Regulation of Interdomain Interactions by Calmodulin in Inducible Nitric-oxide Synthase*

Nitric-oxide synthases (NOSs) catalyze the conversion of l-arginine to nitric oxide and citrulline. There are three NOS isozymes, each with a different physiological role: neuronal NOS, endothelial NOS, and inducible NOS (iNOS). NOSs consist of an N-terminal oxygenase domain and a C-terminal reductase domain, linked by a calmodulin (CaM)-binding region. CaM is required for NO production, but unlike other NOS isozymes, iNOS binds CaM independently of the exogenous Ca2+ concentration. We have co-expressed CaM and the FMN domain of human iNOS, which includes the CaM-binding region. The Ca2+-bound protein complex (CaCaM·FMN) forms an air-stable semiquinone when reduced with NADPH and reduces cytochrome c when reconstituted with the iNOS FAD/NADPH domain. We have solved the crystal structure of the CaCaM·FMN complex in four different conformations, each with a different relative orientation, between the FMN domain and the bound CaM. The CaM-binding region together with bound CaM forms a hinge, pivots on the conserved Arg536, and regulates electron transfer from FAD to FMN and from FMN to heme by adjusting the relative orientation and distance among the three cofactors. In addition, the relative orientations of the N- and C-terminal lobes of CaM are also different among the four conformations, suggesting that the flexibility between the two halves of CaM also contributes to the fine tuning of the orientation/distance between the redox centers. The data demonstrate a possible mode for precise control of electron transfer by altering the distance and orientation of redox centers in a protein displaying domain movement.

The freely diffusible and moderately reactive free radical, nitric oxide (NO), 2 is a biological signal molecule in numerous physiological and pathophysiological processes (for reviews, see Refs. [1][2][3][4]. Nitric-oxide synthases (NOSs) catalyze the NADPH-dependent conversion of L-arginine to NO and L-citrulline (for reviews, see Refs. 5-7). In mammals, three different isoforms have been identified. Neuronal NOS (nNOS) and endothelial NOS (eNOS) are constitutively expressed, and their activities are Ca 2ϩ /CaM-dependent, whereas the inducible NOS (iNOS) is independent of intracellular Ca 2ϩ concentration. These isoforms share ϳ55% sequence identity yet differ in their size, tissue distribution, and regulation. The 165-kDa nNOS is located in neurons in the brain and neuromuscular junctions and is involved in neurotransmission. eNOS has a molecular mass of 133 kDa, is located in vascular endothelial cells, and is involved in vascular homeostasis. iNOS can be found in macrophages and many other tissues, has a molecular mass of 130 kDa, and is expressed only in response to endotoxins or inflammatory cytokines.
All three isoforms of NOS are modular, homodimeric hemoflavoproteins. The N-terminal half of each NOS isozyme is similar to the cytochrome P450 enzyme family and contains iron protoporphyrin IX (heme). It is referred to as the heme domain or the oxygenase domain. This latter domain also contains tetrahydrobiopterin-and arginine-binding sites. The C-terminal half of each isozyme is the flavin-binding domain (or reductase domain) and contains FAD-, FMN-, and NADPH-binding sites, much the same as in NADPH-cytochrome P450 oxidoreductase (CYPOR). These two domains are linked by a CaM-binding region (8). The constitutive isoforms (nNOS and eNOS) are Ca 2ϩ -dependent due to their reversible binding of CaM, providing a mechanism for rapid response in a signaling cascade. On the other hand, iNOS has tightly bound Ca 2ϩ /CaM and is virtually independent of Ca 2ϩ concentration (9). In contrast to the other NOS isozymes, it is regulated at the transcriptional level. As in the case of the P450 (CYP)-CYPOR system, the FAD in the reductase domain accepts a pair of electrons in the form of a hydride ion from NADPH and transfers them one at a time to FMN. FMN, in turn, transfers the electrons again one by one to the heme of the other monomer in the NOS dimer (10 -12). However, the mechanisms of electron transfer and regulation of the FMN domain interactions with its electron acceptor (the heme domain) in NOS and related enzymes, including CYPOR and methionine synthase reductase, are largely unknown. Only recently, studies on this subject have been emerging (13)(14)(15)(16).
