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. 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. Nitric oxide is a ubiquitous cell-signaling molecule, with protean roles in physiology and pathophysiology (1Nathan C. Xie Q.W. Cell. 1994; 78: 915-918Abstract Full Text PDF PubMed Scopus (2740) Google Scholar, 2Ignarro L.J. Annu. Rev. Pharmacol. Toxicol. 1990; 30: 535-560Crossref PubMed Scopus (1220) Google Scholar, 3Moncada S. Palmer R.M. Higgs E.A. Biochem. Pharmacol. 1989; 38: 1709-1715Crossref PubMed Scopus (1099) Google Scholar). Encoded by distinct genes, mammalian NO synthases (NOSs) 1The abbreviations used are: NOS, nitric oxide synthase; cNOS, calcium-dependent NOS, iNOS, calcium-independent NOS; eNOS, the endothelial isoform of cNOS; nNOS, the neuronal isoform of cNOS; CaM, calmodulin, SCR, structurally conserved region; CPR, cytochrome P450 reductase; DTT, dithiothreitol. 1The abbreviations used are: NOS, nitric oxide synthase; cNOS, calcium-dependent NOS, iNOS, calcium-independent NOS; eNOS, the endothelial isoform of cNOS; nNOS, the neuronal isoform of cNOS; CaM, calmodulin, SCR, structurally conserved region; CPR, cytochrome P450 reductase; DTT, dithiothreitol. comprise a family of three calmodulin-dependent biopterohemoflavoproteins that are functionally distinguished by their modes of regulation (4Nathan C. Xie Q.W. J. Biol. Chem. 1994; 269: 13725-13728Abstract Full Text PDF PubMed Google Scholar). The two constitutively expressed isoforms of NOS (cNOSs), first identified in neuronal cells (nNOS) and endothelial cells (eNOS), remain dormant until calcium/calmodulin (Ca2+/CaM) binding is actuated by transient elevations in intracellular Ca2+. This Ca2+-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 Ca2+-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 Ca2+ independence of iNOS (5Cho H.J. Xie Q.W. Calaycay J. Mumford R.A. Swiderek K.M. Lee T.D. Nathan C. J. Exp. Med. 1992; 176: 599-604Crossref PubMed Scopus (559) Google Scholar). Whether a given NOS isoform binds CaM in a Ca2+-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 chimeric eNOS and nNOS, which have had their CaM binding sequences replaced with the corresponding sequence from iNOS, still require Ca2+ for full activity (6Venema R.C. Sayegh H.S. Kent J.D. Harrison D.G. J. Biol. Chem. 1996; 271: 6435-6440Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 7Ruan J. Xie Q. Hutchinson N. Cho H. Wolfe G.C. Nathan C. J. Biol. Chem. 1996; 271: 22679-22686Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Because regulation of enzyme systems by Ca2+/CaM typically involves displacement of an intrinsic autoinhibitory polypeptide (8Jarrett H.W. Madhavan R. J. Biol. Chem. 1991; 266: 362-371Abstract Full Text PDF PubMed Google Scholar,9Brickey D.A. Bann J.G. Fong Y.L. Perrino L. Brennan R.G. Soderling T.R. J. Biol. Chem. 1994; 269: 29047-29054Abstract Full Text PDF PubMed Google Scholar), we hypothesized that the binding of Ca2+/CaM to cNOSs may similarly trigger activation by displacing a control element. Here we identify a multiple amino acid insertion which serves as a control element unique to cNOSs and which molecularly defines Ca2+-dependent isoforms of NOS. Molecular modeling of the FMN binding module of nitric oxide synthase isoforms was done using the Insight and Homology programs from Biosym (BIOSYM/Molecular Simulations, San Diego, CA) running on a silicon graphics Indigo2 workstation. After alignment of NOS sequences with homologous FMN binding proteins of known structure (see “Results”), structurally conserved region (SCR) boxes were created corresponding to conserved regions of secondary structure and regions involved directly in FMN binding. These regions were characterized by high positive scores as evaluated by Dayhoff's mutation matrix (10Dayhoff M.O. Hunt W.C. Hunt L.T. Methods Enzymol. 