Calmodulin-dependent Regulation of Inducible and Neuronal Nitric-oxide Synthase
1998; Elsevier BV; Volume: 273; Issue: 42 Linguagem: Inglês
10.1074/jbc.273.42.27430
ISSN1083-351X
Autores Tópico(s)Analytical Chemistry and Sensors
ResumoNeuronal and endothelial nitric-oxide synthases depend upon Ca2+/calmodulin for activation, whereas the activity of the inducible nitric-oxide synthase is Ca2+-independent, presumably due to tightly bound calmodulin. To study these different mechanisms, a series of chimeras derived from neuronal and inducible nitric- oxide synthases were analyzed. Chimeras containing only the oxygenase domain, calmodulin-binding region, or reductase domain of inducible nitric-oxide synthase did not confer significant Ca2+-independent activity. However, each chimera was more sensitive to Ca2+ than the neuronal isoform. The calmodulin-binding region of inducible nitric-oxide synthase with either its oxygenase or reductase domains resulted in significant, but not total, Ca2+-independent activity. Co-immunoprecipitation experiments showed no calmodulin associated with the former chimera in the absence of Ca2+. Trifluoperazine also inhibited this chimera in the absence of Ca2+. The combined interactions of calmodulin bound to inducible nitric-oxide synthase calmodulin-binding region with the oxygenase domain may be weaker than with the reductase domain. Thus, Ca2+-independent activity of inducible nitric-oxide synthase appears to result from the concerted interactions of calmodulin with both the oxygenase and reductase domains in addition to the canonical calmodulin-binding region. The neuronal isoform is not regulated by a unique autoinhibitory element in its reductase domain. Neuronal and endothelial nitric-oxide synthases depend upon Ca2+/calmodulin for activation, whereas the activity of the inducible nitric-oxide synthase is Ca2+-independent, presumably due to tightly bound calmodulin. To study these different mechanisms, a series of chimeras derived from neuronal and inducible nitric- oxide synthases were analyzed. Chimeras containing only the oxygenase domain, calmodulin-binding region, or reductase domain of inducible nitric-oxide synthase did not confer significant Ca2+-independent activity. However, each chimera was more sensitive to Ca2+ than the neuronal isoform. The calmodulin-binding region of inducible nitric-oxide synthase with either its oxygenase or reductase domains resulted in significant, but not total, Ca2+-independent activity. Co-immunoprecipitation experiments showed no calmodulin associated with the former chimera in the absence of Ca2+. Trifluoperazine also inhibited this chimera in the absence of Ca2+. The combined interactions of calmodulin bound to inducible nitric-oxide synthase calmodulin-binding region with the oxygenase domain may be weaker than with the reductase domain. Thus, Ca2+-independent activity of inducible nitric-oxide synthase appears to result from the concerted interactions of calmodulin with both the oxygenase and reductase domains in addition to the canonical calmodulin-binding region. The neuronal isoform is not regulated by a unique autoinhibitory element in its reductase domain. nitric-oxide synthase (6R,6S)-2-amino-4-hydroxy-6-(l-erythro-1,2-dihydroxypropyl)-5,6,7,8-tetrahydropteridine N ω-nitro-l-arginine methyl ester neuronal NOS endothelial NOS inducible NOS trifluoperazine. NO with an unpaired electron reacts with protein targets primarily through their thio or heme groups and acts as a messenger or modulator molecule in many biological systems (1Dinerman J.L. Lowenstein C.J. Snyder S.H. Circ. Res. 1993; 73: 217-222Crossref PubMed Scopus (329) Google Scholar, 2Schmidt H.H.H.W. Lohmann S.M. Walter U. Biochim. Biophys. Acta. 1993; 1178: 153-175Crossref PubMed Scopus (744) Google Scholar, 3Schmidt H.H.H.W. Walter U. Cell. 1994; 78: 919-925Abstract Full Text PDF PubMed Scopus (1495) Google Scholar, 4Sessa W.C. J. Vasc. Res. 1994; 31: 131-143Crossref PubMed Scopus (400) Google Scholar). It is produced froml-arginine