Identification, characterization, and comparison of the calmodulin-binding domains of the endothelial and inducible nitric oxide synthases

The calmodulin (CaM)-binding regions in bovine endothelial nitric oxide synthase (eNOS) and murine inducible nitric oxide synthase (iNOS) are identified in this study as eNOS residues 493-512 and iNOS residues 501-532. Peptides corresponding to eNOS 493-512 and iNOS 501-532 produce a Ca²⁺-dependent, electrophoretic mobility shift of CaM on 4 M urea gels. The two peptides are also potent inhibitors of the CaM-mediated activation of neuronal nitric oxide synthase and have dissociation constants for CaM binding of 4.0 and 1.5 nM, respectively. Substitution of eNOS and iNOS CaM-bind-ing domains in eNOS/iNOS chimeric proteins produces major alterations in the Ca²⁺ and CaM dependence of the intact enzymes expressed and purified from a baeuIovirus/Sf9 insect cell system. Replacement of aligned iNOS sequence with eNOS 493-512 creates a functional, chimeric iNOS that is both Ca²⁺- and CaM-dependent. Replacement of aligned eNOS sequence with iNOS 501-532 creates a functional, chimeric eNOS that is CaMindependent but that remains Ca²⁺-dependent. Specific amino acid residues critical for CaM binding by eNOS are also identified in this study as Phe-498, Lys-499, and Leu-511 in the bovine eNOS sequence.