Abstract:
NOS3 gene encodes the enzyme endothelial nitric oxide synthase (eNOS) that is
involved in cardiovascular homeostasis. Defects in eNOS have been associated with
many cardiovascular diseases. Missense non-synonymous single nucleotide
polymorphisms (nsSNPs) of NOS3 have the potential to damage eNOS and cause
disease. This study aimed to identify the most damaging missense nsSNPs i.e.,
functionally significant of NOS3 and study their effect on the structure and function of
eNOS through in silico approaches. Deleterious nsSNPs were predicted using
PredictSNP. Out of these nsSNPs, the ones that replace amino acids determined as
conserved by ConSurf were considered to be the most damaging. Upon analyzing the
impact of these 48 nsSNPs on eNOS stability through I-mutant, 44 were found to be
destabilizing. HOPE found the 48 nsSNPs to replace wild-type amino acids with amino
acids having different properties, affecting protein structure and function. 41 nsSNPs
reside in domains and can prevent their functioning. Two nsSNPs can affect domain
functioning through loss of phosphorylation sites predicted there. 48 mutated eNOS
structures corresponding to the nsSNPs were generated using Phyre2 and structurally
compared with native eNOS structure through TM-align. A proline to serine substitution
at position 1002 (rs1563233862) caused the greatest structural deviation from normal
eNOS, followed by a glycine to leucine substitution at position 1115 (rs746892263). 3D
structures of native eNOS and these two mutants were modeled by I-TASSER. The
mutant and native structures were superimposed, showing a clear structural difference
between them. This can be attributed to the loss of important structural properties of
proline and glycine. These findings can be validated by experimental studies. Such
studies can also further explore the effect of these nsSNPs on eNOS and their
involvement in eNOS related diseases. This will help in better understanding of the role
of eNOS in disease and development of therapeutic strategies.
