A candidate Arg-287/Arg-288-centered electrostatic interface between SNIP1 and p65 in osteoarthritis: proteomic identification and computational structure-function analysis.

The physical interfaces governing macromolecular protein-protein interactions dictate critical gene transcription programs, yet the structural basis of regulatory complexes inhibiting NF-κB remains partially resolved. Here, we characterize the macromolecular interaction between Smad nuclear interacting protein 1 (SNIP1) and the transcriptionally active p65 subunit of NF-κB, a regulatory axis whose pathological downregulation was identified via comparative proteomics of human osteoarthritic cartilage. GSE114007 and descriptive single-cell analyses of GSE152805 showed lower SNIP1 expression in diseased cartilage; GSE169077 showed the same direction without statistical significance. Co-immunoprecipitation confirmed the endogenous physical association of SNIP1 and p65 in human chondrocytes. Molecular docking and molecular dynamics (MD) simulations mapped a candidate SNIP1-p65 interface with a negative MM/GBSA endpoint estimate (ΔGbind = -209.6 kJ/mol, 95% CI -381.4 to -37.8 kJ/mol across three trajectory means). The computational models position SNIP1 Arg-287 and Arg-288 within a cationic interface that is sterically incompatible with the DNA-bound conformation of p65. Computational substitution analysis further identified Arg-288 as a candidate hotspot residue, with charge-reversal substitutions predicted to destabilize the complex; this residue-level prediction awaits experimental site-directed mutagenesis. An assumption-based ODE model illustrates the sensitivity of downstream outputs to imposed SNIP1-dependent inhibition without establishing clinical kinetics or residue-specific causality. Overall, the combined data define a testable structural model for SNIP1-mediated regulation of p65 while avoiding causal attribution to Arg-288 before experimental validation.