Selective enhancement of PNOC protein binding to HIV-1 co-receptors CCR5 and CXCR4 by missense mutations: a computational analysis and prediction

The aim of this study was a systematic in silico evaluation of the effect of natural missense mutations in PNOC on the stability and binding energy of its complexes with CCR5 and CXCR4 to identify variants with enhanced affinity, potentially suitable for inhibiting viral entry. Materials and methods . Three-dimensional structures of wild-type PNOC protein complexes with CCR5 and CXCR4 were built using molecular modeling methods with the AlphaFold algorithm. Based on these, mutant PNOC variants localized at the receptor contact sites were modeled using the FoldX program. To quantitatively assess the mutation effects, we calculated the change in the complex stability free energy (∆Stability) and the protein-protein interaction energy (∆Connection), analyzed the change in the number of atomic contacts (∆Contacts), and predicted the functional impact of the substitutions using the PolyPhen-2 algorithm. Results and discussion . The wild-type PNOC was shown to have a higher predicted affinity for CXCR4 compared to CCR5. For the PNOC-CCR5 complex, one candidate (E50K) was identified that fully met the strict selection criteria (decrease in both ∆Stability and ∆Connection, preserved number of contacts, «benign» status). For the PNOC-CXCR4 complex, a broader spectrum of significant mutations was revealed, among which four (F14L, S20N, R23K, V43M) met all the criteria. Mutations with selective action are of particular interest: E50K (improves binding to CCR5 but impairs it for CXCR4) and F14L (the only mutation that improves binding parameters for both receptors). Conclusion . For the first time, a systematic computational analysis of the impact of PNOC missense mutations on its interaction with HIV-1 co-receptors has been conducted. Specific amino acid substitutions (E50K for CCR5; F14L, Q22P, and others for CXCR4) that statistically significantly improve the binding energy and complex stability have been identified. These mutant PNOC forms represent promising candidates for further experimental validation of their ability to inhibit viral binding to the target cell and can be considered as a basis for developing new strategies for antiviral therapy.