Protein Structure and Folding
The ASCC2 CUE domain in the ALKBH3–ASCC DNA repair complex recognizes adjacent ubiquitins in K63-linked polyubiquitin
Alkylation of DNA and RNA is a potentially toxic lesion that can result in mutations and even cell death. In response to alkylation damage, K63-linked polyubiquitin chains are assembled that localize the Alpha-ketoglutarate-dependent dioxygenase alkB homolog 3–Activating Signal Cointegrator 1 Complex Subunit (ASCC) repair complex to damage sites in the nucleus. The protein ASCC2, a subunit of the ASCC complex, selectively binds K63-linked polyubiquitin chains via its coupling of ubiquitin conjugation to ER degradation (CUE) domain.Structural and DNA-binding properties of the cytoplasmic domain of Vibrio cholerae transcription factor ToxR
ToxR represents an essential transcription factor of Vibrio cholerae, which is involved in the regulation of multiple, mainly virulence associated genes. Its versatile functionality as activator, repressor or coactivator suggests a complex regulatory mechanism, whose clarification is essential for a better understanding of the virulence expression system of V. cholerae. Here, we provide structural information elucidating the organization and binding behavior of the cytoplasmic DNA-binding domain of ToxR (cToxR), containing a winged helix–turn–helix (wHTH) motif.Conformational triggers associated with influenza matrix protein 1 polymerization
A central role for the influenza matrix protein 1 (M1) is to form a polymeric coat on the inner leaflet of the host membrane that ultimately provides shape and stability to the virion. M1 polymerizes upon binding membranes, but triggers for conversion of M1 from a water-soluble component of the nucleus and cytosol into an oligomer at the membrane surface are unknown. While full-length M1 is required for virus viability, the N-terminal domain (M1NT) retains membrane binding and pH-dependent oligomerization.Structural characterization of HIV-1 matrix mutants implicated in envelope incorporation
During the late phase of HIV-1 infection, viral Gag polyproteins are targeted to the plasma membrane (PM) for assembly. Gag localization at the PM is a prerequisite for the incorporation of the envelope protein (Env) into budding particles. Gag assembly and Env incorporation are mediated by the N-terminal myristoylated matrix (MA) domain of Gag. Nonconservative mutations in the trimer interface of MA (A45E, T70R, and L75G) were found to impair Env incorporation and infectivity, leading to the hypothesis that MA trimerization is an obligatory step for Env incorporation.Structural details of amyloid β oligomers in complex with human prion protein as revealed by solid-state MAS NMR spectroscopy
Human PrP (huPrP) is a high-affinity receptor for oligomeric amyloid β (Aβ) protein aggregates. Binding of Aβ oligomers to membrane-anchored huPrP has been suggested to trigger neurotoxic cell signaling in Alzheimer’s disease, while an N-terminal soluble fragment of huPrP can sequester Aβ oligomers and reduce their toxicity. Synthetic oligomeric Aβ species are known to be heterogeneous, dynamic, and transient, rendering their structural investigation particularly challenging. Here, using huPrP to preserve Aβ oligomers by coprecipitating them into large heteroassemblies, we investigated the conformations of Aβ(1–42) oligomers and huPrP in the complex by solid-state MAS NMR spectroscopy.Structural basis for the DNA-binding activity of human ARID4B Tudor domain
Human ARID4A and ARID4B are homologous proteins that are important in controlling gene expression and epigenetic regulation but have distinct functions. Previous studies have shown that the N-terminal domain of ARID4A is an unusual interdigitated double Tudor domain with DNA-binding activity. However, how the Tudor domain of ARID4B differs from that of ARID4A remains unknown. Here, we found that the ARID4B Tudor domain has significantly weaker DNA affinity than the ARID4A Tudor domain despite sharing more than 80% sequence identity.Polyphosphates induce amyloid fibril formation of α-synuclein in concentration-dependent distinct manners