with l-citrulline as a co-product in a reaction catalyzed by NOS1that requires NADPH, FAD, FMN, BH4, calmodulin, and heme (1Dinerman J.L. Lowenstein C.J. Snyder S.H. Circ. Res. 1993; 73: 217-222Crossref PubMed Scopus (329) Google Scholar, 2Schmidt H.H.H.W. Lohmann S.M. Walter U. Biochim. Biophys. Acta. 1993; 1178: 153-175Crossref PubMed Scopus (744) Google Scholar, 3Schmidt H.H.H.W. Walter U. Cell. 1994; 78: 919-925Abstract Full Text PDF PubMed Scopus (1495) Google Scholar, 4Sessa W.C. J. Vasc. Res. 1994; 31: 131-143Crossref PubMed Scopus (400) Google Scholar).Three NOS isoforms were originally identified based on the tissue source: nNOS, eNOS, and iNOS (5Marletta M.A. Cell. 1994; 78: 927-930Abstract Full Text PDF PubMed Scopus (808) Google Scholar). All NOSs contain three domains: an oxygenase domain at the N-terminal half and a reductase domain at the C-terminal half connected by a calmodulin-binding region in the middle of the molecule. All isoforms are highly related with at least 50–60% identity and are classified into two categories based on their dependence on Ca2+ for activity. When Ca2+binds to calmodulin, the complex binds to nNOS or eNOS and stimulates nitric oxide production. In contrast, when iNOS expression is induced upon stimulation of cells with cytokines or endotoxins, it is fully active, even when Ca2+ levels in cells are low. The Ca2+-independent activity of iNOS is associated with calmodulin tightly bound to the enzyme (6Cho H.J. Xie Q. Calaycay J. Mumford R.A. Swiderek K.M. Lee T.D. Nathan C. J. Exp. Med. 1992; 176: 599-604Crossref PubMed Scopus (556) Google Scholar).Calmodulin binds proteins through IQ (IQXXXRGXXXR) motifs in a Ca2+-independent manner or through a canonical calmodulin-binding region in a Ca2+-dependent way (7Rhoads A.R. Friedman J.E. FASEB J. 1997; 11: 331-340Crossref PubMed Scopus (734) Google Scholar). However, iNOS does not have a typical IQ motif. Moreover, all of the putative calmodulin-binding regions from nNOS, eNOS, and iNOS satisfy the criteria for properties of a canonical calmodulin-binding region, a basic amphipathic α-helical sequence containing 12 basic and hydrophobic residues (8Vorherr T. James P. Krebs J. Enyedi A. McCormick D.J. Penniston J.T. Carafoli E. Biochemistry. 1990; 29: 355-365Crossref PubMed Scopus (110) Google Scholar). Calmodulin activates nNOS at two points in the electron transfer sequence: electron transfer into the flavins and interdomain electron transfer between the flavins and the hemes (9Abu-Soud H.M. Yoho L.L. Stuehr D.J. J. Biol. Chem. 1994; 269: 32047-32050Abstract Full Text PDF PubMed Google Scholar). The association of calmodulin with nNOS may tether the oxygenase and the reductase domain or some subdomains close together for electron transfer by which calmodulin acts as a molecular switch (10Abu-Soud H.M. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10769-10772Crossref PubMed Scopus (393) Google Scholar).The calmodulin-binding sequence in iNOS is necessary but not sufficient for Ca2+-independent activity (11Ruan J. Xie Q. Hutchinson N.I. Cho H. Wolfe G.C. Nathan C. J. Biol. Chem. 1996; 271: 22679-22686Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 12Venema 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). Replacement of the calmodulin-binding sequence of eNOS or nNOS with the corresponding sequence from iNOS resulted in a chimera that was still Ca2+-dependent. It was proposed that, in addition to the canonical calmodulin-binding region, sites in the reductase domain confer Ca2+-independent binding of calmodulin, which is presumably responsible for the iNOS Ca2+-independent activity. Alternatively, Salerno et al. (13Salerno J.C. Harris D.E. Irizarry K. Patel B. Morales A.J. Smith S.M. Martasek P. Roman L.J. Masters B.S. Jones C.L. Weissman B.A. Lane P. Liu Q. Gross S.S. J. Biol. Chem. 1997; 272: 29769-29777Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar) proposed an autoinhibitory segment in the FMN-binding domains of nNOS and eNOS, but not iNOS. Hence, calmodulin binding may displace this unique autoinhibitory segment, resulting in catalysis. These two models for Ca2+/calmodulin regulation of NOS activities were investigated by the characterization of chimeric enzymes made from nNOS and iNOS.DISCUSSIONSynthetic peptides that bind calmodulin provide important structural information about molecular determinants for specific interactions (31Meador W.E. Means A.R. Quiocho F.A. Science. 