Structural basis of collagen recognition by human osteoclast-associated receptor and design of osteoclastogenesis inhibitors

Human osteoclast-associated receptor (OSCAR) is an immunoglobulin (Ig)-like collagen receptor that is up-regulated on osteoclasts during osteoclastogenesis and is expressed in a range of myeloid cells. As a member of the leukocyte receptor complex family of proteins, OSCAR shares a high degree of sequence and structural homology with other collagen receptors of this family, including glycoprotein VI, leukocyte-associated Ig-like receptor-1, and leukocyte Ig-like receptor B4, but recognizes a unique collagen sequence. Here, we present the crystal structures of OSCAR in its free form and in complex with a triple-helical collagen-like peptide (CLP). These structures reveal that the CLP peptide binds only one of the two Ig-like domains, the membrane-proximal domain (domain 2) of OSCAR, with the middle and trailing chain burying a total of 661 Å2 of solvent-accessible collagen surface. This binding mode is facilitated by the unusual topography of the OSCAR protein, which displays an obtuse interdomain angle and a rotation of domain 2 relative to the membrane-distal domain 1. Moreover, the binding of the CLP to OSCAR appears to be mediated largely by tyrosine residues and conformational changes at a shallow Phe pocket. Furthermore, we investigated CLP peptides as inhibitors of osteoclastogenesis and found that a peptide length of 40 amino acids is required to ensure adequate inhibition of osteoclastogenesis in vitro. These findings provide valuable structural insights into the mode of collagen recognition by OSCAR and into the use of synthetic peptide matrikines for osteoclastogenesis inhibition.

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Assembly and cryo-EM structures of RNA-specific measles virus nucleocapsids provide mechanistic insight into paramyxoviral replication

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1816417116 ◽

2019 ◽

Vol 116 (10) ◽

pp. 4256-4264 ◽

Cited By ~ 14

Author(s):

Ambroise Desfosses ◽

Sigrid Milles ◽

Malene Ringkjøbing Jensen ◽

Serafima Guseva ◽

Jacques-Philippe Colletier ◽

...

Keyword(s):

Conformational Changes ◽

Measles Virus ◽

Rna Binding ◽

Structural Basis ◽

Particle Assembly ◽

Interaction Sites ◽

Assembly Kinetics ◽

High Resolution Structure ◽

Binding Groove

Assembly of paramyxoviral nucleocapsids on the RNA genome is an essential step in the viral cycle. The structural basis of this process has remained obscure due to the inability to control encapsidation. We used a recently developed approach to assemble measles virus nucleocapsid-like particles on specific sequences of RNA hexamers (poly-Adenine and viral genomic 5′) in vitro, and determined their cryoelectron microscopy maps to 3.3-Å resolution. The structures unambiguously determine 5′ and 3′ binding sites and thereby the binding-register of viral genomic RNA within nucleocapsids. This observation reveals that the 3′ end of the genome is largely exposed in fully assembled measles nucleocapsids. In particular, the final three nucleotides of the genome are rendered accessible to the RNA-dependent RNA polymerase complex, possibly enabling efficient RNA processing. The structures also reveal local and global conformational changes in the nucleoprotein upon assembly, in particular involving helix α6 and helix α13 that form edges of the RNA binding groove. Disorder is observed in the bound RNA, localized at one of the two backbone conformational switch sites. The high-resolution structure allowed us to identify putative nucleobase interaction sites in the RNA-binding groove, whose impact on assembly kinetics was measured using real-time NMR. Mutation of one of these sites, R195, whose sidechain stabilizes both backbone and base of a bound nucleic acid, is thereby shown to be essential for nucleocapsid-like particle assembly.

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Structural basis for pH-dependent retrieval of ER proteins from the Golgi by the KDEL receptor

Science ◽

10.1126/science.aaw2859 ◽

2019 ◽

Vol 363 (6431) ◽

pp. 1103-1107 ◽

Cited By ~ 30

Author(s):

Philipp Bräuer ◽

Joanne L. Parker ◽

Andreas Gerondopoulos ◽

Iwan Zimmermann ◽

Markus A. Seeger ◽

...

