A control element in calcium-dependent nitric oxide synthases

NO synthases are classified based on whether calmodulin (CAM) binding is Calcium-dependent (cNOS) or independent tiNeS). The molecular basis for this functional dichotomy is unresolved. Amino acid sequence alignment reveals that cNOSs contain a unique polypeptide insert in their FMN binding domains that is absent from iNOSs and other FMN flavoproteins. Since autoinhibitory domains are typical of CaM-regulated enzymes, we considered whether the polypeptide insert is a CaM displacable autoinhibitory element. In support, three-dimensional molecular modeling indicated that the insert originates from a site immediately adjacent to the CaM binding sequence. Synthetic peptides derived from the 45 amino acid insert of endothelial NOS potently inhibited binding of CaM and activation of cNOSs. This inhibition was associated with peptide binding to NOS, rather than free CaM, and could be reversed by increasing [CAM]. In contrast, insert-derived peptides did not potently inhibit iNOS. Limited trypsinolysis studies revealed that the activation of cNOSs by CaM occurs with displacement of the insert polypeptide - this is the first specific CaM-induced change in NOS conformation to be identified. Our findings suggest that the insert reversibly docks with a site on cNOSs that impedes CaM binding and enzyme activity. This control element molecularly defines cNOSs and offers a target for novel NOS activators and inhibitors. [Supported by HL 50656 & HL 44603 (S.S,(;.), HL 30050 & GM52419 (B.S.S.M.)].