Structural basis of biased T cell receptor recognition of an immunodominant HLA-A2 epitope of the SARS-CoV-2 spike protein

CD8⁺ T cells play an important role in vaccination and immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Although numerous SARS-CoV-2 CD8⁺ T cell epitopes have been identified, the molecular basis underpinning T cell receptor (TCR) recognition of SARS-CoV-2-specific T cells remains unknown. The T cell response directed toward SARS-CoV-2 spike protein–derived S^269–277 peptide presented by the human leukocyte antigen (HLA)-A∗02:01 allomorph (hereafter the HLA-A2^S269–277 epitope) is, to date, the most immunodominant SARS-CoV-2 epitope found in individuals bearing this allele. As HLA-A2^S269–277-specific CD8⁺ T cells utilize biased TRAV12 gene usage within the TCR α-chain, we sought to understand the molecular basis underpinning this TRAV12 dominance. We expressed four TRAV12⁺ TCRs which bound the HLA-A2^S269–277 complex with low micromolar affinity and determined the crystal structure of the HLA-A2^S269–277 binary complex, and subsequently a ternary structure of the TRAV12⁺ TCR complexed to HLA-A2^S269–277. We found that the TCR made extensive contacts along the entire length of the S^269–277 peptide, suggesting that the TRAV12⁺ TCRs would be sensitive to sequence variation within this epitope. To examine this, we investigated cross-reactivity toward analogous peptides from existing SARS-CoV-2 variants and closely related coronaviruses. We show via surface plasmon resonance and tetramer studies that the TRAV12⁺ T cell repertoire cross-reacts poorly with these analogous epitopes. Overall, we defined the structural basis underpinning biased TCR recognition of CD8⁺ T cells directed at an immunodominant epitope and provide a framework for understanding TCR cross-reactivity toward viral variants within the S^269–277 peptide.     

Match_Motif: A rapid computational tool to assist in protein–protein interaction design

In order to generate protein assemblies with a desired function, the rational design of protein–protein binding interfaces is of significant interest. Approaches based on random mutagenesis or directed evolution may involve complex experimental selection procedures. Also, molecular modeling approaches to design entirely new proteins and interactions with partner molecules can involve large computational efforts and screening steps. In order to simplify at least the initial effort for designing a putative binding interface between two proteins the Match_Motif approach has been developed. It employs the large collection of known protein–protein complex structures to suggest interface modifications that may lead to improved binding for a desired input interaction geometry. The approach extracts interaction motifs based on the backbone structure of short (four residues) segments and the relative arrangement with respect to short segments on the partner protein. The interaction geometry is used to search through a database of such motifs in known stable bound complexes. All matches are rapidly identified (within a few seconds) and collected and can be used to guide changes in the interface that may lead to improved binding. In the output, an alternative interface structure is also proposed based on the frequency of occurrence of side chains at a given interface position in all matches and based on sterical considerations. Applications of the procedure to known complex structures and alternative arrangements are presented and discussed. The program, data files, and example applications can be downloaded from https://www.groups.ph.tum.de/t38/downloads/.     

Two distinct classes of cochaperones compete for the EEVD motif in heat shock protein 70 to tune its chaperone activities

Chaperones of the heat shock protein 70 (Hsp70) family engage in protein–protein interactions with many cochaperones. One “hotspot” for cochaperone binding is the EEVD motif, found at the extreme C terminus of cytoplasmic Hsp70s. This motif is known to bind tetratricopeptide repeat domain cochaperones, such as the E3 ubiquitin ligase CHIP. In addition, the EEVD motif also interacts with a structurally distinct domain that is present in class B J-domain proteins, such as DnaJB4. These observations suggest that CHIP and DnaJB4 might compete for binding to Hsp70’s EEVD motif; however, the molecular determinants of such competition are not clear. Using a collection of EEVD-derived peptides, including mutations and truncations, we explored which residues are critical for binding to both CHIP and DnaJB4. These results revealed that some features, such as the C-terminal carboxylate, are important for both interactions. However, CHIP and DnaJB4 also had unique preferences, especially at the isoleucine position immediately adjacent to the EEVD. Finally, we show that competition between these cochaperones is important in vitro, as DnaJB4 limits the ubiquitination activity of the Hsp70–CHIP complex, whereas CHIP suppresses the client refolding activity of the Hsp70–DnaJB4 complex. Together, these data suggest that the EEVD motif has evolved to support diverse protein–protein interactions, such that competition between cochaperones may help guide whether Hsp70-bound proteins are folded or degraded.     

