: 24-30
        • Clore G.M.
        • Iwahara J.
        Theory, practice, and applications of paramagnetic relaxation enhancement for the characterization of transient low-population states of biological macromolecules and their complexes.
        Chem. Rev. 2009; 109: 4108-4139
        • Ubbink M.
        The courtship of proteins. Understanding the encounter complex.
        FEBS Lett. 2009; 583: 1060-1066
        • Dominguez C.
        • Boelens R.
        • Bonvin A.
        HADDOCK. A protein-protein docking approach based on biochemical or biophysical information.
        J. Am. Chem. Soc. 2003; 125: 1731-1737
        • de Vries S.J.
        • van Dijk A.D.
        • Krzeminski M.
        • van Dijk M.
        • Thureau A.
        • Hsu V.
        • Wassenaar T.
        • Bonvin A.M.
        HADDOCK versus HADDOCK. New features and performance of HADDOCK2.0 on the CAPRI targets.
        Proteins. 2007; 69: 726-733
        • Markin C.J.
        • Xiao W.
        • Spyracopoulos L.
        Mechanism for recognition of polyubiquitin chains. Balancing affinity through interplay between multivalent binding and dynamics.
        J. Am. Chem. Soc. 2010; 132: 11247-11258
        • Zhou H.X.
        Loops in proteins can be modeled as worm-like chains.
        J. Phys. Chem. B. 2001; 105: 6763-6766
        • Zhou H.X.
        • Gilson M.K.
        Theory of free energy and entropy in noncovalent binding.
        Chem. Rev. 2009; 109: 4092-4107
        • Bilodeau P.S.
        • Urbanowski J.L.
        • Winistorfer S.C.
        • Piper R.C.
        The Vps27p Hse1p complex binds ubiquitin and mediates endosomal protein sorting.
        Nat. Cell Biol. 2002; 4: 534-539
        • Shih S.C.
        • Katzmann D.J.
        • Schnell J.D.
        • Sutanto M.
        • Emr S.D.
        • Hicke L.
        Epsins and Vps27p/Hrs contain ubiquitin-binding domains that function in receptor endocytosis.
        Nat. Cell Biol. 2002; 4: 389-393
        • Hofmann K.
        • Falquet L.
        A ubiquitin-interacting motif conserved in components of the proteasomal and lysosomal protein degradation systems.
        Trends Biochem. Sci. 2001; 26: 347-350
        • Haririnia A.
        • D'Onofrio M.
        • Fushman D.
        Mapping the interactions between Lys48- and Lys63-linked diubiquitins and a ubiquitin-interacting motif of S5a.
        J. Mol. Biol. 2007; 368: 753-766
        • Datta A.B.
        • Hura G.L.
        • Wolberger C.
        The structure and conformation of Lys63-linked tetraubiquitin.
        J. Mol. Biol. 2009; 392: 1117-1124
        • Sims J.J.
        • Cohen R.E.
        Linkage-specific avidity defines the lysine 63-linked polyubiquitin-binding preference of Rap80.
        Mol. Cell. 2009; 33: 775-783
        • Finkelstein A.V.
        • Janin J.
        The price of lost freedom. Entropy of bimolecular complex formation.
        Protein Eng. 1989; 3: 1-3
        • Grünberg R.
        • Nilges M.
        • Leckner J.
        Flexibility and conformational entropy in protein-protein binding.
        Structure. 2006; 14: 683-693
        • Zhang N.
        • Wang Q.
        • Ehlinger A.
        • Randles L.
        • Lary J.W.
        • Kang Y.
        • Haririnia A.
        • Storaska A.J.
        • Cole J.L.
        • Fushman D.
        • Walters K.J.
        Structure of the S5a:K48-linked diubiquitin complex and its interactions with Rpn13.
        Mol. Cell. 2009; 35: 280-290
        • Rahighi S.
        • Ikeda F.
        • Kawasaki M.
        • Akutsu M.
        • Suzuki N.
        • Kato R.
        • Kensche T.
        • Uejima T.
        • Bloor S.
        • Komander D.
        • Randow F.
        • Wakatsuki S.
        • Dikic I.
        Specific recognition of linear ubiquitin chains by NEMO is important for NF-κB activation.
        Cell. 2009; 136: 1098-1109
        • Lo Y.C.
        • Lin S.C.
        • Rospigliosi C.C.
        • Conze D.B.
        • Wu C.J.
        • Ashwell J.D.
        • Eliezer D.
        • Wu H.
        Structural basis for recognition of diubiquitins by NEMO.
        Mol. Cell. 2009; 33: 602-615
        • Mayers J.R.
        • Fyfe I.
        • Schuh A.L.
        • Chapman E.R.
        • Edwardson J.M.
        • Audhya A.
        ESCRT-0 assembles as a heterotetrameric complex on membranes and binds multiple ubiquitinylated cargoes simultaneously.
        J. Biol. Chem. 2011; 286: 9636-9645
        • Jencks W.P.
        On the attribution and additivity of binding energies.
        Proc. Natl. Acad. Sci. U.S.A. 1981; 78: 4046-4050
        • Barriere H.
        • Nemes C.
        • Du K.
        • Lukacs G.L.
        Plasticity of polyubiquitin recognition as lysosomal targeting signals by the endosomal sorting machinery.
        Mol. Biol. Cell. 2007; 18: 3952-3965
        • Garrus J.E.
        • von Schwedler U.K.
        • Pornillos O.W.
        • Morham S.G.
        • Zavitz K.H.
        • Wang H.E.
        • Wettstein D.A.
        • Stray K.M.
        • Côté M.
        • Rich R.L.
        • Myszka D.G.
        • Sundquist W.I.
        Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding.
        Cell. 2001; 107: 55-65
        • Shields S.B.
        • Oestreich A.J.
        • Winistorfer S.
        • Nguyen D.
        • Payne J.A.
        • Katzmann D.J.
        • Piper R.
        ESCRT ubiquitin-binding domains function cooperatively during MVB cargo sorting.
        J. Cell Biol. 2009; 185: 213-224
        • Slagsvold T.
        • Pattni K.
        • Malerød L.
        • Stenmark H.
        Endosomal and nonendosomal functions of ESCRT proteins.
        Trends Cell Biol. 2006; 16: 317-326