Keyword(s):

Conformational Changes ◽

Secretory Pathway ◽

Cell Function ◽

Interaction Network ◽

Eukaryotic Cell ◽

Structural Basis ◽

Ph Dependent ◽

Kdel Receptor

Selective export and retrieval of proteins between the endoplasmic reticulum (ER) and Golgi apparatus is indispensable for eukaryotic cell function. An essential step in the retrieval of ER luminal proteins from the Golgi is the pH-dependent recognition of a carboxyl-terminal Lys-Asp-Glu-Leu (KDEL) signal by the KDEL receptor. Here, we present crystal structures of the chicken KDEL receptor in the apo ER state, KDEL-bound Golgi state, and in complex with an antagonistic synthetic nanobody (sybody). These structures show a transporter-like architecture that undergoes conformational changes upon KDEL binding and reveal a pH-dependent interaction network crucial for recognition of the carboxyl terminus of the KDEL signal. Complementary in vitro binding and in vivo cell localization data explain how these features create a pH-dependent retrieval system in the secretory pathway.

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CD94-NKG2A recognition of human leukocyte antigen (HLA)-E bound to an HLA class I leader sequence

Journal of Experimental Medicine ◽

10.1084/jem.20072525 ◽

2008 ◽

Vol 205 (3) ◽

pp. 725-735 ◽

Cited By ~ 124

Author(s):

Emma J. Petrie ◽

Craig S. Clements ◽

Jie Lin ◽

Lucy C. Sullivan ◽

Darryl Johnson ◽

...

Keyword(s):

Human Leukocyte Antigen ◽

Conformational Changes ◽

Specific Interaction ◽

Human Leukocyte ◽

Hla Class I ◽

Binding Mode ◽

Leader Sequence ◽

Structural Basis ◽

Peptide Ligand ◽

Leukocyte Antigen

The recognition of human leukocyte antigen (HLA)-E by the heterodimeric CD94-NKG2 natural killer (NK) receptor family is a central innate mechanism by which NK cells monitor the expression of other HLA molecules, yet the structural basis of this highly specific interaction is unclear. Here, we describe the crystal structure of CD94-NKG2A in complex with HLA-E bound to a peptide derived from the leader sequence of HLA-G. The CD94 subunit dominated the interaction with HLA-E, whereas the NKG2A subunit was more peripheral to the interface. Moreover, the invariant CD94 subunit dominated the peptide-mediated contacts, albeit with poor surface and chemical complementarity. This unusual binding mode was consistent with mutagenesis data at the CD94-NKG2A–HLA-E interface. There were few conformational changes in either CD94-NKG2A or HLA-E upon ligation, and such a “lock and key” interaction is typical of innate receptor–ligand interactions. Nevertheless, the structure also provided insight into how this interaction can be modulated by subtle changes in the peptide ligand or by the pairing of CD94 with other members of the NKG2 family. Differences in the docking strategies used by the NKG2D and CD94-NKG2A receptors provided a basis for understanding the promiscuous nature of ligand recognition by NKG2D compared with the fidelity of the CD94-NKG2 receptors.

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Characterization of a new series of non-covalent proteasome inhibitors with exquisite potency and selectivity for the 20S β5-subunit

Biochemical Journal ◽

10.1042/bj20100383 ◽

2010 ◽

Vol 430 (3) ◽

pp. 461-476 ◽

Cited By ~ 104

Author(s):

Christopher Blackburn ◽

Kenneth M. Gigstad ◽

Paul Hales ◽

Khristofer Garcia ◽

Matthew Jones ◽

...

