scholarly journals A model for pH coupling of the SARS-CoV-2 spike protein open/closed equilibrium

2020 ◽  
Author(s):  
Jim Warwicker
Keyword(s):  
Cell Surface ◽  
Aspartic Acid ◽  
Receptor Binding ◽  
Ph Dependence ◽  
Salt Bridge ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Salt Bridges ◽  
Heat Map ◽  
Human Coronaviruses

AbstractSARS-CoV-2, causative agent of the COVID-19 pandemic, is thought to release its RNA genome at either the cell surface or within endosomes, the balance being dependent on spike protein stability, and the complement of receptors, co-receptors and proteases. To investigate possible mediators of pH-dependence, pKa calculations have been made on a set of structures for spike protein ectodomain and fragments from SARS-CoV-2 and other coronaviruses. Dominating a heat map of the aggregated predictions, 3 histidine residues in S2 are consistently predicted as destabilising in pre-fusion (all 3) and post-fusion (2 of 3) structures. Other predicted features include the more moderate energetics of surface salt-bridge interactions, and sidechain-mainchain interactions. Two aspartic acid residues in partially buried salt-bridges have pKas that are calculated to be elevated and destabilising. Notably, the degree of destabilisation is predicted to vary between open and closed receptor binding domain conformations. It is therefore suggested that these groups contribute to a pH-dependence of the open/closed equilibrium. These observations are discussed in the context of SARS-CoV-2 infection, mutagenesis studies, and other human coronaviruses.

scholarly journals A model for pH coupling of the SARS-CoV-2 spike protein open/closed equilibrium

2021 ◽  
Author(s):  
Jim Warwicker
Keyword(s):  
Linoleic Acid ◽  
Severe Acute Respiratory Syndrome ◽  
Cell Surface ◽  
Ph Dependence ◽  
Salt Bridge ◽  
Spike Protein ◽  
Salt Bridges ◽  
Heat Map ◽  
Closed Conformation ◽  
Human Coronaviruses

Abstract Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causative agent of the coronavirus disease 2019 (COVID-19) pandemic, is thought to release its RNA genome at either the cell surface or within endosomes, the balance being dependent on spike protein stability, and the complement of receptors, co-receptors and proteases. To investigate possible mediators of pH-dependence, pKa calculations have been made on a set of structures for spike protein ectodomain and fragments from SARS-CoV-2 and other coronaviruses. Dominating a heat map of the aggregated predictions, three histidine residues in S2 are consistently predicted as destabilizing in pre-fusion (all three) and post-fusion (two of the three) structures. Other predicted features include the more moderate energetics of surface salt–bridge interactions and sidechain–mainchain interactions. Two aspartic acid residues in partially buried salt-bridges (D290–R273 and R355–D398) have pKas that are calculated to be elevated and destabilizing in more open forms of the spike trimer. These aspartic acids are most stabilized in a tightly closed conformation that has been observed when linoleic acid is bound, and which also affects the interactions of D614. The D614G mutation is known to modulate the balance of closed to open trimer. It is suggested that D398 in particular contributes to a pH-dependence of the open/closed equilibrium, potentially coupled to the effects of linoleic acid binding and D614G mutation, and possibly also A570D mutation. These observations are discussed in the context of SARS-CoV-2 infection, mutagenesis studies, and other human coronaviruses.

scholarly journals SARS-CoV-2, an evolutionary perspective of interaction with human ACE2 reveals undiscovered amino acids necessary for complex stability

2020 ◽  
Author(s):  
Vinicio Armijos-Jaramillo ◽  
Justin Yeager ◽  
Claire Muslin ◽  
Yunierkis Perez-Castillo
Keyword(s):  
Receptor Binding ◽  
Evolutionary Dynamics ◽  
Hot Spot ◽  
Critical Role ◽  
Human Cells ◽  
Binding Domain ◽  
Host Cells ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Salt Bridges