Letting go: Deep computational modeling insights into pH-dependent calcium affinity

Calcium and other cofactors can feature as key additions to a molecular interface, to the extent that the cofactor is completely buried in the bound state. How can such an interaction be regulated then? The answer: By facilitating a switch through an allosteric network. Although a number of unbinding mechanisms are being characterized, an extensive computational study by Joswig et al. reveals a detailed model for the pattern recognition receptor langerin.     

Computational design of small molecular modulators of protein–protein interactions with a novel thermodynamic cycle: Allosteric inhibitors of HIV‐1 integrase

Targeting protein–protein interactions for therapeutic development involves designing small molecules to either disrupt or enhance a known PPI. For this purpose, it is necessary to compute reliably the effect of chemical modifications of small molecules on the protein–protein association free energy. Here we present results obtained using a novel thermodynamic free energy cycle, for the rational design of allosteric inhibitors of HIV‐1 integrase (ALLINI) that act specifically in the early stage of the infection cycle. The new compounds can serve as molecular probes to dissect the multifunctional mechanisms of ALLINIs to inform the discovery of new allosteric inhibitors. The free energy protocol developed here can be more broadly applied to study quantitatively the effects of small molecules on modulating the strengths of protein–protein interactions.     

Structural insights into the binding of SARS-CoV-2, SARS-CoV,and hCoV-NL63 spike receptor-binding domain to horse ACE2

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Structural delineation and phase-dependent activation of the costimulatory CD27:CD70 complex

CD27 is a tumor necrosis factor (TNF) receptor, which stimulates lymphocytes and promotes their differentiation upon activation by TNF ligand CD70. Activation of the CD27 receptor provides a costimulatory signal to promote T cell, B cell, and NK cell activity to facilitate antitumor and anti-infection immunity. Aberrant increased and focused expression of CD70 on many tumor cells renders CD70 an attractive therapeutic target for direct tumor killing. However, despite their use as drug targets to treat cancers, the molecular basis and atomic details of CD27 and CD70 interaction remain elusive. Here we report the crystal structure of human CD27 in complex with human CD70. Analysis of our structure shows that CD70 adopts a classical TNF ligand homotrimeric assembly to engage CD27 receptors in a 3:3 stoichiometry. By combining structural and rational mutagenesis data with reported disease-correlated mutations, we identified the key amino acid residues of CD27 and CD70 that control this interaction. We also report increased potency for plate-bound CD70 constructs compared with solution-phase ligand in a functional activity to stimulate T-cells in vitro. These findings offer new mechanistic insight into this critical costimulatory interaction.     

The binding of heparin to spike glycoprotein inhibits SARS-CoV-2 infection by three mechanisms

Heparin, a naturally occurring glycosaminoglycan, has been found to have antiviral activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative virus of COVID-19. To elucidate the mechanistic basis for the antiviral activity of heparin, we investigated the binding of heparin to the SARS-CoV-2 spike glycoprotein by means of sliding window docking, molecular dynamics simulations, and biochemical assays. Our simulations show that heparin binds at long, positively charged patches on the spike glycoprotein, thereby masking basic residues of both the receptor-binding domain (RBD) and the multifunctional S1/S2 site. Biochemical experiments corroborated the simulation results, showing that heparin inhibits the furin-mediated cleavage of spike by binding to the S1/S2 site. Our simulations showed that heparin can act on the hinge region responsible for motion of the RBD between the inactive closed and active open conformations of the spike glycoprotein. In simulations of the closed spike homotrimer, heparin binds the RBD and the N-terminal domain of two adjacent spike subunits and hinders opening. In simulations of open spike conformations, heparin induces stabilization of the hinge region and a change in RBD motion. Our results indicate that heparin can inhibit SARS-CoV-2 infection by three mechanisms: by allosterically hindering binding to the host cell receptor, by directly competing with binding to host heparan sulfate proteoglycan coreceptors, and by preventing spike cleavage by furin. Furthermore, these simulations provide insights into how host heparan sulfate proteoglycans can facilitate viral infection. Our results will aid the rational optimization of heparin derivatives for SARS-CoV-2 antiviral therapy.     

