Computational Hot-Spot Analysis of the SARS-CoV-2 Receptor Binding Domain/ACE2 Complex*

Abstract

Infection and replication of SARS CoV-2 (the virus that causes COVID-19) requires entry to the interior of host cells. In humans, a protein-protein interaction (PPI) between the SARS CoV-2 receptor-binding domain (RBD) and the extracellular peptidase domain of ACE2 on the surface of cells in the lower respiratory tract is an initial step in the entry pathway. Inhibition of the SARS CoV-2 RBD/ACE2 PPI is currently being evaluated as a target for therapeutic and/or prophylactic intervention. However, relatively little is known about the molecular underpinnings of this complex. Employing multiple computational platforms, we predicted "hot-spot" residues in a positive-control PPI (PMI/MDM2) and the CoV-2 RBD/ACE2 complex. Computational alanine scanning mutagenesis was performed to predict changes in Gibbs' free energy that are associated with mutating residues at the positive control (PMI/MDM2) or SARS RBD/ACE2 binding interface to alanine. Additionally, we used the Adaptive Poisson-Boltzmann Solver to calculate macromolecular electrostatic surfaces at the interface of the positive-control PPI and SARS CoV-2/ACE2 PPI. Finally, a comparative analysis of hot-spot residues for SARS-CoV and SARS-CoV-2, in complex with ACE2, is provided. Collectively, this study illuminates predicted hot-spot residues, and clusters, at the SARS CoV-2 RBD/ACE2 binding interface, potentially guiding the development of reagents capable of disrupting this complex and halting COVID-19.

Keywords: ACE2; COVID-19; SARS CoV-2; computational alanine scanning mutagenesis; hot spots; protein-protein interactions.

Figure 1.

(A) Structure of the SARS-CoV-2 RBD / full-length ACE2 complex, colored by subunits. Extacellular ACE2 protease domain (PD) is colored grey; SARS-CoV-2 RBD is colored red. Cell membrane is depicted with a grey rectangle. (B) Interface of the SARS-CoV-2 RBD / ACE2 complex. SARS-CoV-2 RBD is colored red; ACE2 α1-helix is colored purple; ACE2 α2-helix is colored orange; ACE2 β3-β4 loop is colored green.

Figure 2.

(A) Hot-spot residue scores predicted by KFC-2 for the PPI involving PMI and MDM2. (B) Structure of the PMI / MDM2 interface, with predicted PMI or MDM2 hot-spot residues highlighted in blue or red, respectively. (C) Hot-spot residue scores predicted by KFC-2 for the PPI involving SARS-CoV-2 RBD and ACE2. (D) Structure of the SARS-CoV-2 RBD / ACE2 interface, with predicted ACE2 or SARS-CoV-2 RBD hot-spot residues highlighted in grey or red, respectively. (E) Hot-spot residue scores predicted by KFC-2 for the PPI involving SARS-CoV RBD and ACE2. (F) Structure of the SARS-CoV RBD / ACE2 interface, with predicted ACE2 or SARS-CoV RBD hot-spot residues highlighted in grey or red, respectively.

Figure 3.. (A)

(A) Computational alanine scanning mutagenesis data for the PMI / MDM2 complex. (B) Residues in the PMI / MDM2 complex that are predicted to contribute significantly to the free energy of the complex. (C) Computational alanine scanning mutagenesis data for the SARS-CoV-2 RBD / ACE2 complex. (D) Residues in the SARS-CoV-2 RBD / ACE2 complex that are predicted to contribute significantly to the free energy of the complex.

Figure 4.

(A) Structure of the PMI / MDM2 complex. Hot spot residues (F3, W7, L10) are highlighed. (B) Electrostatic potential and topology of PMI and MDM2 surface areas that engage one another in the complex. (C) Strucuture of the ACE2 / SARS-CoV-2 RBD complex. α1-helix is highlighted in blue; β3-β4 loop is highlighted in green. (D) Electrostatic potential and topology of ACE2 and SARS-CoV-2 RBD surface areas that engage one another in the complex. Colored from −25 kT/e (red) to +25 kT/e (blue).

Figure 5.

(A) Residues on the α1-helix (blue), α−2 helix (orange), and β3-β4 (green) on ACE2 (top) and SARS-CoV-2 RBD (bottom) that were identified as stabilizing formation of a complex between these two proteins. Structures and selected residues are overlaid with the electrostatic potential map of protein surfaces at the binding interface. Values in parenthesis are predicted ΔΔG (kcal/mol) values obtained from computational alanine scanning mutagenesis. An asterisk (*) indicates that the residue was predicted by they KFC-2 hot-spot server and BAlaS computational alanine scanning server. (B) Overlay of SARS-CoV (pink) and SARS-CoV-2 (red) RBDs with ACE2 (α−1 helix and β3-β4 loop shown in black). Residues identified as hot-spots are highlighted. Mutation of hot-spot residues identified using KFC-2, for SARS-CoV and SARS-CoV-2, are highlighted with an asterisk (*).