Hydrophobic remodeling of the SARS-CoV-2 RBD large loop attenuates ACE2 engagement and viral fitness
5 affiliations
- 1Department of Biochemistry, Key University Laboratory of Metabolism and Health of Guangdong, SUSTech Homeostatic Medicine Institute, School of Medicine, Institute for Biological Electron Microscopy, Southern University of Science and Technology, Shenzhen, Guangdong, China
- 2Key Laboratory of Zoonoses, Ministry of Agriculture and Rural Affairs, South China Agricultural University, Guangzhou, Guangdong, China
- 3Department of Biochemistry, Institute for Hepatology, National Clinical Research Center for Infectious and Communicable Diseases, Shenzhen Third People’s Hospital, the Second Affiliated Hospital, School of Medicine, Southern University of Science and Technology, Shenzhen, Guangdong, China
- 4Institute of Human Immunology, Shenzhen Medical Academy of Research and Translation (SMART), Shenzhen, Guangdong, China
- 5Center for Artificial Intelligence Driven Medical Research, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China
Abstract
The SARS-CoV-2 Omicron subvariant JN.4 harbors a unique A475V substitution within the receptor binding domain (RBD) but exhibits limited expansion compared to its parallel descendant JN.1. Here, we investigate how hydrophobic remodeling within RBD large loop (residues 475–490) modulates receptor engagement and spike function. Cryo-electron microscopy reveals that A475V induces local remodeling of the receptor-binding motif (RBM) and alters the RBD–ACE2 interaction geometry. Local refinement revealed that the bulky valine substitution disrupts the local interaction network surrounding residue 475, resulting in a 3.5-fold reduction in ACE2-binding affinity compared with BA.2.86. In contrast to the L455S substitution in JN.1, which primarily alters the receptor-binding interface, A475V within the flexible large loop influences both local loop conformation and global spike conformational properties. Systematic mutagenesis identifies A475 and F486 as functionally constrained hydrophobic residues that preserve the structural framework of ACE2 engagement despite extensive sequence variation within the surrounding loop. Together with molecular dynamics simulations and functional assays, our findings establish the RBD 475–490 large loop as a structurally plastic yet functionally constrained element that balances receptor recognition, conformational stability, and antigenic diversification. These results provide a mechanistic framework for understanding region-specific evolutionary constraints that shape SARS-CoV-2 RBD adaptation. Author summary: SARS-CoV-2 continues to modify its spike protein in order to enter human cells efficiently, evade antibodies, and maintain structural stability. Most previous work has emphasized mutations that alter charged or polar interactions at the interface between the receptor binding domain and ACE2. In this study we instead focus on a subtle hydrophobic change in a flexible loop of the receptor binding domain, an alanine to valine substitution at position 475 in the Omicron subvariant JN.4, and ask how it shapes viral properties. Using cryo-EM, quantitative binding measurements, molecular dynamics simulations, and cell-based infection and fusion assays, we find that this single hydrophobic substitution does more than weaken a local contact. It reconfigures the conformation of a mobile large loop spanning residues 475–490, changes the tilt and occupancy of ACE2 on the spike trimer, and ultimately reduces ACE2 binding, membrane fusion, and pseudovirus infectivity. By comparing related variants and a panel of point mutants, we identify positions A475 and F486 as hydrophobic anchor residues that support a flexible yet structurally constrained framework for ACE2 recognition. Our work illustrates how small hydrophobic changes in flexible structural elements can impose measurable fitness costs, providing a possible structural explanation for the limited expansion of some Omicron descendants, including JN.4.
