Mutation-driven parallel evolution in emergence of ACE2-utilizing sarbecoviruses

Figures

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  Figure 1. The SARS-CoV-2 RBD binds to hACE2 via residues located on the three loops. (A) The structure of SARS-CoV-2 RBD and hACE2 complex. The RBM comprising the three loops (designated as RBML1 to RBML3) docks onto the surface of hACE2 (shown in purple; pdb entry 6LZG). (B) LigPlot+ plot of the interaction diagram. Hydrophobic contacts and hydrogen bonding between the two loops (RBML1 and RBML2) of the RBD and the two α-helices (α1 and α2) of hACE2 are shown at the top and the interactions between RBML3 and α1, α13, and the β-hairpin of hACE2 at the bottom. The horizontal dotted line represents the interface, in which the residues involved in direct intermolecular hydrophobic contacts are shown as semicircles with radiating spoke and linked by red dotted lines and hydrogens (<4 Å) are represented by green dashed lines.

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  Figure 2. The SARS-CoV-2 RBB mutants for MD simulations. (A) The designed RBD mutant sequences. SARS-CoV-2 RBDwoIN represents a deletion mutant with corresponding amino acids in RBML1 and RBML2 (marked in red) deleted and “woIN” denotes “without insertions.” SARS-CoV-2 RBDCtoS represents a mutant of two Cys to Ser mutations that remove the SS4 (underlined once and shown in orange). SARS-CoV-2 RBDC21_L3 represents a mutant whose RBML3 is substituted by the equivalent of the CoVZXC21 RBD (marked in green). (B) Structures of the SARS-CoV-2 RBD mutants generated by homology modelling with SARS-CoV-2 RBD (PDB entry 6LZG) as template.

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  Figure 3. The classification and evolutionary conservation of Sarbecovirus RBDs. (A) The phylogeny-based classification. The tree was constructed by MEGA with the NJ method based on p-distance model of amino acid substitutions (NJp). Asterisks shown at nodes indicate the braches supported by up to 50% bootstrap based on 500 replicates. Two subfamilies differentiated by the tree are denoted as RBD-L (shown in red) and RBD-S (shown in green) based on their loop length. RBDs known to use ACE2 are marked by plus signs and those incapable of binding ACE2 by minus signs (data derived from Ren et al., 2008; Hu et al., 2017, 2018; Letko et al., 2020; Roelle et al., 2022; Starr et al., 2022). Hosts of the viruses, including bats, palm civet and human, are shown here. (B) The evolutionary conservation of RBDs analyzed by “Weblogo.” The three loops are boxed in red and positions as tree determinants are indicated by cyan triangles. Top: RBD-S. Bottom: RBD-L. The SS4 in RBD-L is indicated by cyan lines. (C) Comparison of the frequency of new amino acids emergence in RBML3 and its flanking region.

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  Figure 4. Structural and dynamics evidences for ACE2 binding origin. (A) Backbone-RMSDs of SARS-CoV-2 RBD and its deletion mutant shown as a function of time (left). Gyrate of proteins. SARS-CoV-2 RBD and its deletion mutant shown as a function of time (right). (B) ΔCα-RMSF data. SARS-CoV-2 RBDCtoS – SARS-CoV-2 RBD is marked in red and SARS-CoV-2 RBDC21_L3 – SARS-CoV-2 RBD in green (left). Conformational ensembles of RBML2 generated by MD simulations and shown by a “sausage” model with MOLMOL (right). (C) A 20-ns MD simulations showing structural dynamics of RBML2 in the apo state or ACE2-bound sate (left). Snapshots extracted from the MD trajectories at 10 and 15 ns, respectively, showing two distinct conformations in RBML2 (open and closed; middle). Comparison of the RBML2 RMSDs between SARS-CoV-2 RBD and the P491T mutant (right). (D) Structural mapping showing parallel molecular evolution removing steric hindrance and electric charge repulse present in the ancestral state. The clash occurs between Pro-502 of RBDs incapable of binding ACE2 and Lys-353 of ACE2, indicated by a cyan dashed circle, and the electric charge repulse between Asp-496 of the RBDs incapable of binding ACE2 and…

