Comirnaty-Elicited and Convalescent Sera Recognize Different Spike Epitopes

Many of the approved SARS-CoV-2 vaccines are based on a stabilized variant of the spike protein. This raises the question of whether the immune response against the stabilized spike is identical to the immune response that is elicited by the native spike in the case of a SARS-CoV-2 infection. Using a peptide array-based approach, we analysed the binding of antibodies from Comirnaty-elicited, convalescent, and control sera to the peptides covering the spike protein. A total of 37 linear epitopes were identified. A total of 26 of these epitopes were almost exclusively recognized by the convalescent sera. Mapping these epitopes to the spike structures revealed that most of these 26 epitopes are masked in the pre-fusion structure. In particular, in the conserved central helix, three epitopes that are only exposed in the post-fusion conformation were identified. This indicates a higher spike-specific antibody diversity in convalescent sera. These differences could be relevant for the breadth of spike-specific immune response.

Cotranscriptional RNA strand exchange underlies the gene regulation mechanism in a purine-sensing transcriptional riboswitch

RNA folds cotranscriptionally to traverse out-of-equilibrium intermediate structures that are important for RNA function in the context of gene regulation. To investigate this process, here we study the structure and function of the Bacillus subtilis yxjA purine riboswitch, a transcriptional riboswitch that downregulates a nucleoside transporter in response to binding guanine. Although the aptamer and expression platform domain sequences of the yxjA riboswitch do not completely overlap, we hypothesized that a strand exchange process triggers its structural switching in response to ligand binding. In vivo fluorescence assays, structural chemical probing data, and experimentally informed secondary structure modeling suggest the presence of a nascent intermediate central helix. The formation of this central helix in the absence of ligand appears to compete with both the aptamer's P1 helix and the expression platform's transcriptional terminator. All-atom molecular dynamics simulations support the hypothesis that ligand binding stabilizes the aptamer P1 helix against central helix strand invasion, thus allowing the terminator to form. These results present a potential model mechanism to explain how ligand binding can induce downstream conformational changes by influencing local strand displacement processes of intermediate folds that could be at play in multiple riboswitch classes.

In Vitro Viral Evolution Identifies a Critical Residue in the Alphaherpesvirus Fusion Glycoprotein B Ectodomain That Controls gH/gL-Independent Entry

ABSTRACT Herpesvirus entry and spread requires fusion of viral and host cell membranes, which is mediated by the conserved surface glycoprotein B (gB). Upon activation, gB undergoes a major conformational change and transits from a metastable prefusion to a stable postfusion conformation. Although gB is a structural homolog of low-pH-triggered class III fusogens, its fusion activity depends strictly on the presence of the conserved regulatory gH/gL complex and nonconserved receptor binding proteins, which ensure that fusion occurs at the right time and space. How gB maintains its prefusion conformation and how gB fusogenicity is controlled remain poorly understood. Here, we report the isolation and characterization of a naturally selected pseudorabies virus (PrV) gB able to mediate efficient gH/gL-independent virus-cell and cell-cell fusion. We found that the control exerted on gB by the accompanying viral proteins is mediated via its cytosolic domain (CTD). Whereas gB variants lacking the CTD are inactive, a single mutation of a conserved asparagine residue in an alpha-helical motif of the ectodomain recently shown to be at the core of the gB prefusion trimer compensated for CTD absence and uncoupled gB from regulatory viral proteins, resulting in a hyperfusion phenotype. This phenotype was transferred to gB homologs from different alphaherpesvirus genera. Overall, our data propose a model in which the central helix acts as a molecular switch for the gB pre-to-postfusion transition by conveying the structural status of the endo- to the ectodomain, thereby governing their cross talk for fusion activation, providing a new paradigm for herpesvirus fusion regulation. IMPORTANCE The class III fusion protein glycoprotein B (gB) drives membrane fusion during entry and spread of herpesviruses. To mediate fusion, gB requires activation by the conserved gH/gL complex by a poorly defined mechanism. A detailed molecular-level understanding of herpesvirus membrane fusion is of fundamental virological interest and has considerable potential for the development of new therapeutics blocking herpesvirus cell invasion and spread. Using in vitro evolution and targeted mutagenesis of three different animal alphaherpesviruses, we identified a single conserved amino acid in a regulatory helix in the center of the gB ectodomain that enables efficient gH/gL-independent entry and plays a crucial role in the pre-to-postfusion transition of gB. Our results propose that the central helix is a key regulatory element involved in the intrastructural signal transduction between the endo- and ectodomain for fusion activation. This study expands our understanding of herpesvirus membrane fusion and uncovers potential targets for therapeutic interventions.