Keyword(s):

High Throughput Screening ◽

Covalent Binding ◽

Proteasome Inhibitors ◽

Binding Mode ◽

26S Proteasome ◽

Luciferase Reporter ◽

Structural Basis ◽

Core Particle ◽

In Cells

The mammalian 26S proteasome is a 2500 kDa multi-catalytic complex involved in intracellular protein degradation. We describe the synthesis and properties of a novel series of non-covalent di-peptide inhibitors of the proteasome used on a capped tri-peptide that was first identified by high-throughput screening of a library of approx. 350000 compounds for inhibitors of the ubiquitin–proteasome system in cells. We show that these compounds are entirely selective for the β5 (chymotrypsin-like) site over the β1 (caspase-like) and β2 (trypsin-like) sites of the 20S core particle of the proteasome, and over a panel of less closely related proteases. Compound optimization, guided by X-ray crystallography of the liganded 20S core particle, confirmed their non-covalent binding mode and provided a structural basis for their enhanced in vitro and cellular potencies. We demonstrate that such compounds show low nanomolar IC50 values for the human 20S β5 site in vitro, and that pharmacological inhibition of this site in cells is sufficient to potently inhibit the degradation of a tetra-ubiquitin–luciferase reporter, activation of NFκB (nuclear factor κB) in response to TNF-α (tumour necrosis factor-α) and the proliferation of cancer cells. Finally, we identified capped di-peptides that show differential selectivity for the β5 site of the constitutively expressed proteasome and immunoproteasome in vitro and in B-cell lymphomas. Collectively, these studies describe the synthesis, activity and binding mode of a new series of non-covalent proteasome inhibitors with unprecedented potency and selectivity for the β5 site, and which can discriminate between the constitutive proteasome and immunoproteasome in vitro and in cells.

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Structural Basis of Eco1-Mediated Cohesin Acetylation

Scientific Reports ◽

10.1038/srep44313 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 4

Author(s):

William C. H. Chao ◽

Benjamin O. Wade ◽

Céline Bouchoux ◽

Andrew W. Jones ◽

Andrew G. Purkiss ◽

...

Keyword(s):

Mass Spectrometry ◽

Substrate Specificity ◽

Binding Site ◽

Conformational Changes ◽

Sister Chromatid ◽

Molecular Basis ◽

Substrate Recognition ◽

Structural Basis ◽

Chromatid Cohesion

Abstract Sister-chromatid cohesion is established by Eco1-mediated acetylation on two conserved tandem lysines in the cohesin Smc3 subunit. However, the molecular basis of Eco1 substrate recognition and acetylation in cohesion is not fully understood. Here, we discover and rationalize the substrate specificity of Eco1 using mass spectrometry coupled with in-vitro acetylation assays and crystallography. Our structures of the X. laevis Eco2 (xEco2) bound to its primary and secondary Smc3 substrates demonstrate the plasticity of the substrate-binding site, which confers substrate specificity by concerted conformational changes of the central β hairpin and the C-terminal extension.

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A metal ion orients mRNA to ensure accurate 2’-O ribosyl methylation of the first nucleotide of the SARS-CoV-2 genome

10.1101/2021.03.12.435174 ◽

2021 ◽

Author(s):

Thiruselvam Viswanathan ◽

Anurag Misra ◽

Siu-Hong Chan ◽

Shan Qi ◽

Nan Dai ◽

...

Keyword(s):

Conformational Changes ◽

Metal Ion ◽

Immune Surveillance ◽

Binding Mode ◽

Product Formation ◽

Structural Basis ◽

Outbreak Strain ◽

Product Release ◽

The Status ◽

Divalent Metal Ion

AbstractThe SARS-CoV-2 nsp16/nsp10 enzyme complex modifies the 2’-OH of the first transcribed nucleotide of the viral mRNA by covalently attaching a methyl group to it. The 2’-O methylation of the first nucleotide converts the status of mRNA cap from Cap-0 to Cap-1, and thus, helps the virus evade immune surveillance in the host cell. Here, we report two structures of nsp16/nsp10 representing pre- and post-release states of the RNA product (Cap-1). We observe overall widening of the enzyme upon product formation, and an inward twisting motion in the substrate binding region upon product release. These conformational changes reset the enzyme for the next round of catalysis. The structures also identify a unique binding mode and the importance of a divalent metal ion for 2’-O methylation. We also describe underlying structural basis for the perturbed enzymatic activity of a clinical variant of SARS-CoV-2, and a previous SARS-CoV outbreak strain.