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  Figure 5. Evidence for parallel evolution of ACE2-binding RBMs in three Sarbecovirus clades. Phylogenetic relationships of SARS-CoV (strain GZ02), SARS-CoV-2 (Strain Wuhan-Hu-1) and SL-CoVs were reconstructed based on their whole genomes. The relationships were inferred by using the Maximum Likelihood method with the model GTR + G + I. The percentage of trees (≥70%) in which the associated taxa clustered together is shown at the nodes. Proposed insertion or deletion events are denoted by solid or dotted arrows. Right, sequence alignment of RBML1-3 with conservation across the alignment shaded in grey and the determinants for the topology of RBD-based trees in Figure 2B indicated by triangles, in which two directly associated with functional divergence via removing unfavorable interactions are shown in red. Sites involved in parallel substitutions are reconstructed for each nodes, in which the same ancestral amino acids are indicated by green circles and the same derived amino acids evolved through independent changes by red circles. “X” denotes a deficient residue. Structure-and function-based classifications are shown on the right of the alignment: ACE2 (+): RBDs capable of binding ACE2;…

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  Figure 6. Ancestral sequence reconstruction for elucidating the histories of the insertions and point mutations proposed by parallel evolution. (A) Schematic diagram illustrating the structural organization of RBDs, in which the pink region corresponding to the RBM was used to reconstruct ancestral sequences. The putative recombination breakpoint was denoted by an arrow (Starr et al., 2022). Residues were numbered according to the SARS-CoV-2 spike. (B) The projection of the ancestral traits including the insertions and point mutations proposed by parallel evolution onto the genome phylogeny. Green sector graphs represent RBD-S without an insertion and green and red sector graphs represent RBD-L with the insertion. Green and red circles represent an ancestral state and a derived state, respectively.

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  Figure 7. Purification, identification and functional characterization of recombinant RBDs. (A) Purification of refolded BtRBD and BtRBD|GY by SEC. Inset: SDS-PAGE analysis of the purified products, marked by a red arrow. “t6 to t9” denote collection tubes in SEC and “M” denotes protein molecular weight standard. (B) HPLC-Q-TOF-MS determining the molecular mass of BtRBD and BtRBD|GY. (C) Sensorgrams of SARS-CoV-2 RBD binding to the ACE2-immobilized chip surface (left top). The 125 nM analyte concentration was analyzed in duplicate. The concentrations used were 1,000–15.625 nM with two-fold serial dilutions. Sensorgrams of BtRBD to the chip surface (left bottom). The concentrations used were 10,000–625 nM with two-fold serial dilutions. Sensorgrams of BtRBD|GY to the chip surface (right bottom). The concentrations used were 40,000, 10,000, and 2,500 nM. Inset, schematic diagram of SPR experiment, in which the ligand hACE2 was covalently immobilized onto CM5 via its amine groups and the analytes (RBDs) flowed over the surface.

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  Figure 8. The structural basis of the RBML1 of SARS-CoV-2 RBD interacting with hACE2. (A) Gly-446 and Tyr-449 of the RBML1 (colored in cyan) interact with Gln-42 and Asp-38 of hACE2 via three hydrogen bonds (pdb entry 6LZG). In the model of BtRBD, its RBML1 far away from the interface is colored in purple. (B) The lifetimes of the hydrogen bonds during 100-ns MD simulations. The dashed line represents the length threshold (4 Å) of a hydrogen bond.

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  Figure 9. Schematic diagram of the proposed evolutionary histories of ACE2-utilizing spike proteins in sarbecoviruses. For simplicity, only RBDs are shown here. In this evolutionary scenario, two distinct progenitors are developed into SARS-CoV and SARS-CoV-2 clades via parallel insertions and point mutations followed by sequence optimization. ACE2 utilizing and non-utilizing are denoted by minus and plus signs, respectively.