1992; 257: 1251-1255Crossref PubMed Scopus (939) Google Scholar, 34Ikura M. Barbato G. Klee C.B. Bax A. Cell Calcium. 1992; 13: 391-400Crossref PubMed Scopus (51) Google Scholar, 35Ikura M. Clore G.M. Gronenborn A.M. Zhu G. Klee C.B. Bax A. Science. 1992; 256: 632-638Crossref PubMed Scopus (1176) Google Scholar, 36Meador W.E. Means A.R. Quiocho F.A. Science. 1993; 262: 1718-1721Crossref PubMed Scopus (611) Google Scholar, 37Vogel H.J. Zhang M. Mol. Cell. Biochem. 1995; 67: 149-150Google Scholar). However, multiple regions of a protein may interact with calmodulin for effective regulation, and calmodulin binding per se is necessary but not sufficient for activation of many enzymes (38Persechini A. McMillan K. Leakey P. J. Biol. Chem. 1994; 269: 16148-16154Abstract Full Text PDF PubMed Google Scholar, 39Su Z. Blazing M.A. Fan D. George S.E. J. Biol. Chem. 1995; 270: 29117-29122Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Moreover, unique calmodulin-binding sequences with atypical structural properties may be overlooked (40Sonnenburg W.K. Seger D. Kwak K.S. Huang J. Charbonneau H. Beavo J.A. J. Biol. Chem. 1995; 270: 30989-31000Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 41Ye L.-H. Hayakawa K. Kishi H. Imamura M. Nakamura A. Okagaki T. Takagi T. Iwata A. Tanaka T. Kohama K. J. Biol. Chem. 1997; 272: 32182-32189Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Previous results obtained on chimeras with the respective calmodulin-binding sequences from iNOS substituted in eNOS or nNOS showed the sequence was necessary but not sufficient for Ca2+-independent activity or calmodulin binding (11Ruan J. Xie Q. Hutchinson N.I. Cho H. Wolfe G.C. Nathan C. J. Biol. Chem. 1996; 271: 22679-22686Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 12Venema 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). Similar results are presented herein with chimera nNOS-I503–533 activity, which was Ca2+-dependent. However, the sensitivity of nNOS-I503–533 activity to [Ca2+] was greater than that obtained with nNOS. When the calmodulin-binding sequence of nNOS was substituted into iNOS, the chimeric iNOS-N725–755activity was still Ca2+-dependent, but its sensitivity to activation by [Ca2+] was also greater than nNOS. These results support the concept that there are sites in addition to the canonical calmodulin-binding sequence that contribute to Ca2+ regulation of NOS activity.As expected, substitution of the iNOS oxygenase or reductase domains alone into nNOS did not result in Ca2+-independent NOS activity, consistent with the view that the iNOS calmodulin-binding region is necessary, but not sufficient, for Ca2+-independent NOS activity (11Ruan J. Xie Q. Hutchinson N.I. Cho H. Wolfe G.C. Nathan C. J. Biol. Chem. 1996; 271: 22679-22686Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 12Venema 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). However, substitution of either iNOS oxygenase or reductase domains increases the Ca2+ sensitivity of the respective chimeric NOS activities with a greater effect observed with the reductase domain (nNOS-I576–1144). Even the nNOS-I749–1144chimera, containing only the C-terminal half of the iNOS reductase domain, was more sensitive to [Ca2+] for activity than nNOS. The functional interaction between iNOS calmodulin-binding sequence and its reductase domain in terms of Ca2+regulation was also recently reported (42Nishida C.R. Ortiz de Montellano P.R. J. Biol. Chem. 1998; 273: 5566-5571Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). However, the functional interaction with the oxygenase domain is