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The structural basis of huntingtin (Htt) fibril polymorphism, revealed by cryo-EM of exon 1 Htt fibrils

10.1101/2021.09.23.461534 ◽

2021 ◽

Author(s):

Sergey Nazarov ◽

Anass Chiki ◽

Driss Boudeffa ◽

Hilal Lashuel

Keyword(s):

Structural Heterogeneity ◽

Structural Basis ◽

Particle Analysis ◽

Structural Determinants ◽

Huntingtin Protein ◽

Fibril Structure ◽

Exon 1 ◽

High Degree ◽

Insight Into

The lack of detailed insight into the structure of aggregates formed by the huntingtin protein has hampered efforts to develop therapeutics and diagnostics targeting pathology formation in the brain of patients with Huntington's disease. To address this knowledge gap, we investigated the structural properties of in vitro generated fibrils from exon1 of the huntingtin protein by electron cryo-microscopy and single- particle analysis. We show that wildtype and mutant exon1 of the huntingtin protein form non-helical fibrils with a polygultamine amyloid core composed of β-hairpins with unique characteristics that have not been previously observed with other amyloid filaments. The stacks of β-hairpins form long planar β- sheets (protofilaments) with variable stacking angle and occasional out-of-register state of individual β-hairpins. These features and the propensity of protofilament to undergo lateral association results in a high degree of fibril polymorphism, including fibrils composed of varying numbers of protofilaments. Our results also represent the first direct observation of how the flanking domains are organized around the polyglutamine core of the fibril and provide insight into how they might affect huntingtin fibril structure, polymorphism, and stacking of β-hairpins within its core structure. Removal of the first 17 amino acids at the N-terminus resulted in surprising intra-fibril structural heterogeneity and reduced fibril's propensity to lateral associations. Overall, this work provides valuable insights that could guide future mechanistic studies to elucidate the sequence and structural determinants of huntingtin aggregation, as well as cryo- EM and structural studies of fibrils derived from huntingtin proteins and other disease-associated polyglutamine-containing proteins.

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Molecular Dissection of the Semliki Forest Virus Homotrimer Reveals Two Functionally Distinct Regions of the Fusion Protein

Journal of Virology ◽

10.1128/jvi.76.3.1194-1205.2002 ◽

2002 ◽

Vol 76 (3) ◽

pp. 1194-1205 ◽

Cited By ~ 29

Author(s):

Don L. Gibbons ◽

Margaret Kielian

Keyword(s):

Conformational Changes ◽

Semliki Forest Virus ◽

Fusion Reaction ◽

Fusion Peptide ◽

Structural Basis ◽

Reducing Conditions ◽

Target Membrane ◽

Membrane Bound ◽

Harsh Conditions

ABSTRACT Semliki Forest virus (SFV) is an enveloped alphavirus that infects cells via a membrane fusion reaction triggered by the acidic pH of endosomes. In response to low pH, the E1 proteins on the virus membrane undergo a series of conformational changes, resulting in the formation of a stable E1 homotrimer. Little is known about the structural basis of either the E1 conformational changes or the resulting homotrimer or about the mechanism of action of the homotrimer in fusion. Here, the E1 homotrimer was formed in vitro from either virus or soluble E1 ectodomain and then probed by various perturbants, proteases, or glycosidase. The preformed homotrimer was extremely stable to moderately harsh conditions and proteases. By contrast, mild reducing conditions selectively disrupted the N-terminal region of trimeric E1, making it accessible to proteolytic cleavage and producing E1 fragments that retained trimer interactions. Trypsin digestion produced a fragment missing a portion of the N terminus just proximal to the putative fusion peptide. Digestion with elastase produced several fragments with cleavage sites between residues 78 and 102, resulting in the loss of the putative fusion peptide and the release of membrane-bound E1 ectodomain as a soluble trimer. Elastase also cleaved the homotrimer within an E1 loop located near the fusion peptide in the native E1 structure. Mass spectrometry was used to map the C termini of several differentially produced and fully functional E1 ectodomains. Together, our data identify two separate regions of the SFV E1 ectodomain, one responsible for target membrane association and one necessary for trimer interactions.