1983; 91: 524-545Crossref PubMed Scopus (846) Google Scholar). After assignment of coordinates in the SCR regions, the loop regions between the SCR boxes were modeled by searching the Brookhaven protein data base. The crude model structure was relaxed to a sterically and energetically reasonable state using the Discover program (BIOSYM/Molecular Simulations) for molecular mechanics and dynamics calculations. This includes splice repair to remove unrealistic structural features at SCR-loop junctions, end repair to assign reasonable structures to C-terminal and N-terminal extensions, and structural optimization to remove steric overlaps and to reduce the structure to a energetic minimum. Energy minimizations were begun using the steepest descent method; this was replaced by conjugate gradient method as convergence was approached. Rat neuronal cNOS (nNOS) and bovine endothelial cNOS (eNOS) were purified from Escherichia coli harboring pGroELS and pCW vector expression systems for nNOS and eNOS, as described previously (11McMillan K. Bredt D.S. Hirsch D.J. Snyder S.H. Clark J.E. Masters B.S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11141-11145Crossref PubMed Scopus (354) Google Scholar, 12Martasek P. Liu Q. Liu J. Roman L.J. Gross S.S. Sessa W.C. Masters B.S. Biochem. Biophys. Res. Commun. 1996; 219: 359-365Crossref PubMed Scopus (142) Google Scholar). iNOS-rich cytosol was prepared from rat aortic smooth muscle cells, which were isolated from Fisher rat thoracic aortae (13Gross S.S. Levi R. J. Biol. Chem. 1992; 267: 25722-25729Abstract Full Text PDF PubMed Google Scholar) and grown in 75-cm2 culture flasks at passage 10–15. Cells were stimulated for 16 h in culture medium containing a combination of lipopolysaccharide (30 μg/ml) and rat recombinant interferon-γ (50 ng/ml), washed twice with 10 ml of ice-cold phosphate-buffered and with a an 10 ml of phosphate-buffered were at 10 in culture of ice-cold containing a of 10 10 and and by three of in and in a were at for and were at until NOS activity was in at based on the of or the of to as described previously S.S. Methods Enzymol. 1996; PubMed Google Scholar). 10 DTT, calmodulin, and the of peptide in a of were by the of of nNOS, 10 of eNOS, or of rat iNOS-rich was from the of in A at for a of in a The of in A NOS was from was from in which iNOS activity was based on were prepared as of rat iNOS-rich cytosol and a of of and of of to by was as described S.S. Methods Enzymol. 1996; PubMed Google Scholar). was with to a specific activity of using the method Biochem. J. PubMed Scopus Google binding were in using with were for with of containing DTT, and was by were DTT, 10 and of NOS in a were for at and binding was by were washed twice with of ice-cold containing and and was to and were in a binding was in that 10 In studies of the effect of effect of peptide binding to NOS, binding was in the of peptide and of NOS. was similarly in were first by of of nNOS, of 10 DTT, and for a of CaM was at with or of the of bovine of [3H]N G-nitro-l-arginine binding were in as described previously P. K. J. Liu Q. Gross S.S. Masters B.S. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar), using of nNOS and the of of the calmodulin-dependent was in a at based on of of the A. Biochem. J. 1991; PubMed Scopus Google Scholar). bovine calmodulin, and in a were by the of of and activity was for at 10 in inhibition of activity. Limited proteolysis was on containing of recombinant nNOS purified from cells (11McMillan K. Bredt D.S. Hirsch D.J. Snyder S.H. Clark J.E. Masters B.S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11141-11145Crossref PubMed Scopus (354) Google Scholar) or of recombinant eNOS purified from coli P. Liu Q. Liu J. Roman L.J. Gross S.S. Sessa W.C. Masters B.S. Biochem. Biophys. Res. Commun. 1996; 219: 359-365Crossref PubMed Scopus (142) Google Scholar). were at for in a DTT, 10 with or 10 was by the of of of NOS. were and and proteolysis was by with an of were on an gradient and by with molecular of was by at the using and of N-terminal sequence were prepared as and to to acid was on an protein Synthetic peptides were from CA) and other was evaluated by high and and in with from the by amino acid from Rat recombinant culture and culture were from were from lipopolysaccharide coli were from calmodulin was from and was from were from or Nitric oxide synthases are enzymes in which a of has in the of to proteins B.S. K. E.A. Roman L.J. P. J. 1996; PubMed Scopus Google Q. Gross S.S. Methods Enzymol. 