CaM regulates a wide range of cellular functions through its reversible Ca 2ϩ -dependent binding to target proteins, including NOS. CaM regulates NOS activity by controlling the rates of electron transfer between the two flavin cofactors and between FMN and heme (13,(17)(18)(19)(20)(21)(22)(23). The mechanism by which CaM regulates the electron flux in NOS isozymes has been under intensive investigation (19, 24 -26), and recent studies have focused on the movement of the FMN domain and its interactions with the FAD domain and the oxygenase domain in NOS (13,14,16,27) and in other diflavin enzymes (15,28). In addition, there are three elements in the reductase domain of constitutive NOSs that are influenced by the CaM binding: the autoregulatory region (AR, a ϳ40 residue insert in the FMN domain), the C-terminal extension, and, to a lesser extent, the ␤-finger (or SI, a small insert in the FAD/connecting domain) (13, 29 -31), all of which are lacking in iNOS.
Although the crystal structure of a complete NOS holoenzyme has not been obtained, structures of various individual domains of the NOS proteins have been determined, including the oxygenase domains of all three NOS isozymes (32)(33)(34)(35) and the isolated FAD/NADPH domain (36) and the reductase domain of nNOS (37). In addition, the structure of CaM bound to a 20-residue peptide corresponding to the eNOS CaM-binding region has been determined (38), as has the structure of CaM bound to a 23-residue peptide corresponding to the nNOS CaM-binding region (Protein Data Bank code 2O60). However, no structural information is available regarding domain-domain interactions of Ca 2ϩ /CaM with either the FMN domain or the heme domain.
Here we report the crystal structure of a complex between Ca 2ϩ /CaM and the FMN domain of human iNOS, including the CaM-binding region (hereafter referred to simply as iNOS CaCaM⅐FMN). The complex has been crystallized in four different conformations, demonstrating the flexible nature of domain-domain interactions between Ca 2ϩ /CaM and the FMN domain. In addition, the structure shows detailed interactions between Ca 2ϩ /CaM and the CaM-binding region of iNOS, revealing a structural basis for the irreversible tight binding of Ca 2ϩ /CaM to iNOS. Finally, we propose a model of Ca 2ϩ /CaM regulation of electron transfer flux in iNOS and constitutive NOSs by combining the structure of iNOS CaCaM⅐FMN with known structural information of the NOS individual domains and related diflavin enzymes.

EXPERIMENTAL PROCEDURES
Construction of Expression Plasmids-The plasmids for expression of the recombinant human FAD/NADPH (referred to as the FAD domain), CaM⅐FMN, and CaM⅐FMN/FAD/ NADPH (CaM⅐FMN-FAD) domains were constructed as described by Yamamoto et al. (39). The full reductase domain consists of residues from Glu 504 to Leu 1153 , the CaM⅐FMN domain contains Glu 504 -Ser 715 , and the FAD domain contains Arg 687 -Leu 1153 . With the exception of the FAD domain, all proteins were co-expressed with human CaM as previously described (39). These constructs utilized the pCWori ϩ vector and supported expression of untagged proteins. To facilitate purification of the CaM⅐FMN domain, a His 6 tag was attached to the N terminus of the protein by replacing the vector sequences with those of pCWeNOS (40) (a gift from Dr. Otiz de Montellano) that contained an N-terminal His tag for expression of human eNOS, since both plasmids utilized NdeI and XbaI sites to insert the respective NOS proteins. The full-length human iNOS reductase domain (CaM⅐FMN-FAD) and both the tagged and untagged CaM⅐FMN proteins were co-expressed with human CaM (pKK-CaM) in Escherichia coli BL21 cells as previously described (39). In all cases, the cells were grown in a modified 2ϫ YT medium containing 50 g/ml ampicillin and 10 g/ml tetracycline. Protein expression was induced at an A 600 of 0.8 with 0.6 mM isopropyl 1-thio-␤-Dgalactopyranoside. After supplementing the growth medium with 0.5 g/ml riboflavin, the bacterial culture was allowed to grow overnight at 28°C. The cells were harvested by centrifugation and stored at Ϫ80°C until needed.