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Structural basis for arginine methylation-independent recognition of PIWIL1 by TDRD2

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1711486114 ◽

2017 ◽

Vol 114 (47) ◽

pp. 12483-12488 ◽

Cited By ~ 12

Author(s):

Heng Zhang ◽

Ke Liu ◽

Natsuko Izumi ◽

Haiming Huang ◽

Deqiang Ding ◽

...

Keyword(s):

Binding Mode ◽

Arginine Methylation ◽

P Element ◽

Structural Basis ◽

Dependent Manner ◽

Transposon Silencing ◽

Tudor Domain ◽

Genome Protection ◽

Pirna Pathway

The P-element–induced wimpy testis (PIWI)-interacting RNA (piRNA) pathway plays a central role in transposon silencing and genome protection in the animal germline. A family of Tudor domain proteins regulates the piRNA pathway through direct Tudor domain–PIWI interactions. Tudor domains are known to fulfill this function by binding to methylated PIWI proteins in an arginine methylation-dependent manner. Here, we report a mechanism of methylation-independent Tudor domain–PIWI interaction. Unlike most other Tudor domains, the extended Tudor domain of mammalian Tudor domain-containing protein 2 (TDRD2) preferentially recognizes an unmethylated arginine-rich sequence from PIWI-like protein 1 (PIWIL1). Structural studies reveal an unexpected Tudor domain-binding mode for the PIWIL1 sequence in which the interface of Tudor and staphylococcal nuclease domains is primarily responsible for PIWIL1 peptide recognition. Mutations disrupting the TDRD2–PIWIL1 interaction compromise piRNA maturation via 3′-end trimming in vitro. Our work presented here reveals the molecular divergence of the interactions between different Tudor domain proteins and PIWI proteins.

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A metal ion orients SARS-CoV-2 mRNA to ensure accurate 2′-O methylation of its first nucleotide

Nature Communications ◽

10.1038/s41467-021-23594-y ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Thiruselvam Viswanathan ◽

Anurag Misra ◽

Siu-Hong Chan ◽

Shan Qi ◽

Nan Dai ◽

...

Keyword(s):

Conformational Changes ◽

Metal Ion ◽

Immune Surveillance ◽

Binding Mode ◽

Product Formation ◽

Host Cells ◽

Structural Basis ◽

Outbreak Strain ◽

The Status ◽

Divalent Metal Ion

AbstractThe SARS-CoV-2 nsp16/nsp10 enzyme complex modifies the 2′-OH of the first transcribed nucleotide of the viral mRNA by covalently attaching a methyl group to it. The 2′-O methylation of the first nucleotide converts the status of mRNA cap from Cap-0 to Cap-1, and thus, helps the virus evade immune surveillance in host cells. Here, we report two structures of nsp16/nsp10 representing pre- and post-release states of the RNA product (Cap-1). We observe overall widening of the enzyme upon product formation, and an inward twisting motion in the substrate binding region upon product release. These conformational changes reset the enzyme for the next round of catalysis. The structures also identify a unique binding mode and the importance of a divalent metal ion for 2′-O methylation. We also describe underlying structural basis for the perturbed enzymatic activity of a clinical variant of SARS-CoV-2, and a previous SARS-CoV outbreak strain.

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