SARS-CoV-2 is a recently emerged coronavirus that binds angiotensin-converting enzyme 2 (ACE2) for cell entry via its receptor-binding domain (RBD) on a surface-expressed spike glycoprotein. Studies show that despite its similarities to severe acute respiratory syndrome (SARS) coronavirus, there are critical differences in key RBD residues when compared to SARS-CoV-2. Here we present a short in silico study, showing that SARS-like bat coronavirus Rs3367 shares a high conservation with SARS-CoV-2 in important RBD residues for ACE2 binding: SARS-CoV-2’s Phe486, Thr500, Asn501 and Tyr505; implicated in receptor-binding strength and host-range determination. These features were not shared with other studied bat coronaviruses belonging to the betacoronavirus genus, including RaTG13, the closest reported bat coronavirus to SARS-CoV-2’s spike protein. Sequence and phylogeny analyses were followed by the computation of a reliable model of the RBD of SARS-like bat coronavirus Rs3367, which allowed structural insight of the conserved residues. Superimposition of this model on the SARS-CoV-2 ACE2-RBD complex revealed critical ACE2 contacts are also maintained. In addition, residue Asn488Rs3367 interacted with a previously defined pocket on ACE2 composed of Tyr41, Lys353 and Asp355. When compared to available SARS-CoV-2 crystal structure data, Asn501SARS-CoV-2 showed a different interaction with the ACE2 pocket. Taken together, this study offers molecular insights on RBD-receptor interactions with implications for vaccine design.
To date, the closest identified coronavirus to SARS-CoV-2 with bat origin is RaTG13 CoV, which shares a 96 % identity at whole-genome-sequence level and more than 93.1 % identity in the spike glycoprotein region [4]. Alongside other reported coronaviruses of the sarbecovirus subgenus such as SARS-CoV and SL CoV RsSHC014, RaTG13 has been shown to use receptor ACE2 for entry [19]. Despite its high sequence identity to SARS-CoV-2, RaTG13 and other studied CoVs showed little conservation in key ACE2-binding reported residues within the spike-protein RBD.
Although SARS-like bat coronavirus Rs3367 has a 77.70 % identity to SARS-CoV-2 in the spike sequence, it showed a high conservation in the studied ACE2-binding residues. This bat coronavirus shares a 99.9 % sequence identity with bat coronavirus WIV1, which was confirmed to use human, bat and civet ACE2 for cell entry [22]. Phylogeny spike-protein analysis revealed this strain clustered closely with other ACE2-using bat SL CoVs, where many shared the bat host Rhinolophus sinicus (Fig. 2).
Here, we reported using sequence data and through spike RBD structural modelling, a previously identified SARS-like bat coronavirus [22], which shares conserved structural features with SARS-CoV-2 in critical residues known from SARS-CoV studies to mediate ACE2-spike binding interactions [13, 14].
A study looking at SARS-CoV-2’s RBD concluded its interactions with ACE2 are stronger than those between SARS-CoV and ACE2 [45], where researchers defined Phe486 as a key residue, which has the ability to reach into a deep hydrophobic pocket in ACE2, and has a major role in conferring binding strength to this receptor [45]. Here we have shown that not only is this key residue conserved in Rs3367 at a sequence level, but its three-dimensional conformation also points to a conservation in its interaction with Met82ACE2, alongside conserved interactions with other key ACE2 residues, which include Gln42 and Lys353 as shown by the in silico structural studies of superimposing the RBDs of Rs3367 and SARS-CoV-2 (Fig. 4).
Surface ACE2 analyses with the RBDs of SARS-CoV-2 and Rs3367 have shown another pocket composed of Lys353, Asp355 and Tyr41 is important in the receptor-binding interactions of both Rs3367 and SARS-CoV-2, views which have been confirmed in SARS-CoV-2 by x-ray crystallography data [44]. We found that SARS-like bat CoV Rs3367 has an interaction with a critical ACE2 pocket, which differs from that of SARS-CoV-2 (Fig. 5b). This pocket has been previously defined as a viral hotspot for ACE2 interaction, where a study conducted in 2011 concluded its structure confers important energy contributions to ACE2-viral RBD interactions in SARS-CoV and NL63-CoV [46]. Tyr41 corresponds to a histidine residue (His41) in the ACE2 receptor of several studied bat species [47], and has been proposed to be responsible for the weak binding of human SARS-CoV, where mutation of this residue to a tyrosine greatly increases receptor activity [47], implicating this pocket in human infectivity. Furthermore, when Asn501SARS-CoV-2 was mutated to a threonine, this significantly reduced ACE2-binding affinity [19], indicating the importance of this residue in receptor binding. This residue is also conserved in SL bat CoV Rs3367 and together with the presented in silico data, feature important ACE2-RBD interactions, which may have implications in the context of vaccine design.
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