SARS-CoV-2 S protein:ACE2 interaction reveals novel allosteric targets

The Spike (S) protein is the main handle for SARS-CoV-2 to enter host cells via surface ACE2 receptors. How ACE2 binding activates proteolysis of S protein is unknown. Here, using amide hydrogen-deuterium exchange mass spectrometry and molecular dynamics simulations, we have mapped the S:ACE2 interaction interface and uncovered long-range allosteric propagation of ACE2 binding to sites necessary for host-mediated proteolysis of S protein, critical for viral host entry. Unexpectedly, ACE2 binding enhances dynamics at a distal S1/S2 cleavage site and flanking protease docking site ~27 Å away while dampening dynamics of the stalk hinge (central helix and heptad repeat) regions ~130 Å away. This highlights that the stalk and proteolysis sites of the S protein are dynamic hotspots in the pre-fusion state. Our findings provide a dynamics map of the S:ACE2 interface in solution and also offer mechanistic insights into how ACE2 binding is allosterically coupled to distal proteolytic processing sites and viral-host membrane fusion. Our findings highlight protease docking sites flanking the S1/S2 cleavage site, fusion peptide and heptad repeat 1 (HR1) as alternate allosteric hotspot targets for potential therapeutic development.

Structural Aspects and Prediction of Calmodulin-Binding Proteins

Calmodulin (CaM) is an important intracellular protein that binds Ca2+ and functions as a critical second messenger involved in numerous biological activities through extensive interactions with proteins and peptides. CaM’s ability to adapt to binding targets with different structures is related to the flexible central helix separating the N- and C-terminal lobes, which allows for conformational changes between extended and collapsed forms of the protein. CaM-binding targets are most often identified using prediction algorithms that utilize sequence and structural data to predict regions of peptides and proteins that can interact with CaM. In this review, we provide an overview of different CaM-binding proteins, the motifs through which they interact with CaM, and shared properties that make them good binding partners for CaM. Additionally, we discuss the historical and current methods for predicting CaM binding, and the similarities and differences between these methods and their relative success at prediction. As new CaM-binding proteins are identified and classified, we will gain a broader understanding of the biological processes regulated through changes in Ca2+ concentration through interactions with CaM.

Ultra-Large-Scale Ab Initio Quantum Chemical Computation of Bio-Molecular Systems: The Case of Spike Protein of SARS-CoV-2 Virus

<p>The COVID-19 pandemic poses a severe threat to human health with an unprecedented social and economic disruption. <i>Spike (S) glycoprotein</i> of the SARS-CoV-2 virus is pivotal in understanding the virus anatomy, since it initiates the first contact with the ACE2 receptor in the human cell. We report results of <i>ab initio</i> computation of the spike protein, the largest <i>ab initio</i> quantum chemical computation to date on any bio-molecular system, using a <i>divide and conquer strategy</i> by focusing on individual structural domains. In this approach we divided the S-protein into seven structural domains: N-terminal domain (NTD), receptor binding domain (RBD), subdomain 1 (SD1), subdomain 2 (SD2), fusion peptide (FP), heptad repeat 1 with central helix (HR1-CH) and connector domain (CD). The entire Chain A has 14,488 atoms including the hydrogen atoms but excluding the amino acids with missing coordinates based on the PDB data (ID: 6VSB). The results include structural refinement, <i>ab initio</i> calculation of intra-molecular bonding mechanism, 3- dimensional non-local inter-amino acid interaction with implications for the inter-domain interaction. Details of the electronic structure, interatomic bonding, partial charge distribution and the role played by hydrogen bond network are discussed. Extension of such calculation to the interface between the S-protein binding domain and ACE2 receptor can provide a pathway for computational understanding of mutations and the design of therapeutic drugs to combat the COVID-19 pandemic. </p>

Ultra-Large-Scale Ab Initio Quantum Chemical Computation of Bio-Molecular Systems: The Case of Spike Protein of SARS-CoV-2 Virus

<p>The COVID-19 pandemic poses a severe threat to human health with an unprecedented social and economic disruption. <i>Spike (S) glycoprotein</i> of the SARS-CoV-2 virus is pivotal in understanding the virus anatomy, since it initiates the first contact with the ACE2 receptor in the human cell. We report results of <i>ab initio</i> computation of the spike protein, the largest <i>ab initio</i> quantum chemical computation to date on any bio-molecular system, using a <i>divide and conquer strategy</i> by focusing on individual structural domains. In this approach we divided the S-protein into seven structural domains: N-terminal domain (NTD), receptor binding domain (RBD), subdomain 1 (SD1), subdomain 2 (SD2), fusion peptide (FP), heptad repeat 1 with central helix (HR1-CH) and connector domain (CD). The entire Chain A has 14,488 atoms including the hydrogen atoms but excluding the amino acids with missing coordinates based on the PDB data (ID: 6VSB). The results include structural refinement, <i>ab initio</i> calculation of intra-molecular bonding mechanism, 3- dimensional non-local inter-amino acid interaction with implications for the inter-domain interaction. Details of the electronic structure, interatomic bonding, partial charge distribution and the role played by hydrogen bond network are discussed. Extension of such calculation to the interface between the S-protein binding domain and ACE2 receptor can provide a pathway for computational understanding of mutations and the design of therapeutic drugs to combat the COVID-19 pandemic. </p>