AbstractThe emergence of SARS-CoV-2 has resulted in more than 200,000 infections and nearly 9,000 deaths globally so far. This novel virus is thought to have originated from an animal reservoir, and acquired the ability to infect human cells using the SARS-CoV cell receptor hACE2. In the wake of a global pandemic it is essential to improve our understanding of the evolutionary dynamics surrounding the origin and spread of a novel infectious disease. One way theory predicts selection pressures should shape viral evolution is to enhance binding with host cells. We first assessed evolutionary dynamics in select betacoronavirus spike protein genes to predict where these genomic regions are under directional or purifying selection between divergent viral lineages at various scales of relatedness. With this analysis, we determine a region inside the receptor-binding domain with putative sites under positive selection interspersed among highly conserved sites, which are implicated in structural stability of the viral spike protein and its union with human receptor hACE2. Next, to gain further insights into factors associated with coronaviruses recognition of the human host receptor, we performed modeling studies of five different coronaviruses and their potential binding to hACE2. Modeling results indicate that interfering with the salt bridges at hot spot 353 could be an effective strategy for inhibiting binding, and hence for the prevention of coronavirus infections. We also propose that a glycine residue at the receptor binding domain of the spike glycoprotein can have a critical role in permitting bat variants of the coronaviruses to infect human cells.

scholarly journals Predicted pH-dependent stability of SARS-CoV-2 spike protein trimer from interfacial acidic groups

2021 ◽  
Author(s):  
Vanessa R Lobo ◽  
Jim Warwicker
Keyword(s):  
Electron Microscopy ◽  
Receptor Binding ◽  
Ph Dependence ◽  
Spike Protein ◽  
Ph Effects ◽  
Large Set ◽  
Acidic Ph ◽  
Receptor Binding Domain ◽  
Cryo Electron Microscopy ◽  
Ph Dependent

Transition between receptor binding domain (RBD) up and down forms of the SARS-CoV-2 spike protein trimer is coupled to receptor binding and is one route by which variants can alter viral properties. It is becoming apparent that key roles in the transition are played by pH and a more compact closed form, termed locked. Calculations of pH-dependence are made for a large set of spike trimers, including locked form trimer structures that have recently become available. Several acidic sidechains become sufficiently buried in the locked form to give a predicted pH-dependence in the mild acidic range, with stabilisation of the locked form as pH reduces from 7.5 to 5, consistent with emerging characterisation by cryo-electron microscopy. The calculated pH effects in pre-fusion spike trimers are modulated mainly by aspartic acid residues, rather than the more familiar histidine role at mild acidic pH. These acidic sidechains are generally surface located and weakly interacting when not in a locked conformation. In this model, their replacement (perhaps with asparagine) would remove the pH-dependent destabilisation of locked spike trimer conformations, and increase their recovery at neutral pH. This would provide an alternative or supplement to the insertion of disulphide linkages for stabilising spike protein trimers, with potential relevance for vaccine design.

scholarly journals Effects of common mutations in the SARS-CoV-2 Spike RBD and its ligand, the human ACE2 receptor on binding affinity and kinetics

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Michael I Barton ◽  
Stuart A MacGowan ◽  
Mikhail A Kutuzov ◽  
Omer Dushek ◽  
Geoffrey John Barton ◽  
...  
Keyword(s):  
Cell Surface ◽  
Binding Site ◽  
Receptor Binding ◽  
Immune Escape ◽  
Surface Protein ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Cell Surface Protein ◽  
Kinetics Analysis ◽  
Protein Receptor

