Structural effects of spike protein D614G mutation in SARS-CoV-2

A single mutation from aspartate to glycine at position 614 has dominated all circulating variants of the severe acute respiratory syndrome coronavirus 2. D614G mutation induces structural changes in the spike (S) protein that strengthen the virus infectivity. Here, we use molecular dynamics simulations to dissect the effects of mutation and 630-loop rigidification on S-protein structure. The introduction of the mutation orders the 630-loop structure and thereby induces global structural changes toward the cryoelectron microscopy structure of the D614G S-protein. The ordered 630-loop weakens local interactions between the 614^th residue and others in contrast to disordered structures in the wild-type protein. The mutation allosterically alters global interactions between receptor-binding domains, forming an asymmetric and mobile down conformation and facilitating transitions toward up conformation. The loss of salt bridge between D614 and K854 upon the mutation generally stabilizes S-protein protomer, including the fusion peptide proximal region that mediates membrane fusion. Understanding the molecular basis of D614G mutation is crucial as it dominates in all variants of concern, including Delta and Omicron.     

Mutational sensitivity of D614G in spike protein of SARS-CoV-2 in Jordan

BackgroundSpike protein is the surface glycoprotein of the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) necessary for the entry of the virus via the transmembrane receptors of the human respiratory cells causing COVID-19 disease.AimHere, we aimed to predict the three-dimensional monomer structure of spike protein of SARS-CoV-2 from 20 Jordanian nasopharyngeal samples and to determine the percentage of single amino acid variants (SAV) in the spike protein of SARS-CoV-2.MethodsThe output of the Protein Homology/analogY Recognition Engine V 2.0 (Phyre2) found four single amino acid variants in the spike gene.ResultsThe first variant represented by 5% of samples that showed tyrosine deletion at Y144 located in the N terminal domain. The second and the dominant variant, represented by 62%, showed aspartate a coil amino acid substitution to glycine an extracellular amino acid at D614G located in the spike recognition binding site. The third variant, represented by 5%, showed aspartate substitution to tyrosine at D1139Y, and the fourth variant, represented by 5% glycine substitution to serine at G1167S.ConclusionOur results have shown low mutational sensitivity in all variants except to D614G the one with the most likely neutral mutational sensitivity that all variants might not explicitly affect the function of spike glycoprotein. However, D614G might change the viral conformational plasticity and hence a potential viral fitness gain but one must be cautious about drawing any concrete conclusions about the severity of symptoms and viral transmission from genomic data only.General significanceStudying mutations such as D614G in deep is essential to control the pandemic in terms of immune systems, antibodies, or even vaccines.     

D614G substitution at the hinge region enhances the stability of trimeric SARS-CoV-2 spike protein

Mutations in the spike protein of SARS-CoV-2 are the major causes for the modulation of ongoing COVID-19 infection. Currently, the D614G substitution in the spike protein has become dominant worldwide. It is associated with higher infectivity than the ancestral (D614)variant. We demonstrate using Gaussian network model-based normal mode analysis that the D614G substitution occurs at the hinge region that facilitates domain-domain motions between receptor binding domain and S2 region of the spike protein. Computer-aided mutagenesis and inter-residue energy calculations reveal that contacts involving D614 are energetically frustrated. However, contacts involving G614 are energetically favourable, implying the substitution strengthens residue contacts that are formed within as well as between protomers. We also find that the free energy difference (ΔΔG) between two variants is -2.6 kcal/mol for closed and -2.0 kcal/mol for 1-RBD up conformation. Thus, the thermodynamic stability has increased upon D614G substitution. Whereas the reverse mutation in spike protein structures having G614 substitution has resulted in the free energy differences of 6.6 kcal/mol and 6.3 kcal/mol for closed and 1-RBD up conformations, respectively, indicating that the overall thermodynamic stability has decreased. These results suggest that the D614G substitution modulates the flexibility of spike protein and confers enhanced thermodynamic stability irrespective of conformational states. This data concurs with the known information demonstrating increased availability of the functional form of spikeprotein trimer upon D614G substitution.     