Differential binding of SARS-CoV-2 Spike protein variants to its cognate receptor hACE2 using molecular modeling based binding analysis

The current emergence of novel coronavirus, SARS-CoV-2 and its ceaseless expansion worldwide has posed a global health emergency that has adversely affected the humans. With the entire world striving to understand the newly emerged virus, differences in morbidity and infection rate of SARS-CoV-2 have been observed across varied geographic areas, which have been ascribed to viral mutation and evolution over time. The homotrimeric Spike (S) glycoprotein on the viral envelope surface is responsible for binding, priming, and initiating infection in the host. Our phylogeny analysis of 1947 sequences of S proteins indicated there is a change in amino acid (aa) from aspartate (Group-A) to glycine (Group-B) at position 614, near the receptor- binding domain (RBD; aa positions 331-524). The two variants are reported to be in circulation, disproportionately across the world, with Group-A dominant in Asia and Group-B in North America. The trimeric, monomeric, and RBD of S protein of both the variant groups (A & B) were modeled using the Swiss-Model server and were docked with the human receptor angiotensin-converting enzyme 2 (hACE2) employing the PatchDock server and visualized in PyMol. Group-A S protein''s RBD bound imperceptibly to the two binding clefts of the hACE2 protein, on the other hand, Group-B S protein''s RBD perfectly interacted inside the binding clefts of hACE2, with higher number of hydrogen and hydrophobic interactions. This implies that the S protein''s amino acid at position 614 near the core RBD influences its interaction with the cognate hACE2 receptor, which may induce its infectivity that should be explored further with molecular and biochemical studies.     

Open Modification Searching of SARS-CoV-2–Human Protein Interaction Data Reveals Novel Viral Modification Sites

The outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus 2019 disease, has led to an ongoing global pandemic since 2019. Mass spectrometry can be used to understand the molecular mechanisms of viral infection by SARS-CoV-2, for example, by determining virus–host protein–protein interactions through which SARS-CoV-2 hijacks its human hosts during infection, and to study the role of post-translational modifications. We have reanalyzed public affinity purification–mass spectrometry data using open modification searching to investigate the presence of post-translational modifications in the context of the SARS-CoV-2 virus–host protein–protein interaction network. Based on an over twofold increase in identified spectra, our detected protein interactions show a high overlap with independent mass spectrometry-based SARS-CoV-2 studies and virus–host interactions for alternative viruses, as well as previously unknown protein interactions. In addition, we identified several novel modification sites on SARS-CoV-2 proteins that we investigated in relation to their interactions with host proteins. A detailed analysis of relevant modifications, including phosphorylation, ubiquitination, and S-nitrosylation, provides important hypotheses about the functional role of these modifications during viral infection by SARS-CoV-2.     

Linking structural features of protein complexes and biological function

Protein–protein interaction (PPI) establishes the central basis for complex cellular networks in a biological cell. Association of proteins with other proteins occurs at varying affinities, yet with a high degree of specificity. PPIs lead to diverse functionality such as catalysis, regulation, signaling, immunity, and inhibition, playing a crucial role in functional genomics. The molecular principle of such interactions is often elusive in nature. Therefore, a comprehensive analysis of known protein complexes from the Protein Data Bank (PDB) is essential for the characterization of structural interface features to determine structure–function relationship. Thus, we analyzed a nonredundant dataset of 278 heterodimer protein complexes, categorized into major functional classes, for distinguishing features. Interestingly, our analysis has identified five key features (interface area, interface polar residue abundance, hydrogen bonds, solvation free energy gain from interface formation, and binding energy) that are discriminatory among the functional classes using Kruskal-Wallis rank sum test. Significant correlations between these PPI interface features amongst functional categories are also documented. Salt bridges correlate with interface area in regulator-inhibitors (r = 0.75). These representative features have implications for the prediction of potential function of novel protein complexes. The results provide molecular insights for better understanding of PPIs and their relation to biological functions.     