The interaction between the SARS-CoV-2 virus Spike protein receptor binding domain (RBD) and the ACE2 cell surface protein is required for viral infection of cells. Mutations in the RBD are present in SARS-CoV-2 variants of concern that have emerged independently worldwide. For example, the B.1.1.7 lineage has a mutation (N501Y) in its Spike RBD that enhances binding to ACE2. There are also ACE2 alleles in humans with mutations in the RBD binding site. Here we perform a detailed affinity and kinetics analysis of the effect of five common RBD mutations (K417N, K417T, N501Y, E484K, and S477N) and two common ACE2 mutations (S19P and K26R) on the RBD/ACE2 interaction. We analysed the effects of individual RBD mutations and combinations found in new SARS-CoV-2 Alpha (B.1.1.7), Beta (B.1.351), and Gamma (P1) variants. Most of these mutations increased the affinity of the RBD/ACE2 interaction. The exceptions were mutations K417N/T, which decreased the affinity. Taken together with other studies, our results suggest that the N501Y and S477N mutations enhance transmission primarily by enhancing binding, the K417N/T mutations facilitate immune escape, and the E484K mutation enhances binding and immune escape.

scholarly journals Role of Extracellular Vimentin in Cancer-Cell Functionality and Its Influence on Cell Monolayer Permeability Changes Induced by SARS-CoV-2 Receptor Binding Domain

2021 ◽  
Vol 22 (14) ◽  
pp. 7469
Author(s):  
Divyendu Goud Thalla ◽  
Philipp Jung ◽  
Markus Bischoff ◽  
Franziska Lautenschläger
Keyword(s):  
Cell Surface ◽  
Receptor Binding ◽  
Cell Monolayer ◽  
Binding Domain ◽  
Spike Protein ◽  
Outer Cell ◽  
Receptor Binding Domain ◽  
Monolayer Permeability ◽  
Mcf 7

The cytoskeletal protein vimentin is secreted under various physiological conditions. Extracellular vimentin exists primarily in two forms: attached to the outer cell surface and secreted into the extracellular space. While surface vimentin is involved in processes such as viral infections and cancer progression, secreted vimentin modulates inflammation through reduction of neutrophil infiltration, promotes bacterial elimination in activated macrophages, and supports axonal growth in astrocytes through activation of the IGF-1 receptor. This receptor is overexpressed in cancer cells, and its activation pathway has significant roles in general cellular functions. In this study, we investigated the functional role of extracellular vimentin in non-tumorigenic (MCF-10a) and cancer (MCF-7) cells through the evaluation of its effects on cell migration, proliferation, adhesion, and monolayer permeability. Upon treatment with extracellular recombinant vimentin, MCF-7 cells showed increased migration, proliferation, and adhesion, compared to MCF-10a cells. Further, MCF-7 monolayers showed reduced permeability, compared to MCF-10a monolayers. It has been shown that the receptor binding domain of SARS-CoV-2 spike protein can alter blood–brain barrier integrity. Surface vimentin also acts as a co-receptor between the SARS-CoV-2 spike protein and the cell-surface angiotensin-converting enzyme 2 receptor. Therefore, we also investigated the permeability of MCF-10a and MCF-7 monolayers upon treatment with extracellular recombinant vimentin, and its modulation of the SARS-CoV-2 receptor binding domain. These findings show that binding of extracellular recombinant vimentin to the cell surface enhances the permeability of both MCF-10a and MCF-7 monolayers. However, with SARS-CoV-2 receptor binding domain addition, this effect is lost with MCF-7 monolayers, as the extracellular vimentin binds directly to the viral domain. This defines an influence of extracellular vimentin in SARS-CoV-2 infections.

scholarly journals Biological and Clinical Consequences of Integrin Binding via a Rogue RGD Motif in the SARS CoV-2 Spike Protein

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 146
Author(s):  
Lee Makowski ◽  
William Olson-Sidford ◽  
John W. Weisel
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Signaling Pathways ◽  
Receptor Binding ◽  
Binding Domain ◽  
Cell Entry ◽  
Spike Protein ◽  
Patient Specific ◽  
Receptor Binding Domain ◽  
Rgd Motif