Protein Purification-The human iNOS CaM⅐FMN-FAD complex, the FAD domain, and untagged CaM⅐FMN complex were purified as described previously (39). The His-tagged Ca 2ϩ /CaM⅐FMN complex was purified using Ni 2ϩ -nitrilotriacetic acid-agarose chromatography. Briefly, the cell pellet was resuspended in a solution containing 50 mM Tris-HCl, pH 7.4, 2 mM calcium chloride, 5% glycerol, 1 g/ml deoxyribonuclease, 1 g/ml ribonuclease, 0.25 g/ml leupeptin, 1 g/ml aprotinin, 5 g/ml lysozyme, and 5 mM phenylmethylsulfonyl fluoride and lysed by sonication. Clear supernatant was obtained from the homogenate by centrifugation at 100,000 ϫ g and loaded onto a Ni 2ϩ -nitrilotriacetic acid-agarose column equilibrated with 50 mM Tris-HCl, pH 7.4, 5 mM imidazole, and 5% glycerol. The column was washed with the equilibration buffer followed by elution with a gradient of 5-100 mM imidazole in the same buffer. Enzyme was pooled according to purity on SDS-PAGE. Pure fractions were dialyzed against 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, and glycerol was added to a final concentration of 20% before freezing at Ϫ80°C. About 15 mg of the CaM⅐FMN complex was obtained from 10 g of bacterial cell pellet. All three proteins were purified as complexes with Ca 2ϩ /CaM.
Reconstitution Experiment and Activity Measurements-The spectra of the reconstituted system were obtained with 14 M iNOS CaCaM⅐FMN, 0.28 M iNOS FAD domain in 50 mM Hepes buffer, pH 7.0, 500 M CaCl 2 before and 400 s after the addition of 100 M NADPH. The enzyme concentrations were determined optically from absorbance at ⑀ 457 ϭ 10.2 mM Ϫ1 cm Ϫ1 for the iNOS FAD protein, ⑀ 457 nm ϭ 11.2 mM Ϫ1 cm Ϫ1 for iNOS CaCaM⅐FMN complex, and ⑀ 457 nm ϭ 22 mM Ϫ1 cm Ϫ1 for the iNOS CaCaM⅐FMN-FAD protein (39). All spectral analyses and activity measurements were performed with the untagged CaCaM⅐FMN complex protein, unless stated otherwise. Cytochrome c reductase activities were measured in 50 mM Hepes buffer, pH 7.0, 500 M CaCl 2 , 20 M cytochrome c, and 100 M NADPH at 25°C. 0.25 M iNOS FAD/NADPH was mixed with different concentrations of iNOS CaCaM⅐FMN ranging from 0.125 to 1.5 M. The reaction was monitored by an increase in absorbance at 550 nm using an extinction coefficient difference of 21 mM Ϫ1 cm Ϫ1 between the reduced and oxidized forms. Activity was plotted against ratio of the iNOS CaCaM⅐FMN protein (iFMN) to the FAD/NADPH domain.
Crystallization and Data Collection of the iNOS CaCaM⅐ FMN Complex-Purified His-tagged CaCaM-bound CaM⅐ FMN complex was dialyzed against 50 mM Hepes buffer, pH 7.6, and 0.1 M NaCl and concentrated to 15 mg/ml, and dithiothreitol was added to a final concentration of 5 mM. Crystals were grown by vapor diffusion using the hanging drop method, by mixing a 1:1 ratio of the enzyme and the precipitant solution (18% polyethylene glycol 8000, 0.1 M sodium acetate, pH 5.0, and 0.5 M NaCl) and equilibrating the protein drop against the precipitant solution at 19°C. Thin yellow plate-shaped crystals appeared within 2-3 days and grew to their maximum size over a week. Diffraction data were collected at 100 K using an inhouse R-AXIS IV ϩϩ equipped with a MicroMax 007 generator. The cryoprotectant solution was composed of the precipitant solution with an additional 7% polyethylene glycol 8000 and 5% glycerol. Crystals were in the P2 1 space group with cell dimensions of a ϭ 36.6 Å, b ϭ 160.8 Å, c ϭ 127.8 Å, and ␤ ϭ 90.4°and contained four complex molecules in an asymmetric unit.