1996; PubMed Google Scholar). be an N-terminal domain and a C-terminal by a binding sequence for CaM E.A. K. Masters B.S. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). binding electron between the and D.J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The domains have binding for and the domain has binding for and Bredt D.S. C. Snyder S.H. 1991; PubMed Scopus Google Scholar) were first to the between the C-terminal of NOS and P450 conserved regions corresponding to and binding The of NOS isoforms and are in homologous to the which are proteins that as electron in T.D. Biochem. Sci. 1991; Full Text PDF PubMed Scopus Google Scholar). The and binding domains are related to and other related have been and by Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). regions in are involved in binding the FMN the first of is to the and is immediately by the of the of FMN binding regions was identified in nNOS by Bredt D.S. C. Snyder S.H. 1991; PubMed Scopus Google Scholar), of has a corresponding in The first in alignment of the NOS FMN binding domain with the was the of regions in NOS This was by the of conserved secondary structural in NOS, by their to the corresponding in by mutation matrix (10Dayhoff M.O. Hunt W.C. Hunt L.T. Methods Enzymol. 1983; 91: 524-545Crossref PubMed Scopus (846) Google Scholar). alignment of a of NOS, CPR, and the of regions involved in FMN binding. is that a insertion of amino has in mammalian cNOSs. A corresponding insert is found in the FMN binding region of the cNOSs from Scholar) Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google are amino contain regions of to mammalian cNOS this alignment to of known and related that cNOSs an the corresponding region of iNOS sequences sequences an insert the FMN binding of the amino acid insertion with Ca2+/CaM This insertion the between cNOSs and iNOS amino acid their The cNOS FMN module are in and have an of positive This is of the eNOS which the between the cNOS their is that structural has which may the two or three on in the nNOS of the region to a binding site. The of that eNOS and nNOS contain at two The of structures for to the insertion in three to the calmodulin binding site. molecular have been for the FMN binding domains of iNOS, and CPR, which could be relaxed to a sterically and energetically reasonable After of FMN binding domain steric overlaps were and were or than that of in the structure of iNOS and are on the of the structural of the FMN binding of the NOS isoforms. the structure of P450 has been at R. Masters and of Scholar). The module The a PubMed Scopus Google Scholar), is a with the FMN binding site Homology that two in iNOS, and are in with the FMN serves as a the corresponding structure of nNOS is not of the eNOS be on in with the insertion from the of the the FMN binding site. to the of a loop with an structure the of the FMN binding are to a conformation for the insertion we a the structure is to and The CaM binding site is immediately adjacent to the N-terminal of the FMN binding domain in CaM the CaM site be in a conformation Biochem. Sci. 1990; Full Text PDF PubMed Scopus Google Scholar, L. S. PubMed Scopus Google steric suggest that directly from the FMN binding of show of the FMN binding domains of iNOS and eNOS with CaM based on L. S. PubMed Scopus Google the N-terminal of the FMN are between the end of the site and the of the of the at end of this are to the of CaM and the FMN This that are are the the of CaM and the of the of the CaM site. The of CaM to the FMN domain is with to they of by to the of the of is that are than the the insertion is through the sequence of the in three the model to be directly adjacent to the CaM binding site. The model that CaM binding be sterically by the that the insertion in than of this model strongly suggest that the insert as a control element, the between Ca2+/CaM control and the of the insertion and the of the CaM binding site to the insertion and the steric which for the insert is that of an polypeptide which is by CaM binding. from