# Structural Insights into the Receptor-Binding Domain of Bat Coronavirus HKU5-CoV-2: Implications for Zoonotic Transmission via ACE2

## TL;DR

This study explores how the bat coronavirus HKU5 binds to human cells via ACE2 and suggests it may be more infectious than SARS-CoV-2, with potential for new antiviral treatments.

## Contribution

The study provides novel structural insights into HKU5's binding to ACE2 and identifies a peptide mutant that could inhibit this interaction.

## Key findings

- HKU5 shows stronger ACE2 binding affinity than SARS-CoV-2, suggesting higher zoonotic potential.
- A specific mutant peptide demonstrated strong and sustained binding to HKU5, potentially inhibiting ACE2 interaction.
- QM/MM calculations confirmed mutant-1's favorable interaction with HKU5, supporting its therapeutic potential.

## Abstract

Bats are recognized as carriers of many coronaviruses, some of which are capable of infecting people. The virus HKU5 (Bat Merbecovirus HKU5) may possess the capability to attach to the human cell receptor angiotensin-converting enzyme 2 (ACE2), which is also utilized by viruses such as SARS-CoV and SARS-CoV-2 for cellular entry. This study employed computational modeling and simulations to elucidate the attachment of the HKU5 virus to ACE2 and the strength of its binding. We also engineered small protein-like molecules, known as peptides, to evaluate their capacity to inhibit this interaction. Our findings indicate that HKU5 exhibits a stronger affinity for ACE2 compared to SARS-CoV-2, implying a greater potential for human cell infection. Among the evaluated peptides, a specific mutant variant demonstrated notably robust and sustained affinity for HKU5, indicating its potential to inhibit the virus’s attachment to ACE2. These findings enhance our comprehension of the infection risk associated with HKU5 and establish a basis for the development of novel antiviral therapies to prevent potential cross-species transmission.

The zoonotic potential of bat coronaviruses, especially HKU5, is a significant issue because of their capacity to utilize human angiotensin-converting enzyme 2 (ACE2) as a receptor for cellular entry. This study offers structural insights into the binding kinetics of HKU5 (Bat Merbecovirus HKU5) receptor-binding domain (RBD) spike protein with human ACE2 through a multiscale computational method. This study employed structural modeling, 300-nanosecond (ns) molecular dynamics (MD) simulations, alanine-scanning mutagenesis, and computational peptide design to investigate ACE2 recognition by the HKU5 RBD and its interactions with peptides. The root mean square deviation (RMSD) investigation of HKU5–ACE2 complexes indicated that HKU5 exhibited greater flexibility than SARS-CoV-2, with RMSD values reaching a maximum of 1.2 nm. Free energy analysis, Molecular Mechanics/Generalized Born Surface Area (MM/GBSA), indicated a more robust binding affinity of HKU5 to ACE2 (ΔGTotal = −21.61 kcal/mol) in contrast to SARS-CoV-2 (ΔGTotal = −5.82 kcal/mol), implying that HKU5 binding with ACE2 had higher efficiency. Additionally, a peptide was designed from the ACE2 interface, resulting in the development of 380 single-site mutants by mutational alterations. The four most promising mutant peptides were selected for 300-nanosecond (ns) MD simulations, subsequently undergoing quantum chemical calculations (DFT) to evaluate their electronic characteristics. MM/GBSA of −37.83 kcal/mol indicated that mutant-1 exhibits the most favorable binding with HKU5, hence potentially inhibiting ACE2 interaction. Mutant-1 formed hydrogen bonds involving Glu74, Ser202, Ser204, and Asn152 residues of HKU5. Finally, QM/MM calculations on the peptide–HKU5 complexes showed the most favorable ΔE_interaction of −170.47 (Hartree) for mutant-1 peptide. These findings offer a thorough comprehension of receptor-binding dynamics and are crucial for evaluating the zoonotic risk associated with HKU5-CoV and guiding the design of receptor-targeted antiviral treatments.