Structure Determination-The structure was determined by the molecular replacement method using the Phaser program (41). The FMN domain of rat nNOS reductase structure (37) (Protein Data Bank code 1TLL) was used as the search model for the FMN domain, whereas two CaM structures (Protein Data Bank code 1NIW (38) and Protein Data Bank code 2FOT (42)) were used as the search model for the CaM domain. Initially, the four FMN domains were sequentially located by Phaser. For the subsequent search for the four CaM molecules, different CaM models were tested, and five independent solutions were obtained. From the inspection of the electron density maps, these solutions belonged to three N-terminal and two C-terminal lobes, together completing two CaMs and the N-terminal lobe of the third CaM molecule. However, the fourth CaM and the C-terminal lobe of the third CaM could not be located. These partial solutions were combined, and additional rigid body refinements were carried out, yielding an R factor of 50.7%. Further search with Phaser resulted in locating all but the C-terminal lobe of the last CaM molecule. At this stage, the resulting difference Fourier map clearly showed most of the ␣-helices of the C-terminal lobe. Furthermore, these four CaM molecules appeared to form two pairs, each pair having a similar conformation. Therefore, the C-terminal lobe of the last CaM was manually built to fit the F o Ϫ F c densities using the conformation of its partner CaM molecule. At this stage, further rigid body refinements with CNS (43) yielded R crystal of 33.3% and R free of 39.4% (5% of the data were used for the R free calculation). Full refinements were performed with CNS alternating with manual model rebuilding and adjustments using the program COOT (44). The final R values are 23.7% for R crystal and 31.0% for R free . Statistics for data collection and structure refinement are summarized in Table 1.
Modeling of the Entire iNOS Reductase Structure and Docking of the Oxygenase Domain and the Reductase Domain-The closed conformation of the iNOS reductase domain was generated by superimposing the FMN domain of the iNOS CaCaM⅐FMN structure onto the structure of the rat nNOS reductase domain, which lacks the CaM-binding region (Protein Data Bank code 1TLL (37). The iNOS closed form structure was generated by simply deleting the autoregulatory helix and modifying the long ␤-finger of the rat nNOS structure after the corresponding loop of the rat CYPOR structure (45). The C-terminal tail structure was not modified, since the nNOS structure has a tail four residues shorter than the iNOS tail. A putative open conformation of the structure of the iNOS reductase domain was generated by superimposing the FMN domain of the iNOS CaCaM⅐FMN structure onto the FMN domain of the open conformation observed for the hinge-shortened mutant CYPOR structure (15), Mol A (the least open form of Protein Data Bank code 3ES9). Docking of the two halves of the iNOS molecule was performed using GRAMM-X (46). The open conformation of the entire reductase domain (CaCaM⅐FMN-FAD) was assigned as the ligand molecule, and the oxygenase domain dimer structure (Protein Data Bank code 4NOS (32)) was used as the receptor. Any four of 26 conserved positive surface residues of the oxygenase domain dimer (13 residues/monomer) and any two of 11 conserved negative surface residues on the FMN domain were required to be at the interface of the docked molecule. The top 10 solutions were inspected. The criteria for choosing a suitable solution were as follows. 1) There should be no overlap when the second reductase domain corresponding to the second monomer is generated using the molecular 2-fold symmetry of the oxygenase domain dimer. This is because, although only one reductase domain was used to dock the oxygenase domain dimer, the entire iNOS molecule is a dimer of both the reductase and oxygenase (heme) domains. 2) The distance between Gln 502 of the oxygenase domain and Ile 513 of the CaM-binding region (i.e. the 11-residue linker between the two halves in each monomer) should be less than ϳ35 Å, even if the linker adopts a completely stretched polypeptide conformation, which is unlikely. Only one solution satisfied these conditions. The same solution was obtained when the number of surface charged residues on the ligand molecule used for the boundary conditions was increased from 11 to 27 residues (by adding 16 negative residues of CaM, allowing the CaM surface to interact with the oxygenase domain) or from 11 to 34 residues (i.e. 11 from the FMN domain, 16 from CaM, and 7 from the FAD domain),

Structure of a Complex between Human iNOS and CaM
allowing both CaM and the FAD domain to contact the oxygenase domain.