Related
- Saccharomyces boulardii reduces food intake and attenuates weight gain in diet-induced obese mice: links to gut microbial, metabolic, and immune remodeling
- Tirzepatide Attenuates Pulmonary Vascular Remodeling in Pulmonary Arterial Hypertension: Association with the SIRT1/H4K12la/c-Jun Axis
- KAT3 Shuttling Between Neuronal Identity and Activity-Dependent Plasticity Programs Drives Large-Scale Chromatin Remodeling
- Large-scale integrated single-cell analysis reveals molecular subtypes and microenvironmental remodeling in cutaneous T cell lymphoma
- Multi-site Cleavage of Amyloid-β by a Minimal 5-mer Catalytic Peptide: Mimicking Serine Protease Activity via Hydrophobic Anchoring
BibTeX
@article{wang2026hydrophobic,
title = {{Hydrophobic remodeling of the SARS-CoV-2 RBD large loop attenuates ACE2 engagement and viral fitness}},
author = {Aojie Wang and Huimin Guo and Haonan Yang and Haojin Chen and Anna Imojara and Bin Ju and Zheng Zhang and Renhong Yan},
journal = {PLOS Pathogens},
year = {2026},
month = oct,
doi = {10.1371/journal.ppat.1014742},
url = {https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1014742},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Hydrophobic remodeling of the SARS-CoV-2 RBD large loop attenuates ACE2 engagement and viral fitness
AU - Aojie Wang
AU - Huimin Guo
AU - Haonan Yang
AU - Haojin Chen
AU - Anna Imojara
AU - Bin Ju
AU - Zheng Zhang
AU - Renhong Yan
PY - 2026
DA - 2026/10/26
JO - PLOS Pathogens
DO - 10.1371/journal.ppat.1014742
UR - https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1014742
AB - The SARS-CoV-2 Omicron subvariant JN.4 harbors a unique A475V substitution within the receptor binding domain (RBD) but exhibits limited expansion compared to its parallel descendant JN.1. Here, we investigate how hydrophobic remodeling within RBD large loop (residues 475–490) modulates receptor engagement and spike function. Cryo-electron microscopy reveals that A475V induces local remodeling of the receptor-binding motif (RBM) and alters the RBD–ACE2 interaction geometry. Local refinement revealed that the bulky valine substitution disrupts the local interaction network surrounding residue 475, resulting in a 3.5-fold reduction in ACE2-binding affinity compared with BA.2.86. In contrast to the L455S substitution in JN.1, which primarily alters the receptor-binding interface, A475V within the flexible large loop influences both local loop conformation and global spike conformational properties. Systematic mutagenesis identifies A475 and F486 as functionally constrained hydrophobic residues that preserve the structural framework of ACE2 engagement despite extensive sequence variation within the surrounding loop. Together with molecular dynamics simulations and functional assays, our findings establish the RBD 475–490 large loop as a structurally plastic yet functionally constrained element that balances receptor recognition, conformational stability, and antigenic diversification. These results provide a mechanistic framework for understanding region-specific evolutionary constraints that shape SARS-CoV-2 RBD adaptation. Author summary: SARS-CoV-2 continues to modify its spike protein in order to enter human cells efficiently, evade antibodies, and maintain structural stability. Most previous work has emphasized mutations that alter charged or polar interactions at the interface between the receptor binding domain and ACE2. In this study we instead focus on a subtle hydrophobic change in a flexible loop of the receptor binding domain, an alanine to valine substitution at position 475 in the Omicron subvariant JN.4, and ask how it shapes viral properties. Using cryo-EM, quantitative binding measurements, molecular dynamics simulations, and cell-based infection and fusion assays, we find that this single hydrophobic substitution does more than weaken a local contact. It reconfigures the conformation of a mobile large loop spanning residues 475–490, changes the tilt and occupancy of ACE2 on the spike trimer, and ultimately reduces ACE2 binding, membrane fusion, and pseudovirus infectivity. By comparing related variants and a panel of point mutants, we identify positions A475 and F486 as hydrophobic anchor residues that support a flexible yet structurally constrained framework for ACE2 recognition. Our work illustrates how small hydrophobic changes in flexible structural elements can impose measurable fitness costs, providing a possible structural explanation for the limited expansion of some Omicron descendants, including JN.4.
SN - 1553-7374
SN - 1553-7366
ER -