The spike protein of SARS-CoV-2 induces endothelial inflammation through integrin α5β1 and NF-κB signaling

Vascular endothelial cells (ECs) form a critical interface between blood and tissues that maintains whole-body homeostasis. In COVID-19, disruption of the EC barrier results in edema, vascular inflammation, and coagulation, hallmarks of this severe disease. However, the mechanisms by which ECs are dysregulated in COVID-19 are unclear. Here, we show that the spike protein of SARS-CoV-2 alone activates the EC inflammatory phenotype in a manner dependent on integrin ⍺5β1 signaling. Incubation of human umbilical vein ECs with whole spike protein, its receptor-binding domain, or the integrin-binding tripeptide RGD induced the nuclear translocation of NF-κB and subsequent expression of leukocyte adhesion molecules (VCAM1 and ICAM1), coagulation factors (TF and FVIII), proinflammatory cytokines (TNFα, IL-1β, and IL-6), and ACE2, as well as the adhesion of peripheral blood leukocytes and hyperpermeability of the EC monolayer. In addition, inhibitors of integrin ⍺5β1 activation prevented these effects. Furthermore, these vascular effects occur in vivo, as revealed by the intravenous administration of spike, which increased expression of ICAM1, VCAM1, CD45, TNFα, IL-1β, and IL-6 in the lung, liver, kidney, and eye, and the intravitreal injection of spike, which disrupted the barrier function of retinal capillaries. We suggest that the spike protein, through its RGD motif in the receptor-binding domain, binds to integrin ⍺5β1 in ECs to activate the NF-κB target gene expression programs responsible for vascular leakage and leukocyte adhesion. These findings uncover a new direct action of SARS-CoV-2 on EC dysfunction and introduce integrin ⍺5β1 as a promising target for treating vascular inflammation in COVID-19.     

Activation of the SARS-CoV-2 NSP14 3′–5′ exoribonuclease by NSP10 and response to antiviral inhibitors

Understanding the core replication complex of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential to the development of novel coronavirus-specific antiviral therapeutics. Among the proteins required for faithful replication of the SARS-CoV-2 genome are nonstructural protein 14 (NSP14), a bifunctional enzyme with an N-terminal 3′-to-5′ exoribonuclease (ExoN) and a C-terminal N7-methyltransferase, and its accessory protein, NSP10. The difficulty in producing pure and high quantities of the NSP10/14 complex has hampered the biochemical and structural study of these important proteins. We developed a straightforward protocol for the expression and purification of both NSP10 and NSP14 from Escherichia coli and for the in vitro assembly and purification of a stoichiometric NSP10/14 complex with high yields. Using these methods, we observe that NSP10 provides a 260-fold increase in k_cat/K_m in the exoribonucleolytic activity of NSP14 and enhances protein stability. We also probed the effect of two small molecules on NSP10/14 activity, remdesivir monophosphate and the methyltransferase inhibitor S-adenosylhomocysteine. Our analysis highlights two important factors for drug development: first, unlike other exonucleases, the monophosphate nucleoside analog intermediate of remdesivir does not inhibit NSP14 activity; and second, S-adenosylhomocysteine modestly activates NSP14 exonuclease activity. In total, our analysis provides insights for future structure–function studies of SARS-CoV-2 replication fidelity for the treatment of coronavirus disease 2019.     

Basic residues at the C-gate of DNA gyrase are involved in DNA supercoiling

DNA gyrase is a type II topoisomerase that is responsible for maintaining the topological state of bacterial and some archaeal genomes. It uses an ATP-dependent two-gate strand-passage mechanism that is shared among all type II topoisomerases. During this process, DNA gyrase creates a transient break in the DNA, the G-segment, to form a cleavage complex. This allows a second DNA duplex, known as the T-segment, to pass through the broken G-segment. After the broken strand is religated, the T-segment is able to exit out of the enzyme through a gate called the C-gate. Although many steps of the type II topoisomerase mechanism have been studied extensively, many questions remain about how the T-segment ultimately exits out of the C-gate. A recent cryo-EM structure of Streptococcus pneumoniae GyrA shows a putative T-segment in close proximity to the C-gate, suggesting that residues in this region may be important for coordinating DNA exit from the enzyme. Here, we show through site-directed mutagenesis and biochemical characterization that three conserved basic residues in the C-gate of DNA gyrase are important for DNA supercoiling activity, but not for ATPase or cleavage activity. Together with the structural information previously published, our data suggest a model in which these residues cluster to form a positively charged region that facilitates T-segment passage into the cavity formed between the DNA gate and C-gate.     