unique. These results support the concept that structural elements in addition to the specific calmodulin-binding sequences contribute to Ca2+ regulation of NOS activities.It was recently proposed that an autoinhibitory sequence exists in the reductase domain of nNOS and eNOS (13Salerno J.C. Harris D.E. Irizarry K. Patel B. Morales A.J. Smith S.M. Martasek P. Roman L.J. Masters B.S. Jones C.L. Weissman B.A. Lane P. Liu Q. Gross S.S. J. Biol. Chem. 1997; 272: 29769-29777Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). These Ca2+-dependent NOSs contain unique sequences in their respective FMN-binding domains not shared by iNOS (residues 820–880 in nNOS). Synthetic peptides derived from this insert in eNOS inhibited calmodulin binding and Ca2+ activation by binding to either eNOS or nNOS. However, the insert from nNOS lacks a RRKRK motif thought to be important for autoinhibition in eNOS (13Salerno J.C. Harris D.E. Irizarry K. Patel B. Morales A.J. Smith S.M. Martasek P. Roman L.J. Masters B.S. Jones C.L. Weissman B.A. Lane P. Liu Q. Gross S.S. J. Biol. Chem. 1997; 272: 29769-29777Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). In contrast to results obtained with the synthetic peptides from eNOS, synthetic peptides derived from the putative autoinhibitory region of nNOS provided only modest inhibition of calmodulin binding to nNOS and no significant inhibition of nNOS activity. Our results are also not consistent with a proposed autoinhibitory function for this insert in nNOS. For example, chimera nNOS-I576–1144 contains the reductase domain of iNOS, but its activity is inhibited in the absence of Ca2+. Also, chimeras containing the nNOS reductase domain with iNOS canonical calmodulin-binding region and oxygenase domain (nNOS-I1–533 and nNOS-I1–575) showed significant Ca2+-independent activities (50–73%). Nishida and Ortiz de Montellano (42Nishida C.R. Ortiz de Montellano P.R. J. Biol. Chem. 1998; 273: 5566-5571Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar) recently reported that substitution of the reductase domain of nNOS into iNOS resulted in the retention of significant Ca2+-independent activity. The evidence that the insert in the FMN domain may function as an autoinhibitory element for eNOS is more compelling (13Salerno J.C. Harris D.E. Irizarry K. Patel B. Morales A.J. Smith S.M. Martasek P. Roman L.J. Masters B.S. Jones C.L. Weissman B.A. Lane P. Liu Q. Gross S.S. J. Biol. Chem. 1997; 272: 29769-29777Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar) and may point to a structural and functional distinction between nNOS and eNOS. Differences in the Ca2+ regulation of a class of structurally related, calmodulin-dependent enzymes (skeletal and smooth muscle myosin light chain kinases) have been previously noted (43Gao Z.H. Krebs J. VanBerkum M.F.A. Tang W.-J. Maune J.F. Means A.R. Stull J.T. Beckingham K. J. Biol. Chem. 1993; 268: 20096-20104Abstract Full Text PDF PubMed Google Scholar, 44Persechini A. Gansz K.J. Paresi R.J. Biochemistry. 1996; 35: 224-228Crossref PubMed Scopus (46) Google Scholar).Ca2+-dependent activation of nNOS was associated with calmodulin binding, a finding similar to many previously published results. TFP inhibits the activities of other Ca2+/calmodulin-regulated enzymes, e.g. myosin light chain kinase, phosphodiesterase, calcineurin, etc. (45Ingebritsen T.S. Foulkes J.G. Cohen P. Eur. J. Biochem. 1983; 132: 263-267Crossref PubMed Scopus (109) Google Scholar, 46Stewart A.A. Ingebritsen T.S. Cohen P. Eur. J. Biochem. 1983; 132: 289-295Crossref PubMed Scopus (142) Google Scholar, 47Gupta R.C. Khandelwal R.L. Sulakhe P.V. Mol. Cell. Biochem. 1990; 97: 43-52PubMed Google Scholar, 48Gong C.X. Shaikh S. Grundke-Iqbal I. Iqbal K. Brain Res. 1996; 741: 95-102Crossref PubMed Scopus (45) Google Scholar). Previous reports showed that TFP abolishes nNOS but not iNOS activity, which is confirmed herein (49Bredt D.S. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 682-685Crossref PubMed Scopus (3114) Google Scholar, 50Bredt D.S. Hwang P.M. Glatt C.E. Lowenstein C. Reed R.R. Snyder S.H. Nature. 