Polyphosphates (polyPs), chains of phosphate residues found in species across nature from bacteria to mammals, were recently reported to accelerate the amyloid fibril formation of many proteins. How polyPs facilitate this process, however, remains unknown. To gain insight into their mechanisms, we used various physicochemical approaches to examine the effects of polyPs of varying chain lengths on ultrasonication-dependent α-synuclein (α-syn) amyloid formation. Although orthophosphate and diphosphate exhibited a single optimal concentration of amyloid formation, triphosphate and longer-chain phosphates exhibited two optima, with the second at a concentration lower than that of orthophosphate or diphosphate.Seeded fibrils of the germline variant of human λ-III immunoglobulin light chain FOR005 have a similar core as patient fibrils with reduced stability
Systemic antibody light chains (AL) amyloidosis is characterized by deposition of amyloid fibrils derived from a particular antibody light chain. Cardiac involvement is a major risk factor for mortality. Using MAS solid-state NMR, we studied the fibril structure of a recombinant light chain fragment corresponding to the fibril protein from patient FOR005, together with fibrils formed by protein sequence variants that are derived from the closest germline (GL) sequence. Both analyzed fibril structures were seeded with ex-vivo amyloid fibrils purified from the explanted heart of this patient.Determinants of replication protein A subunit interactions revealed using a phosphomimetic peptide
Replication protein A (RPA) is a eukaryotic ssDNA-binding protein and contains three subunits: RPA70, RPA32, and RPA14. Phosphorylation of the N-terminal region of the RPA32 subunit plays an essential role in DNA metabolism in processes such as replication and damage response. Phosphorylated RPA32 (pRPA32) binds to RPA70 and possibly regulates the transient RPA70-Bloom syndrome helicase (BLM) interaction to inhibit DNA resection. However, the structural details and determinants of the phosphorylated RPA32–RPA70 interaction are still unknown.Hydrogen/deuterium exchange memory NMR reveals structural epitopes involved in IgE cross-reactivity of allergenic lipid transfer proteins
Identification of antibody-binding epitopes is crucial to understand immunological mechanisms. It is of particular interest for allergenic proteins with high cross-reactivity as observed in the lipid transfer protein (LTP) syndrome, which is characterized by severe allergic reactions. Art v 3, a pollen LTP from mugwort, is frequently involved in this cross-reactivity, but no antibody-binding epitopes have been determined so far. To reveal human IgE-binding regions of Art v 3, we produced three murine high-affinity mAbs, which showed 70–90% coverage of the allergenic epitopes from mugwort pollen–allergic patients.Structural characterization of anti-CCL5 activity of the tick salivary protein evasin-4
Ticks, as blood-sucking parasites, have developed a complex strategy to evade and suppress host immune responses during feeding. The crucial part of this strategy is expression of a broad family of salivary proteins, called Evasins, to neutralize chemokines responsible for cell trafficking and recruitment. However, structural information about Evasins is still scarce, and little is known about the structural determinants of their binding mechanism to chemokines. Here, we studied the structurally uncharacterized Evasin-4, which neutralizes a broad range of CC-motif chemokines, including the chemokine CC-motif ligand 5 (CCL5) involved in atherogenesis.The chemokine X-factor: Structure-function analysis of the CXC motif at CXCR4 and ACKR3
The human chemokine family consists of 46 protein ligands that induce chemotactic cell migration by activating a family of 23 G protein–coupled receptors. The two major chemokine subfamilies, CC and CXC, bind distinct receptor subsets. A sequence motif defining these families, the X position in the CXC motif, is not predicted to make significant contacts with the receptor, but instead links structural elements associated with binding and activation. Here, we use comparative analysis of chemokine NMR structures, structural modeling, and molecular dynamic simulations that suggested the X position reorients the chemokine N terminus.Cooperative dynamics across distinct structural elements regulate PTP1B activity