Identification of a novel fold type in CPA/AT transporters by ab-initio structure prediction

Members of the CPA/AT transporter superfamily show significant structural variability. All previously known members consist of an inverted duplicated repeat unit that folds into two separate domains, the core and the scaffold domain. Crucial for its transporting function, the central helix in the core domain is a noncanonical transmembrane helix, which can either be in the form of a broken helix or a reentrant helix. Here, we expand the structural knowledge of the CPA/AT family by using contact-prediction-based protein modelling. We show that the N-terminal domains of the Pfam families; PSE (Cons_hypoth698 PF03601), Lysine exporter (PF03956) and LrgB (PF04172) families have a previously unseen reentrant-helix-reentrant fold. The close homology between PSE and the Sodium-citrate symporter (2HCT) suggests that the new fold originates from the truncation of an ancestral reentrant protein, caused by the loss of the C-terminal reentrant helix. To compensate for the lost reentrant helix one external loop moves into the membrane to form the second reentrant helix, highlighting the adaptability of the CPA/AT transporters. This study also demonstrates that the most recent deep-learning-based modelling methods have become a useful tool to gain biologically relevant structural, evolutionary and functional insights about protein families.

SARS-CoV-2 S protein ACE2 interaction reveals novel allosteric targets

The Spike (S) protein is the main handle for SARS-CoV-2 to enter host cells through surface ACE2 receptors. How ACE2 binding activates proteolysis of S protein is unknown. Here, we have mapped the S:ACE2 interface and uncovered long-range allosteric propagation of ACE2 binding to sites critical for viral host entry. Unexpectedly, ACE2 binding enhances dynamics at a distal S1/S2 cleavage site and flanking protease docking site ~27 Å away while dampening dynamics of the stalk hinge (central helix and heptad repeat) regions ~ 130 Å away. This highlights that the stalk and proteolysis sites of the S protein are dynamic hotspots in the pre-fusion state. Our findings provide a mechanistic basis for S:ACE2 complex formation, critical for proteolytic processing and viral-host membrane fusion and highlight protease docking sites flanking the S1/S2 cleavage site, fusion peptide and heptad repeat 1 (HR1) as allosterically exposed cryptic hotspots for potential therapeutic development.One Sentence SummarySARS-CoV-2 spike protein binding to receptor ACE2 allosterically enhances furin proteolysis at distal S1/S2 cleavage sites

Development of CpG-adjuvanted stable prefusion SARS-CoV-2 spike antigen as a subunit vaccine against COVID-19

The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 is a worldwide health emergency. The immense damage done to public health and economies has prompted a global race for cures and vaccines. In developing a COVID-19 vaccine, we applied technology previously used for MERS-CoV to produce a prefusion-stabilized SARS-CoV-2 spike protein by adding two proline substitutions at the top of the central helix (S-2P). To enhance immunogenicity and mitigate the potential vaccine-induced immunopathology, CpG 1018, a Th1-biasing synthetic toll-like receptor 9 (TLR9) agonist was selected as an adjuvant candidate. S-2P was combined with various adjuvants, including CpG 1018, and administered to mice to test its effectiveness in eliciting anti-SARS-CoV-2 neutralizing antibodies. S-2P in combination with CpG 1018 and aluminum hydroxide (alum) was found to be the most potent immunogen and induced high titer of spike-specific antibodies in sera of immunized mice. The neutralizing abilities in pseudotyped lentivirus reporter or live wild-type SARS-CoV-2 were measured with reciprocal inhibiting dilution (ID50) titers of 5120 and 2560, respectively. In addition, the antibodies elicited were able to cross-neutralize pseudovirus containing the spike protein of the D614G variant, indicating the potential for broad spectrum protection. A marked Th-1 dominant response was noted from cytokines secreted by splenocytes of mice immunized with CpG 1018 and alum. No vaccine-related serious adverse effects were found in the dose-ranging study in rats administered single- or two-dose regimens with up to 50 μg of S-2P combined with CpG 1018 alone or CpG 1018 with alum. These data support continued development of CHO-derived S-2P formulated with CpG 1018/alum as a candidate vaccine to prevent COVID-19 disease.