1991; 351: 714-718Crossref PubMed Scopus (2163) Google Scholar, 51Lyons C.R. Orloff G.J. Cunningham J.M. J. Biol. Chem. 1992; 267: 6370-6374Abstract Full Text PDF PubMed Google Scholar). However, inhibition of nNOS activity by TFP in the presence of Ca2+ is not associated with dissociation of calmodulin. The inactivation of nNOS by TFP may be similar to the inactivation of another calmodulin-dependent enzyme, myosin light chain kinase. TFP does not cause dissociation of calmodulin from the kinase, and inhibition is thought to be due to a distortion of the calmodulin structure necessary for activation (52Newton D.L. Burke Jr., T.R. Rice K.C. Klee C.B. Biochemistry. 1983; 22: 5472-5476Crossref PubMed Scopus (41) Google Scholar,53Cook W.J. Walter L.J. Walter M.R. Biochemistry. 1994; 33: 15259-15265Crossref PubMed Scopus (124) Google Scholar).Two chimeras responded differently to EGTA and TFP in terms of activity and calmodulin binding. In the case of nNOS-I504–1144, EGTA alone or in combination with TFP did not result in dissociation of calmodulin, and there was no effect on enzyme activity. The iNOS segment containing amino acids 484–726 with the calmodulin-binding region and the N-terminal portion of the reductase domain may contribute to Ca2+-independent binding of calmodulin (11Ruan J. Xie Q. Hutchinson N.I. Cho H. Wolfe G.C. Nathan C. J. Biol. Chem. 1996; 271: 22679-22686Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). This high affinity binding of calmodulin may account for one of the mechanisms responsible for Ca2+-independent activity of iNOS. It is conceivable that calmodulin binds to the calmodulin-binding region of iNOS with the N- and C-terminal lobes of calmodulin in close opposition forming a tunnel that engulfs the amphipathic α-helix of the iNOS calmodulin-binding sequence. This proposed model is similar to the known structure of calmodulin bound to a similar calmodulin-binding sequence from myosin light chain kinase (31Meador W.E. Means A.R. Quiocho F.A. Science. 1992; 257: 1251-1255Crossref PubMed Scopus (939) Google Scholar, 35Ikura M. Clore G.M. Gronenborn A.M. Zhu G. Klee C.B. Bax A. Science. 1992; 256: 632-638Crossref PubMed Scopus (1176) Google Scholar, 54Krueger J.K. Olah G.A. Rokop S.E. Zhi G. Stull J.T. Trewhella J. Biochemistry. 1997; 36: 6017-6023Crossref PubMed Scopus (62) Google Scholar). Using a synthetic peptide of the calmodulin-binding sequence from nNOS, Zhang et al. (55Zhang M. Yuan T. Aramini J.M. Vogel H.J. J. Biol. Chem. 1995; 270: 20901-20907Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) showed that the structure of bound calmodulin was similar to that obtained with calmodulin bound to the myosin light chain kinase sequence. Although a similar study has not yet been performed with the calmodulin-binding sequence of iNOS, it is not unreasonable to expect a similar structure. Because calmodulin binding to a synthetic peptide of the calmodulin-binding sequence of iNOS is Ca2+-dependent (12Venema 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, 56Anagli J. Hofmann F. Quadroni M. Vorherr T. Carafoli E. Eur. J. Biochem. 1995; 233: 701-708Crossref PubMed Scopus (41) Google Scholar), additional interactions with the reductase domain may result in Ca2+-independent binding of calmodulin to iNOS. However, nNOS-I504–1144 activity was increased in the presence of Ca2+. Upon binding of Ca2+/calmodulin to nNOS, the oxygenase and reductase domains are tethered to project an effective electron transfer pathway for producing nitric oxide (9Abu-Soud H.M. Yoho L.L. Stuehr D.J. J. Biol. Chem. 1994; 269: 32047-32050Abstract Full Text PDF PubMed Google Scholar, 10Abu-Soud H.M. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10769-10772Crossref PubMed Scopus (393) Google Scholar). Moreover, this tethering also increases the electron transfer rate from NADPH to flavin centers by a factor of 10 and 20 in eNOS and nNOS, respectively (9Abu-Soud H.M. Yoho L.L. Stuehr D.J. J. Biol. Chem. 1994; 269: 32047-32050Abstract Full Text PDF PubMed Google Scholar, 57Chen P.F. Tsai A.L. Berka V. Wu K.K. J. Biol. Chem. 1996; 271: 14631-14635Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Conceivably, the binding of Ca2+ to the calmodulin bound to nNOS-I504–1144 may cause a conformational change sufficient to increase enzyme activity, resulting in a more efficient electron transfer pathway. These results are analogous to the effect of Ca2+ on the activity of myosin light chain kinase in which calmodulin was cross-linked (58Zot H.G. Puett D. J. Biol. Chem. 1989; 264: 15552-15555Abstract Full Text PDF PubMed Google Scholar). In the absence of Ca2+, the kinase had 50% of the activity measured in the presence of Ca2+. Evidence was presented that the association of calmodulin was sufficient to stimulate enzyme activity, and the binding of Ca2+ to calmodulin increased catalytic efficiency.Results obtained with nNOS-I1–533, which contains the calmodulin-binding region and oxygenase domain of iNOS, were different. In the absence of Ca2+, the nNOS-I1–533chimera had 50% of the activity measured in the presence of Ca2+, similar to results obtained with nNOS-I504–1144. However, calmodulin was not co-immunoprecipitated with the enzyme in the absence of Ca2+. One possible explanation for these results is that calmodulin binding is not necessary for activity. Activity was detected in the reconstituted activity of eNOS oxygenase and reductase domains expressed separately in the absence of calmodulin compared with its presence; however, the activity was low (57Chen P.F. Tsai A.L. Berka V. Wu K.K. J. Biol. Chem. 1996; 271: 14631-14635Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). A likely explanation is that calmodulin may bind in the presence of EGTA, but its affinity is lower compared with its binding affinity with iNOS and nNOS-I504–1144. Washing of the immunoprecipitated protein may be sufficient to remove the weakly bound calmodulin. This idea is supported by the ability of TFP to inhibit the activity of nNOS-I1–533 in the presence of EGTA but not Ca2+. When calmodulin is bound to the calmodulin-binding region of iNOS, there may be an additional interaction with the oxygenase domain that is stronger in the presence of Ca2+. However, full Ca2+-independent activity of iNOS requires calmodulin binding to the canonical calmodulin-binding region as well as to both the reductase and oxygenase domains. Additional investigations involving biophysical and structural studies are necessary to verify this model. NO with an unpaired electron reacts with protein targets primarily through their thio or heme groups and acts as a messenger or modulator molecule in many biological systems (1Dinerman J.L. Lowenstein C.J. Snyder S.H. Circ. Res. 1993; 73: 217-222Crossref PubMed Scopus (329) Google Scholar, 2Schmidt H.H.H.W. Lohmann S.M. Walter U. Biochim. Biophys. Acta. 1993; 1178: 153-175Crossref PubMed Scopus (744) Google Scholar, 3Schmidt H.H.H.W. Walter U. Cell. 1994; 78: 919-925Abstract Full Text PDF PubMed Scopus (1495) Google Scholar, 4Sessa W.C. J. Vasc. Res. 1994; 31: 131-143Crossref PubMed Scopus (400) Google Scholar). It is produced froml-arginine with l-citrulline as a co-product in a reaction catalyzed by NOS1that requires NADPH, FAD, FMN, BH4, calmodulin, and heme (1Dinerman J.L. Lowenstein C.J. Snyder S.H. Circ. Res. 1993; 73: 217-222Crossref PubMed Scopus (329) Google Scholar, 2Schmidt H.H.H.W. Lohmann S.M. Walter U. Biochim. Biophys. Acta. 1993; 1178: 153-175Crossref PubMed Scopus (744) Google Scholar, 3Schmidt H.H.H.W. Walter U. Cell. 1994; 78: 919-925Abstract Full Text PDF PubMed Scopus (1495) Google Scholar, 4Sessa W.C. J. Vasc. Res. 1994; 31: 131-143Crossref PubMed Scopus (400) Google Scholar). Three NOS isoforms were originally identified based on the tissue source: nNOS, eNOS, and iNOS (5Marletta M.A. Cell. 1994; 78: 927-930Abstract Full Text PDF PubMed Scopus (808) Google Scholar). All NOSs contain three domains: an oxygenase domain at the N-terminal half