Protein-tyrosine phosphatase 1B (PTP1B) is the canonical enzyme for investigating how distinct structural elements influence enzyme catalytic activity. Although it is recognized that dynamics are essential for PTP1B function, the data collected thus far have not resolved whether distinct elements are dynamically coordinated or, alternatively, whether they fulfill their respective functions independently. To answer this question, we performed a comprehensive 13C-methyl relaxation study of Ile, Leu, and Val (ILV) residues of PTP1B, which, because of its substantially increased sensitivity, provides a comprehensive understanding of the influence of protein motions on different time scales for enzyme function.Hairpin RNA-induced conformational change of a eukaryotic-specific lysyl-tRNA synthetase extension and role of adjacent anticodon-binding domain
Human lysyl-tRNA synthetase (hLysRS) is essential for aminoacylation of tRNALys. Higher eukaryotic LysRSs possess an N-terminal extension (Nterm) previously shown to facilitate high-affinity tRNA binding and aminoacylation. This eukaryote-specific appended domain also plays a critical role in hLysRS nuclear localization, thus facilitating noncanonical functions of hLysRS. The structure is intrinsically disordered and therefore remains poorly characterized. Findings of previous studies are consistent with the Nterm domain undergoing a conformational transition to an ordered structure upon nucleic acid binding.Discovery and mechanistic studies of cytotoxic cyclotides from the medicinal herb Hybanthus enneaspermus
Cyclotides are plant-derived peptides characterized by an ∼30-amino acid–long cyclic backbone and a cystine knot motif. Cyclotides have diverse bioactivities, and their cytotoxicity has attracted significant attention for its potential anticancer applications. Hybanthus enneaspermus (Linn) F. Muell is a medicinal herb widely used in India as a libido enhancer, and a previous study has reported that it may contain cyclotides. In the current study, we isolated 11 novel cyclotides and 1 known cyclotide (cycloviolacin O2) from H.A terminal α3-galactose modification regulates an E3 ubiquitin ligase subunit in Toxoplasma gondii
Skp1, a subunit of E3 Skp1/Cullin-1/F-box protein ubiquitin ligases, is modified by a prolyl hydroxylase that mediates O2 regulation of the social amoeba Dictyostelium and the parasite Toxoplasma gondii. The full effect of hydroxylation requires modification of the hydroxyproline by a pentasaccharide that, in Dictyostelium, influences Skp1 structure to favor assembly of Skp1/F-box protein subcomplexes. In Toxoplasma, the presence of a contrasting penultimate sugar assembled by a different glycosyltransferase enables testing of the conformational control model.Mechanism of allosteric inhibition in the Plasmodium falciparum cGMP-dependent protein kinase
Most malaria deaths are caused by the protozoan parasite Plasmodium falciparum. Its life cycle is regulated by a cGMP-dependent protein kinase (PfPKG), whose inhibition is a promising antimalaria strategy. Allosteric kinase inhibitors, such as cGMP analogs, offer enhanced selectivity relative to competitive kinase inhibitors. However, the mechanisms underlying allosteric PfPKG inhibition are incompletely understood. Here, we show that 8-NBD-cGMP is an effective PfPKG antagonist. Using comparative NMR analyses of a key regulatory domain, PfD, in its apo, cGMP-bound, and cGMP analog–bound states, we elucidated its inhibition mechanism of action.Small molecule AX-024 reduces T cell proliferation independently of CD3ϵ/Nck1 interaction, which is governed by a domain swap in the Nck1-SH3.1 domain
Activation of the T cell receptor (TCR) results in binding of the adapter protein Nck (noncatalytic region of tyrosine kinase) to the CD3ϵ subunit of the TCR. The interaction was suggested to be important for the amplification of TCR signals and is governed by a proline-rich sequence (PRS) in CD3ϵ that binds to the first Src homology 3 (SH3) domain of Nck (Nck-SH3.1). Inhibition of this protein/protein interaction ameliorated inflammatory symptoms in mouse models of multiple sclerosis, psoriasis, and asthma.Probing the correlation between ligand efficacy and conformational diversity at the α1A-adrenoreceptor reveals allosteric coupling of its microswitches
