An Autoinhibitory Control Element Defines Calcium-regulated Isoforms of Nitric Oxide Synthase

Abstract

Nitric oxide synthases (NOSs) are classified functionally, based on whether calmodulin binding is Ca 2؉ -dependent (cNOS) or Ca 2؉ -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 [ 3 H]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.Nitric oxide is a ubiquitous cell-signaling molecule, with protean roles in physiology and pathophysiology (1-3). Encoded by distinct genes, mammalian NO synthases (NOSs) 1 comprise a family of three calmodulin-dependent biopterohemoflavoproteins that are functionally distinguished by their modes of regulation (4). The two constitutively expressed isoforms of NOS (cNOSs), first identified in neuronal cells (nNOS) and endothelial cells (eNOS), remain dormant until calcium/calmodulin (Ca 2ϩ /CaM) binding is actuated by transient elevations in intracellular Ca 2ϩ . This Ca 2ϩ -dependent mode of regulation provides pulses of NO for moment-to-moment modulation of vascular tone and neurosignaling. In contrast, activity of the immunostimulant-induced isoform of NOS (iNOS) is Ca 2ϩ -independent, providing continuous high output NO generation for host defense. A remarkably high affinity for CaM, even at basally low levels of intracellular calcium, is responsible for the Ca 2ϩ independence of iNOS (5). Whether a given NOS isoform binds CaM in a Ca 2ϩ -dependent or -independent manner has been assumed to be a property solely of the amino acid sequence specified by a 20 -25-amino acid CaM binding site. However, this restrictive view is challenged by findings that ...

Identification of Caveolin-1-interacting Sites in Neuronal Nitric-oxide Synthase

“…In the absence of Ca 2ϩ /CaM, the autoinhibitory and C-terminal control elements repress electron transfers within and from the reductase domain in both nNOS and eNOS (11)(12)(13)(14)(15)(16)(17). The nNOS control element deletion mutants (⌬40 and ⌬C33) retained activity even in the absence of Ca 2ϩ /CaM (16,17) (Table II).…”

“…Deletion of either element affects the Ca 2ϩ /CaM sensitivity of nNOS and eNOS and allows electron transfers to proceed even in the absence of CaM (11)(12)(13)(14)(15)(16)(17). To investigate how caveolin binding regulates these CaM-independent electron transfers, we examined two deletion mutants of nNOS: ⌬40, in which 40 amino acids have been deleted from the FMN binding subdomain and ⌬C33, in which 33 amino acids have been truncated from the C-terminal end (Fig.…”

“…For example, an autoinhibitory loop exists within the FMN binding subdomains of nNOS and eNOS, but not iNOS. Peptides derived from this putative autoinhibitory domain in eNOS inhibited CaM binding and Ca 2ϩ activation of both nNOS and eNOS (11). Deletion of the autoinhibitory domains of eNOS and nNOS activated the reductase domains in the absence of CaM and affected Ca 2ϩ /CaM-dependent NO formation (12)(13)(14)(15)(16).…”

Arginase II Contributes to the Ca 2+ /CaMKII/eNOS Axis by Regulating Ca 2+ Concentration Between the Cytosol and Mitochondria in a p32‐Dependent Manner

“…Activated calmodulin (Ca 2+ /CaM) from increased cytosolic Ca 2+ level binds to the canonical CaM‐binding domain in eNOS that promotes the alignment of the oxygenase and reductase domains of eNOS and prevents eNOS Thr495 phosphorylation by protein kinase C, leading to efficient NO synthesis. Ca 2+ /CaM can also activate Ca 2+ /calmodulin‐dependent protein kinase II (CaMKII), which participates in the phosphorylation of eNOS Ser1177 to increase NO release 20, 21. Intracellular Ca 2+ can be transported and stored in endoplasmic (sarcoplasmic) reticulum and mitochondria.…”

Chimeric Enzymes of Cytochrome P450 Oxidoreductase and Neuronal Nitric-oxide Synthase Reductase Domain Reveal Structural and Functional Differences

“…2). In addition to the CaM binding sequence, an autoregulatory insert was identified in the FMN binding region of the constitutively expressed isoforms, nNOS and eNOS, but not in the inducible isoform, iNOS (8). This autoregulatory insert obstructs transfer of electrons into the heme domain in the absence of CaM.…”