D614G mutation in the SARS-CoV-2 spike protein enhances viral fitness by desensitizing it to temperature-dependent denaturation

The D614G mutation in the spike protein of SARS-CoV-2 alters the fitness of the virus, leading to the dominant form observed in the COVID-19 pandemic. However, the molecular basis of the mechanism by which this mutation enhances fitness is not clear. Here we demonstrated by cryo-electron microscopy that the D614G mutation resulted in increased propensity of multiple receptor-binding domains (RBDs) in an upward conformation poised for host receptor binding. Multiple substates within the one RBD-up or two RBD-up conformational space were determined. According to negative staining electron microscopy, differential scanning calorimetry, and differential scanning fluorimetry, the most significant impact of the mutation lies in its ability to eliminate the unusual cold-induced unfolding characteristics and to significantly increase the thermal stability under physiological pH. The D614G spike variant also exhibited exceptional long-term stability when stored at 37 °C for up to 2 months. Our findings shed light on how the D614G mutation enhances the infectivity of SARS-CoV-2 through a stabilizing mutation and suggest an approach for better design of spike protein-based conjugates for vaccine development.     

Evaluating the Effects of SARS-CoV-2 Spike Mutation D614G on Transmissibility and Pathogenicity

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Detection of the SARS-CoV-2 D614G mutation using engineered Cas12a guide RNA

Detection of pathogens with single-nucleotide variations is indispensable for the disease tracing, but remains technically challenging. The D614G mutation in the SARS-CoV-2 spike protein is known to markedly enhance viral infectivity but is difficult to detect. Here, we report an effective approach called “synthetic mismatch integrated crRNA guided Cas12a detection” (symRNA-Cas12a) to detect the D614 and G614 variants effectively. Using this method, we systemically screened a pool of crRNAs that contain all the possible nucleotide substitutions covering the -2 to +2 positions around the mutation and identify one crRNA that can efficiently increase the detection specificity by 13-fold over the ancestral crRNA. With this selected crRNA, the symRNA-Cas12a assay can detect as low as 10 copies of synthetic mutant RNA and the results are confirmed to be accurate by Sanger sequencing. Overall, we have developed the symRNA-Cas12a method to specifically, sensitively and rapidly detect the SARS-CoV-2 D614G mutation.     

Mutations in SARS-CoV-2 variants of concern link to increased spike cleavage and virus transmission

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D614G Spike Mutation Increases SARS CoV-2 Susceptibility to Neutralization

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Making sense of the diverse ligand recognition by NKG2D

NKG2D recognizes multiple diverse ligands. Despite recent efforts in determining the crystal structures of NKG2D-ligand complexes, the principle governing this receptor-ligand recognition and hence the criteria for identifying unknown ligands of NKG2D remain central issues to be resolved. Here we compared the molecular recognition between NKG2D and three of the known ligands, UL16 binding protein (ULBP), MHC class I-like molecule, and retinoic acid early inducible gene as observed in the ligand-complexed crystal structures. The comparison shows that while the receptor uses a common interface region to bind the three diverse ligands, each ligand forms a distinct, but overlapping, set of hydrogen bonds, hydrophobic interactions, and salt bridges, illustrating the underlying principle of NKG2D-ligand recognition being the conservation in overall shape complementarity and binding energy while permitting variation in ligand sequence through induced fit recognition. To further test this hypothesis and to distinguish between diverse recognition and promiscuous ligand binding, four ULBP3 interface mutations, H21A, E76A, R82M, and D169A, were generated to each disrupt a single hydrogen bond or salt bridge. All mutant ULBP3 displayed reduced receptor binding, suggesting a specific, rather than promiscuous, receptor-ligand recognition. Mutants with severe loss of binding affect the receptor interactions that are mostly buried. Finally, a receptor-ligand recognition algorithm was developed to assist the identification of diverse NKG2D ligands based on evaluating the potential hydrogen bonds, hydrophobic interactions, and salt bridges at the receptor-ligand interface.     

Structural diversity of the SARS-CoV-2 Omicron spike

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Computational epitope map of SARS-CoV-2 spike protein

The primary immunological target of COVID-19 vaccines is the SARS-CoV-2 spike (S) protein. S is exposed on the viral surface and mediates viral entry into the host cell. To identify possible antibody binding sites, we performed multi-microsecond molecular dynamics simulations of a 4.1 million atom system containing a patch of viral membrane with four full-length, fully glycosylated and palmitoylated S proteins. By mapping steric accessibility, structural rigidity, sequence conservation, and generic antibody binding signatures, we recover known epitopes on S and reveal promising epitope candidates for structure-based vaccine design. We find that the extensive and inherently flexible glycan coat shields a surface area larger than expected from static structures, highlighting the importance of structural dynamics. The protective glycan shield and the high flexibility of its hinges give the stalk overall low epitope scores. Our computational epitope-mapping procedure is general and should thus prove useful for other viral envelope proteins whose structures have been characterized.     