The role of S477N mutation in the molecular behavior of SARS-CoV-2 spike protein: An in-silico perspective

The attachment of SARA-CoV-2 happens between ACE2 and the receptor binding domain (RBD) on the spike protein. Mutations in this domain can affect the binding affinity of the spike protein for ACE2. S477N, one of the most common mutations reported in the recent variants, is located in the RBD. Today's computational approaches in biology, especially during the SARS-CoV-2 pandemic, assist researchers in predicting a protein's behavior in contact with other proteins in more detail. In this study, we investigated the interactions of the S477N-hACE2 in silico to find the impact of this mutation on its binding affinity for ACE2 and immunity responses using dynamics simulation, protein–protein docking, and immunoinformatics methods. Our computational analysis revealed an increased binding affinity of N477 for ACE2. Four new hydrogen and hydrophobic bonds in the mutant RBD-ACE2 were formed (with S19 and Q24 of ACE2), which do not exist in the wild type. Also, the protein spike structure in this mutation was associated with an increase in stabilization and a decrease in its fluctuations at the atomic level. N477 mutation can be considered as the cause of increased escape from the immune system through MHC-II.     

Structural insights into the role of the WW2 domain on tandem WW–PPxY motif interactions of oxidoreductase WWOX

Class I WW domains are present in many proteins of various functions and mediate protein interactions by binding to short linear PPxY motifs. Tandem WW domains often bind peptides with multiple PPxY motifs, but the interplay of WW–peptide interactions is not always intuitive. The WW domain–containing oxidoreductase (WWOX) harbors two WW domains: an unstable WW1 capable of PPxY binding and stable WW2 that cannot bind PPxY. The WW2 domain has been suggested to act as a WW1 domain chaperone, but the underlying mechanism of its chaperone activity remains to be revealed. Here, we combined NMR, isothermal calorimetry, and structural modeling to elucidate the roles of both WW domains in WWOX binding to its PPxY-containing substrate ErbB4. Using NMR, we identified an interaction surface between these two domains that supports a WWOX conformation compatible with peptide substrate binding. Isothermal calorimetry and NMR measurements also indicated that while binding affinity to a single PPxY motif is marginally increased in the presence of WW2, affinity to a dual-motif peptide increases 10-fold. Furthermore, we found WW2 can directly bind double-motif peptides using its canonical binding site. Finally, differential binding of peptides in mutagenesis experiments was consistent with a parallel N- to C-terminal PPxY tandem motif orientation in binding to the WW1–WW2 tandem domain, validating structural models of the interaction. Taken together, our results reveal the complex nature of tandem WW-domain organization and substrate binding, highlighting the contribution of WWOX WW2 to both protein stability and target binding.     

两种表达系统来源新冠病毒RBD蛋白免疫小鼠后的IgG抗体水平对比研究

当前新冠肺炎疫情仍在全球蔓延,给中国及世界的公共卫生安全和经济发展带来了严峻挑战。SARS-CoV-2病毒入侵机体的关键过程是刺突蛋白受体结合域RBD通过结合宿主细胞ACE2受体实现感染,而此过程与病原快速检测、疫苗以及药物干预等主要抗病毒策略的研究与开发密切相关。因此,本研究旨在比较哺乳细胞和昆虫细胞重组表达来源的新冠病毒刺突蛋白受体结合结构域RBD免疫小鼠后产生IgG抗体水平的变化,评估不同来源和不同剂量抗原的抗体滴度水平和持续时间,希望将有助于疫苗、药物以及病原检测等相关研究。通过SDS-PAGE和质谱技术鉴定出昆虫和哺乳动物细胞表达系统制备的bRBD和hRBD蛋白质的分子量略有差异,主要为糖基化修饰不同所致;流式细胞术检测发现,bRBD和hRBD与ACE2受体过表达细胞结合率分别为88.5%和92.7%,表明二者均具有较好的活性;利用Quick Antibody免疫佐剂通过肌肉注射对Balb/c小鼠进行免疫接种,设置10μg、20μg 2个免疫抗原的剂量,共接种2次,并在初次免疫后第2、4、6、8、12、24周从尾部取血,分离获得血清;酶联免疫吸附结果显示,10μg和20μg的...