G protein–coupled receptors (GPCRs) use a series of conserved microswitches to transmit signals across the cell membrane via an allosteric network encompassing the ligand-binding site and the G protein-binding site. Crystal structures of GPCRs provide snapshots of their inactive and active states, but poorly describe the conformational dynamics of the allosteric network that underlies GPCR activation. Here, we analyzed the correlation between ligand binding and receptor conformation of the α1A-adrenoreceptor, a GPCR that stimulates smooth muscle contraction in response to binding noradrenaline.Structure and allosteric activity of a single-disulfide conopeptide from Conus zonatus at human α3β4 and α7 nicotinic acetylcholine receptors
Conopeptides are neurotoxic peptides in the venom of marine cone snails and have broad therapeutic potential for managing pain and other conditions. Here, we identified the single-disulfide peptides Czon1107 and Cca1669 from the venoms of Conus zonatus and Conus caracteristicus, respectively. We observed that Czon1107 strongly inhibits the human α3β4 (IC50 15.7 ± 3.0 μm) and α7 (IC50 77.1 ± 0.05 μm) nicotinic acetylcholine receptor (nAChR) subtypes, but the activity of Cca1669 remains to be identified.The streptococcal multidomain fibrillar adhesin CshA has an elongated polymeric architecture
The cell surfaces of many bacteria carry filamentous polypeptides termed adhesins that enable binding to both biotic and abiotic surfaces. Surface adherence is facilitated by the exquisite selectivity of the adhesins for their cognate ligands or receptors and is a key step in niche or host colonization and pathogenicity. Streptococcus gordonii is a primary colonizer of the human oral cavity and an opportunistic pathogen, as well as a leading cause of infective endocarditis in humans. The fibrillar adhesin CshA is an important determinant of S.The major subunit of widespread competence pili exhibits a novel and conserved type IV pilin fold
Type IV filaments (T4F), which are helical assemblies of type IV pilins, constitute a superfamily of filamentous nanomachines virtually ubiquitous in prokaryotes that mediate a wide variety of functions. The competence (Com) pilus is a widespread T4F, mediating DNA uptake (the first step in natural transformation) in bacteria with one membrane (monoderms), an important mechanism of horizontal gene transfer. Here, we report the results of genomic, phylogenetic, and structural analyses of ComGC, the major pilin subunit of Com pili.The dynein light chain 8 (LC8) binds predominantly “in-register” to a multivalent intrinsically disordered partner
Dynein light chain 8 (LC8) interacts with intrinsically disordered proteins (IDPs) and influences a wide range of biological processes. It is becoming apparent that among the numerous IDPs that interact with LC8, many contain multiple LC8-binding sites. Although it is established that LC8 forms parallel IDP duplexes with some partners, such as nucleoporin Nup159 and dynein intermediate chain, the molecular details of these interactions and LC8's interactions with other diverse partners remain largely uncharacterized.Structural insights into TAZ2 domain–mediated CBP/p300 recruitment by transactivation domain 1 of the lymphopoietic transcription factor E2A
The E-protein transcription factors guide immune cell differentiation, with E12 and E47 (hereafter called E2A) being essential for B-cell specification and maturation. E2A and the oncogenic chimera E2A-PBX1 contain three transactivation domains (ADs), with AD1 and AD2 having redundant, independent, and cooperative functions in a cell-dependent manner. AD1 and AD2 both mediate their functions by binding to the KIX domain of the histone acetyltransferase paralogues CREB-binding protein (CBP) and E1A-binding protein P300 (p300).Methionine sulfoxide reductase B from Corynebacterium diphtheriae catalyzes sulfoxide reduction via an intramolecular disulfide cascade
Corynebacterium diphtheriae is a human pathogen that causes diphtheria. In response to immune system-induced oxidative stress, C. diphtheriae expresses antioxidant enzymes, among which are methionine sulfoxide reductase (Msr) enzymes, which are critical for bacterial survival in the face of oxidative stress. Although some aspects of the catalytic mechanism of the Msr enzymes have been reported, several details still await full elucidation. Here, we solved the solution structure of C. diphtheriae MsrB (Cd-MsrB) and unraveled its catalytic and oxidation-protection mechanisms.