and a reductase domain at the C-terminal half connected by a calmodulin-binding region in the middle of the molecule. All isoforms are highly related with at least 50–60% identity and are classified into two categories based on their dependence on Ca2+ for activity. When Ca2+binds to calmodulin, the complex binds to nNOS or eNOS and stimulates nitric oxide production. In contrast, when iNOS expression is induced upon stimulation of cells with cytokines or endotoxins, it is fully active, even when Ca2+ levels in cells are low. The Ca2+-independent activity of iNOS is associated with calmodulin tightly bound to the enzyme (6Cho H.J. Xie Q. Calaycay J. Mumford R.A. Swiderek K.M. Lee T.D. Nathan C. J. Exp. Med. 1992; 176: 599-604Crossref PubMed Scopus (556) Google Scholar). Calmodulin binds proteins through IQ (IQXXXRGXXXR) motifs in a Ca2+-independent manner or through a canonical calmodulin-binding region in a Ca2+-dependent way (7Rhoads A.R. Friedman J.E. FASEB J. 1997; 11: 331-340Crossref PubMed Scopus (734) Google Scholar). However, iNOS does not have a typical IQ motif. Moreover, all of the putative calmodulin-binding regions from nNOS, eNOS, and iNOS satisfy the criteria for properties of a canonical calmodulin-binding region, a basic amphipathic α-helical sequence containing 12 basic and hydrophobic residues (8Vorherr T. James P. Krebs J. Enyedi A. McCormick D.J. Penniston J.T. Carafoli E. Biochemistry. 1990; 29: 355-365Crossref PubMed Scopus (110) Google Scholar). Calmodulin activates nNOS at two points in the electron transfer sequence: electron transfer into the flavins and interdomain electron transfer between the flavins and the hemes (9Abu-Soud H.M. Yoho L.L. Stuehr D.J. J. Biol. Chem. 1994; 269: 32047-32050Abstract Full Text PDF PubMed Google Scholar). The association of calmodulin with nNOS may tether the oxygenase and the reductase domain or some subdomains close together for electron transfer by which calmodulin acts as a molecular switch (10Abu-Soud H.M. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10769-10772Crossref PubMed Scopus (393) Google Scholar). The calmodulin-binding sequence in iNOS is necessary but not sufficient for Ca2+-independent activity (11Ruan J. Xie Q. Hutchinson N.I. Cho H. Wolfe G.C. Nathan C. J. Biol. Chem. 1996; 271: 22679-22686Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 12Venema 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). Replacement of the calmodulin-binding sequence of eNOS or nNOS with the corresponding sequence from iNOS resulted in a chimera that was still Ca2+-dependent. It was proposed that, in addition to the canonical calmodulin-binding region, sites in the reductase domain confer Ca2+-independent binding of calmodulin, which is presumably responsible for the iNOS Ca2+-independent activity. Alternatively, Salerno et al. (13Salerno J.C. Harris D.E. Irizarry K. Patel B. Morales A.J. Smith S.M. Martasek P. Roman L.J. Masters B.S. Jones C.L. Weissman B.A. Lane P. Liu Q. Gross S.S. J. Biol. Chem. 1997; 272: 29769-29777Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar) proposed an autoinhibitory segment in the FMN-binding domains of nNOS and eNOS, but not iNOS. Hence, calmodulin binding may displace this unique autoinhibitory segment, resulting in catalysis. These two models for Ca2+/calmodulin regulation of NOS activities were investigated by the characterization of chimeric enzymes made from nNOS and iNOS. DISCUSSIONSynthetic peptides that bind calmodulin provide important structural information about molecular determinants for specific interactions (31Meador W.E. Means A.R. Quiocho F.A. Science. 1992; 257: 1251-1255Crossref PubMed Scopus (939) Google Scholar, 34Ikura M. Barbato G. Klee C.B. Bax A. Cell Calcium. 1992; 13: 391-400Crossref PubMed Scopus (51) Google Scholar, 35Ikura M. Clore G.M. Gronenborn A.M. Zhu G. Klee C.B. Bax A. Science. 1992; 256: 632-638Crossref PubMed Scopus (1176) Google Scholar, 36Meador W.E. Means A.R. Quiocho F.A. 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