FMN of the Recombinant iNOS CaCaM⅐FMN Forms Air-stable Semiquinone-
The visible absorption spectra of the CaMbound CaM⅐FMN protein and its semiquinone form were analyzed (supplemental Fig. S1). The spectrum of the oxidized form has the characteristic flavin peaks at 385 and 457 nm. In order to test if iNOS CaCaM⅐FMN was functional, a reconstitution experiment was performed in which the untagged iNOS CaCaM⅐FMN protein was reconstituted with the iNOS NADPH/FAD domain in a 50:1 ratio, and then an excess of NADPH was added. Supplemental Fig. S1 shows the formation of a broad peak at 596 nm, which is characteristic of the FMN neutral semiquinone, an intermediate in the iNOS-catalyzed reaction. Since the concentration of the FAD domain is very low compared with the CaCaM⅐FMN protein, any increase in absorbance that is due to FAD will not contribute to the spectrum of the reconstituted system. Therefore, the peak at 596 nm is almost solely due to the neutral FMN semiquinone. In the absence of the iNOS FAD/NADPH domain, the spectrum of the FMN domain did not change upon the addition of NADPH. This indicates that, as in the full-length reductase domain, electrons are transferred from NADPH to the FMN domain via the FAD domain, although the two domains are not covalently linked.
The Isolated iNOS CaCaM⅐FMN Protein Is Capable of Reducing Cytochrome c When Reconstituted with the FAD/NADPH Domain-The efficiency of electron transfer between the two separate domains has been analyzed (supplemental Fig. S2). The cytochrome c reductase activity in the reconstitution system, iNOS CaCaM⅐FMN plus iNOS FAD/NADPH, was compared with that of the intact iNOS reductase, CaCaM⅐FMN-FAD. The cytochrome c reductase activity in the reconstituted system was 4.9 M/min, with equimolar concentrations of 0.25 M each protein. The corresponding value for the intact CaCaM⅐FMN-FAD domain was 265.0 M/min. These data indicate that the cytochrome c reductase activity of the reconstituted system is 1.8% of the full-length iNOS reductase. This value is in agreement with the results of a similar study carried out with human CYPOR. In that study, reconstitution of the separate FMN and FAD/NADPH domains of human CYPOR gave 1.6% of the wild-type activity in the reduction of cytochrome c (47). The rate-limiting step in the reconstituted system is the electron transfer between the FAD and FMN domains (39) Therefore, this observation indicates that the rate of electron transfer between the separated FAD and FMN domains is much less effective than the rate of intramolecular electron transfer in the full-length iNOS reductase protein. The cytochrome c reductase activity of the reconstituted system also increased with an increase in the concentration of the CaCaM⅐FMN protein (supplemental Fig. S2), indicating that the cytochrome c reduction in the reconstituted system is indeed dependent upon bimolecular collision between the two proteins. Taken together, the isolated iNOS CaCaM⅐FMN domain is fully functional and is capable of accepting electrons from NADPH via the FAD domain and donating them to cytochrome c.
