Computational modeling of NOS3 interactions with Cav‐1: Influence of exon7 Glu298Asp single nucleotide polymorphism

Caveolar associations are important for endothelial nitric oxide synthase (NOS3) function and NOS3/Cav‐1 interaction plays a major role in NOS3 activity. Given the recent demonstration that caveolar associations are important for normal NOS3 performance and the established importance of NOS3 exon7 Glu298Asp variation for disease risk in humans, we recently investigated the impact of this polymorphism with respect to endothelial cell caveolar localizations. Immunoprecipitation and confocal microscopy studies revealed lower basal Cav‐1/NOS3 association for the variant allele. We also observed diminished NOS3 enrichment in caveolar membrane fractions (NOS3/Cav‐1 ratios in caveolar membrane fractions: 2.40±0.05, 1.65±0.05 & 1.41±0.12 in Glu/Glu, Glu/Asp & Asp/Asp (p<0.05)). Using a computational modeling approach we tested the hypothesis that NOS3 variant protein has altered structural geometry. Key amino acid residues were identified using NCBI database and literature. Molecular modeling revealed that the distance between important NOS3 binding sites and AA‐298 were lower for Asp298 as compared to Glu298. Changes in geometric distance were greatest in the caveolin‐1 binding region with Asp298. These findings suggest a biochemical mechanism for the clinical consequences of NOS3 Glu298Asp SNP, and support altered NOS3 coordination with Cav‐1 as a contributing mechanism for enhanced disease risk.