to other and CaM in of and amino this binding of the insertion to CaM in NOS isoforms CaM could the polypeptide insert from a site by binding domain or through of the autoinhibitory insert of cNOSs was evaluated using a of polypeptide corresponding to as the of the eNOS insert were in from to as in and peptide were on NOS activity are in of polypeptide of the cNOS eNOS and nNOS activity. The were from the eNOS insertion and the and eNOS, were effect on peptides were on iNOS, inhibition was with and inhibition of iNOS activity by peptides was and of Because CaM is to iNOS and has a remarkably with even (5Cho H.J. Xie Q.W. Calaycay J. Mumford R.A. Swiderek K.M. Lee T.D. Nathan C. J. Exp. Med. 1992; 176: 599-604Crossref PubMed Scopus (559) Google Scholar), inhibition CaM and and of from the insert in the FMN binding domain of cNOSs and for on NOS activity and in a FMN binding domain insert of effect of peptide on NOS activity and activity were using purified recombinant nNOS and eNOS, or are of to binding was of of NOS for at with or [3H]N G-nitro-l-arginine and the are of of NOS activity were using purified recombinant nNOS and eNOS, or are of binding was of of NOS for at with or [3H]N G-nitro-l-arginine and the are of in a and of from the insert in the FMN binding domain of cNOSs and for on NOS activity and binding. of the cNOS polypeptide insert and the CaM suggested from molecular that the insert may CaM binding. this involves of the insert of the insert similarly and interfere with CaM binding. in inhibition binding to nNOS was with insert-derived polypeptide peptide for CaM binding that for nNOS activation. for peptide from to 10 and as the was to to amino of nNOS activity and CaM binding by insert-derived peptides was reversed by CaM (see and for findings with of a mode of the of peptides for CaM binding in is by in CaM were used to binding activity of CaM binding by peptide could not be by the peptides could interfere with CaM binding to NOS by with NOS or CaM NOS is the binding target for insert peptides is by binding of peptide in the of NOS, was at that of CaM binding to nNOS not the which cNOSs in for CaM of D.J. Biochem. Biophys. PubMed Scopus Google Scholar), was not by of insert-derived peptides that potently inhibit nNOS activity (see peptides the from with nNOS In this is from with NOS by of a of in for a peptide to CaM from binding site on nNOS, at a with NOS. this transient could of peptide with CaM as as NOS. These findings suggest that the binding domain of the eNOS autoinhibitory element on nNOS overlaps or the CaM binding Previously described with for NOS the arginine site in a manner that be as a in or binding affinity for the arginine [3H]N is that the cNOS insert peptides inhibit NOS activity and CaM binding with a rather than in [3H]N G-nitro-l-arginine binding of the insert peptides is by a of inhibition of NOS activity or CaM binding with of amino in derived from on the FMN binding domain of which are distinct from the insert polypeptide of the insert peptide from an binding site on cNOSs by CaM of the insert to This was by the of peptide of nNOS and eNOS, in the and of CaM E.A. K. Masters B.S. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar) showed that of in rat nNOS, a site at the CaM binding sequence. at this site has as an for of distinct and CaM not to of the we similarly that of nNOS at a with the CaM binding site. we found a of with molecular of and corresponding to C-terminal and N-terminal CaM was to nNOS, by of was from and a novel site was at this site of molecular of and Molecular by the to be This is by at a site the insert peptide which a C-terminal of this originates from the of nNOS is by our that is the of the C-terminal domain is not by of the N-terminal domain not of at is by sequence of 10 N-terminal amino In with our a of nNOS in the of CaM, that a site the CaM binding site at S. A. Biochem. J. 1996; PubMed Scopus Google Scholar). is from in the of CaM, CaM is the CaM binding site is we that CaM the FMN domain insert peptide of A is from of eNOS CaM is not of eNOS peptides of molecular of and This is by at the CaM binding site