An autoinhibitory control element defines calcium-regulated isoforms of nitric oxide synthase

John C. Salerno, Dawn E. Harris, Kris Irizarry, Binesh Patelf, Arturo J. Morales, Susan M.E. Smith, Pavel Martasek, Linda J. Roman, Bettie Sue S. Masters, Caroline L. Jones, Ben A. Weissman, Paul Lane, Qing Liu, Steven S. Gross

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201 Scopus citations

Abstract

Nitric oxide synthases (NOSs) are classified functionally, based on whether calmodulin binding is Ca2+-dependent (cNOS) or Ca2+-independent (iNOS). This key dichotomy has not been defined at the molecular level. Here we show that cNOS isoforms contain a unique polypeptide insert in their FMN binding domains which is not shared with iNOS or other related flavoproteins. Previously identified autoinhibitory domains in calmodulin-regulated enzymes raise the possibility that the polypeptide insert is the autoinhibitory domain of cNOSs. Consistent with this possibility, three-dimensional molecular modeling suggested that the insert originates from a site immediately adjacent to the calmodulin binding sequence. Synthetic peptides derived from the 45-amino acid insert of endothelial NOS were found to potently inhibit binding of calmodulin and activation of cNOS isoforms. This inhibition was associated with peptide binding to NOS, rather than free calmodulin, and inhibition could be reversed by increasing calmodulin concentration. In contrast, insert-derived peptides did not interfere with the arginine site of cNOS, as assessed from [3H]N(G)-nitro-L-arginine binding, nor did they potently effect iNOS activity. Limited proteolysis studies showed that calmodulin's ability to gate electron flow through cNOSs is associated with displacement of the insert polypeptide; this is the first specific calmodulin-induced change in NOS conformation to be identified. Together, our findings strongly suggest that the insert is an autoinhibitory control element, docking with a site on cNOSs which impedes calmodulin binding and enzymatic activation. The autoinhibitory control element molecularly defines cNOSs and offers a unique target for developing novel NOS activators and inhibitors.

Original languageEnglish (US)
Pages (from-to)29769-29777
Number of pages9
JournalJournal of Biological Chemistry
Volume272
Issue number47
DOIs
StatePublished - Nov 21 1997

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Salerno, J. C., Harris, D. E., Irizarry, K., Patelf, B., Morales, A. J., Smith, S. M. E., Martasek, P., Roman, L. J., Masters, B. S. S., Jones, C. L., Weissman, B. A., Lane, P., Liu, Q., & Gross, S. S. (1997). An autoinhibitory control element defines calcium-regulated isoforms of nitric oxide synthase. Journal of Biological Chemistry, 272(47), 29769-29777. https://doi.org/10.1074/jbc.272.47.29769