Although ACE2 (angiotensin converting enzyme 2) is considered the primary receptor for CoV-2 cell entry, recent reports suggest that alternative pathways may contribute. This paper considers the hypothesis that viral binding to cell-surface integrins may contribute to the high infectivity and widespread extra-pulmonary impacts of the SARS-CoV-2 virus. This potential is suggested on the basis of the emergence of an RGD (arginine-glycine-aspartate) sequence in the receptor-binding domain of the spike protein. RGD is a motif commonly used by viruses to bind cell-surface integrins. Numerous signaling pathways are mediated by integrins and virion binding could lead to dysregulation of these pathways, with consequent tissue damage. Integrins on the surfaces of pneumocytes, endothelial cells and platelets may be vulnerable to CoV-2 virion binding. For instance, binding of intact virions to integrins on alveolar cells could enhance viral entry. Binding of virions to integrins on endothelial cells could activate angiogenic cell signaling pathways; dysregulate integrin-mediated signaling pathways controlling developmental processes; and precipitate endothelial activation to initiate blood clotting. Such a procoagulant state, perhaps together with enhancement of platelet aggregation through virions binding to integrins on platelets, could amplify the production of microthrombi that pose the threat of pulmonary thrombosis and embolism, strokes and other thrombotic consequences. The susceptibility of different tissues to virion–integrin interactions may be modulated by a host of factors, including the conformation of relevant integrins and the impact of the tissue microenvironment on spike protein conformation. Patient-specific differences in these factors may contribute to the high variability of clinical presentation. There is danger that the emergence of receptor-binding domain mutations that increase infectivity may also enhance access of the RGD motif for integrin binding, resulting in viral strains with ACE2 independent routes of cell entry and novel integrin-mediated biological and clinical impacts. The highly infectious variant, B.1.1.7 (or VUI 202012/01), includes a receptor-binding domain amino acid replacement, N501Y, that could potentially provide the RGD motif with enhanced access to cell-surface integrins, with consequent clinical impacts.

scholarly journals Stereotypic neutralizing VH antibodies against SARS-CoV-2 spike protein receptor binding domain in COVID-19 patients and healthy individuals

2021 ◽  
pp. eabd6990
Author(s):  
Sang Il Kim ◽  
Jinsung Noh ◽  
Sujeong Kim ◽  
Younggeun Choi ◽  
Duck Kyun Yoo ◽  
...  
Keyword(s):  
B Cells ◽  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
De Novo ◽  
Binding Domain ◽  
Host Cells ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Healthy Individuals

Stereotypic antibody clonotypes exist in healthy individuals and may provide protective immunity against viral infections by neutralization. We observed that 13 out of 17 patients with COVID-19 had stereotypic variable heavy chain (VH) antibody clonotypes directed against the receptor-binding domain (RBD) of SARS-CoV-2 spike protein. These antibody clonotypes were comprised of immunoglobulin heavy variable (IGHV)3-53 or IGHV3-66 and immunoglobulin heavy joining (IGHJ)6 genes. These clonotypes included IgM, IgG3, IgG1, IgA1, IgG2, and IgA2 subtypes and had minimal somatic mutations, which suggested swift class switching after SARS-CoV-2 infection. The different immunoglobulin heavy variable chains were paired with diverse light chains resulting in binding to the RBD of SARS-CoV-2 spike protein. Human antibodies specific for the RBD can neutralize SARS-CoV-2 by inhibiting entry into host cells. We observed that one of these stereotypic neutralizing antibodies could inhibit viral replication in vitro using a clinical isolate of SARS-CoV-2. We also found that these VH clonotypes existed in six out of 10 healthy individuals, with IgM isotypes predominating. These findings suggest that stereotypic clonotypes can develop de novo from naïve B cells and not from memory B cells established from prior exposure to similar viruses. The expeditious and stereotypic expansion of these clonotypes may have occurred in patients infected with SARS-CoV-2 because they were already present.

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