and at a to be which between the CaM binding site and insert peptide of and to the CaM binding site is in the model in this site to be a at the of the from the FMN binding site. of in nNOS at the site homologous to eNOS may be by the of a eNOS In binding of CaM this by providing a site. the proteins and the from of eNOS, two are at and These are by of the the in the insert peptide at at of the N-terminal at of and between of eNOS and of nNOS (see that CaM binding similarly the insert peptide in cNOS CaM binding not the CaM binding site from by on nNOS and eNOS, which are A of to the of in the FMN domain insert as the which are by CaM binding. of by CaM binding could by an or by displacement through binding domain of the insert strongly a for activation of NO we have that cNOSs a polypeptide insert in their FMN binding that is unique to NOS isoforms which are by transient CaM adjacent to the CaM binding an to CaM binding and NOS and CaM Together, strongly that the insertion in cNOSs is an autoinhibitory control element. that inhibition of NOS by the insert of key on CaM binding the cNOS by of the polypeptide to binding on cNOSs in an high the state in the of The of activity with purified eNOS or nNOS, in the of and of CaM of Liu and S. and S. S. may from a low of the cNOS The control that CaM the insert binding to cNOS; this a affinity for of the insert from iNOS the steric to CaM binding, to the binding of CaM at low levels of Ca2+. of corresponding to the CaM binding on eNOS and iNOS, and of in which the CaM binding sequence of NOS isoform is with the corresponding of have that affinity and of CaM binding is by on NOS in to the CaM binding sequence (6Venema R.C. Sayegh H.S. Kent J.D. Harrison D.G. J. Biol. Chem. 1996; 271: 6435-6440Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 7Ruan J. Xie Q. Hutchinson N. Cho H. Wolfe G.C. Nathan C. J. Biol. Chem. 1996; 271: 22679-22686Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). These have been as the of an CaM binding region on iNOS that binding. by our is that the of the autoinhibitory polypeptide from iNOS to CaM affinity at low Ca2+ that iNOS from an protein by of the are suggested by a inhibition of activity in the of of the eNOS The CaM binding on iNOS and cNOS are related to a region the of CPR, and may have from a region in a data suggest that binding of the peptide may at two site binds the A site sequences as and that in the N-terminal of the eNOS and nNOS peptides that contain sequences inhibit NOS activity and CaM binding. between the first and of the insertion be by the sequences of peptide and with of and eNOS The insert peptide an of and which for in the bovine eNOS that may the affinity of insert peptides for binding on cNOSs and on of NOS activation In this is that muscle an nNOS splice in which the insert peptide is by H. Bredt D.S. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar), providing for specific remain to be The and of the of with the polypeptide on the of the enzyme are not of could the of the FMN CaM, and In a site on the domain of an of could as a binding site for the regions on the polypeptide insert which the conformation of that CaM binding and activation of cNOS is associated with displacement of the is not known the of the polypeptide in conformation electron by the polypeptide with between the and domains or a conformation which not electron could in or by domain electron are by the ability of to and with at an of K. 1992; PubMed Scopus Google a in could a in electron of the peptide may not be the by which CaM binding the conformation of as CaM in of In we have identified a novel control element in cNOSs which as a for the of peptide inhibitors. The of in this element the first of a specific change in NOS and may be a of the of NOS. and D.J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) that the of CaM to control electron is unique to A between cNOS and other proteins is the of a CaM the cNOS cNOS may not be unique in this may the of other CaM systems in which the is through binding domain or rather than for a site. The and of and are responsible for the studies that this to