Uncovering cryptic pockets in the SARS-CoV-2 spike glycoprotein

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Prefusion spike protein conformational changes are slower in SARS-CoV-2 than in SARS-CoV-1

Within the last 2 decades, severe acute respiratory syndrome coronaviruses 1 and 2 (SARS-CoV-1 and SARS-CoV-2) have caused two major outbreaks; yet, for reasons not fully understood, the coronavirus disease 2019 pandemic caused by SARS-CoV-2 has been significantly more widespread than the 2003 SARS epidemic caused by SARS-CoV-1, despite striking similarities between these two viruses. The SARS-CoV-1 and SARS-CoV-2 spike proteins, both of which bind to host cell angiotensin-converting enzyme 2, have been implied to be a potential source of their differential transmissibility. However, the mechanistic details of prefusion spike protein binding to angiotensin-converting enzyme 2 remain elusive at the molecular level. Here, we performed an extensive set of equilibrium and nonequilibrium microsecond-level all-atom molecular dynamics simulations of SARS-CoV-1 and SARS-CoV-2 prefusion spike proteins to determine their differential dynamic behavior. Our results indicate that the active form of the SARS-CoV-2 spike protein is more stable than that of SARS-CoV-1 and the energy barrier associated with the activation is higher in SARS-CoV-2. These results suggest that not only the receptor-binding domain but also other domains such as the N-terminal domain could play a crucial role in the differential binding behavior of SARS-CoV-1 and SARS-CoV-2 spike proteins.     

SARS-CoV-2 spike protein dictates syncytium-mediated lymphocyte elimination

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus is highly contagious and causes lymphocytopenia, but the underlying mechanisms are poorly understood. We demonstrate here that heterotypic cell-in-cell structures with lymphocytes inside multinucleate syncytia are prevalent in the lung tissues of coronavirus disease 2019 (COVID-19) patients. These unique cellular structures are a direct result of SARS-CoV-2 infection, as the expression of the SARS-CoV-2 spike glycoprotein is sufficient to induce a rapid (~45.1 nm/s) membrane fusion to produce syncytium, which could readily internalize multiple lines of lymphocytes to form typical cell-in-cell structures, remarkably leading to the death of internalized cells. This membrane fusion is dictated by a bi-arginine motif within the polybasic S1/S2 cleavage site, which is frequently present in the surface glycoprotein of most highly contagious viruses. Moreover, candidate anti-viral drugs could efficiently inhibit spike glycoprotein processing, membrane fusion, and cell-in-cell formation. Together, we delineate a molecular and cellular rationale for SARS-CoV-2 pathogenesis and identify novel targets for COVID-19 therapy.Subject terms: Cell biology, Molecular biology     

Mechanical activation of spike fosters SARS-CoV-2 viral infection

The outbreak of SARS-CoV-2 (SARS2) has caused a global COVID-19 pandemic. The spike protein of SARS2 (SARS2-S) recognizes host receptors, including ACE2, to initiate viral entry in a complex biomechanical environment. Here, we reveal that tensile force, generated by bending of the host cell membrane, strengthens spike recognition of ACE2 and accelerates the detachment of spike’s S1 subunit from the S2 subunit to rapidly prime the viral fusion machinery. Mechanistically, such mechano-activation is fulfilled by force-induced opening and rotation of spike’s receptor-binding domain to prolong the bond lifetime of spike/ACE2 binding, up to 4 times longer than that of SARS-S binding with ACE2 under 10 pN force application, and subsequently by force-accelerated S1/S2 detachment which is up to ~10³ times faster than that in the no-force condition. Interestingly, the SARS2-S D614G mutant, a more infectious variant, shows 3-time stronger force-dependent ACE2 binding and 35-time faster force-induced S1/S2 detachment. We also reveal that an anti-S1/S2 non-RBD-blocking antibody that was derived from convalescent COVID-19 patients with potent neutralizing capability can reduce S1/S2 detachment by 3 × 10⁶ times under force. Our study sheds light on the mechano-chemistry of spike activation and on developing a non-RBD-blocking but S1/S2-locking therapeutic strategy to prevent SARS2 invasion.Subject terms: Molecular biology, Structural biology     

The effect of spike mutations on SARS-CoV-2 neutralization

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