The Overall Structure of CaCaM⅐FMN Adopts an Open Conformation-The structure of the complex between Ca 2ϩ / CaM and the FMN domain of iNOS (which includes the CaMbinding region) shows two completely separate domains, one for the FMN domain and the other for the CaM domain (Fig. 1,  A and B). The CaM-binding region of the iNOS polypeptide (residues Leu 515 -Ser 535 ) forms a tight ␣-helix (Fig. 1C), and the CaM molecule with bound Ca 2ϩ (hereafter CaM refers to Ca 2ϩ -bound CaM, unless otherwise stated) wraps around the helix (Fig. 1A). There are only a few van der Waal's interactions between the two domains (total contact area ϳ200 Å 2 ). The individual domains in the four molecules found in the asymmetric unit are essentially identical, with r.m.s. deviations ranging from 0.17 to 0.25 Å for the FMN domain and from 0.37 to 0.83 Å for the CaM domain. Although the iNOS FMN domain that was used for crystallization was constructed and expressed as a His 6 -tagged polypeptide containing residues 504 -715 of human iNOS, in all four molecules, the N terminus starts from residue 511 or 512, and the C terminus ends at residue 698 or 699, depending on the individual molecule in the asymmetric unit. Thus, the first 13-15 residues (including the His 6 tag) and C-terminal 16 -17 residues are not visible in the final structure, suggesting that both termini are very flexible. This is not surprising, since these termini are parts of the flexible linkers that are present in the oxygenase domain (N terminus) and the NADPH/FAD/connecting domain (C terminus). It is also possible that the N-terminal 14 or 16 residues might have been cleaved by proteolysis during the crystallization process, since there are many tryptic sites within the first 8 residues (Lys 505 -Arg 511 ). No attempt to sequence the crystalline protein was  OCTOBER 30, 2009 • VOLUME 284 • NUMBER 44

JOURNAL OF BIOLOGICAL CHEMISTRY 30711
made. For the CaM molecule, all but the first two N-terminal residues and the last residue at the C terminus were observed in three molecules. In molecule 4, however, parts of helices VII and VIII and the loop between them had high temperature factors, and therefore, several side chains (Glu 119 , Arg 126 , Ile 130 , Glu 139 , and Val 142 ) were not included in the final refinement.
The structure of the FMN domain is essentially identical to the structures of the corresponding domains of rat CYPOR (Protein Data Bank code 1AMO (45)) and the reductase domain of rat nNOS (Protein Data Bank code 1TLL (37)), although the sequence identities are 50 -60% with r.m.s. deviation values of 1.20 and 0.97 Å for a total of 148 C␣ atoms, respectively. As for the structure of CaM, four Ca 2ϩ ions are tightly bound to each CaM, although the protein was dialyzed extensively against buffer that did not contain any added Ca 2ϩ ions, and the crystallization medium did not contain any exogenous Ca 2ϩ ions. However, the protein had been exposed to Ca 2ϩ ions during the purification procedure, and no extra procedure to eliminate tightly bound Ca 2ϩ (e.g. an EGTA treatment) was performed after the purification of the protein. This is consistent with the fact that iNOS binds CaM independently of Ca 2ϩ concentration (9). Each of the four Ca 2ϩ ions in the structure is coordinated to three carboxyl groups of Asp or Glu, one Asn or Gln (the amide oxygen), and one Thr or Tyr (mostly the carbonyl oxygen of the main chain). The sixth ligand is a water molecule. This arrangement is consistent with the structures of CaM found in other complexes of Ca 2ϩ /CaM and its recognition peptides, such as in eNOS-CaM (38) and myosin light chain kinase (48).
What would be the structure of iNOS bound to the Ca 2ϩ -free form of CaM, although it is an unlikely situation in the normal physiological condition? It has been shown by various biophysical methods that CaM binds to a 20-residue peptide derived from human iNOS (circular dichroism and proton NMR methods (49)) and the N-terminal 70-residue truncated human iNOS (fluorescence resonance energy transfer method (50)), virtually independently of Ca 2ϩ concentration. Therefore, it is most likely that the structure of iNOS bound to Ca 2ϩ -free CaM will also adopt the same conformation as iNOS bound to the Ca 2ϩ -bound CaM. However, in the absence of Ca 2ϩ or in extremely low concentrations, it is entirely possible that the loops between helices of CaM that coordinate Ca 2ϩ ions might adopt conformations different from and less tight than those observed in the current structure.
Arg 536 Forms a Flexible Hinge between the Two Domains-Arg 536 of iNOS is located at the end of the CaM-binding region and forms the junction between this region and the FMN domain of the iNOS polypeptide. It is the only residue that interacts with both the CaM molecule and the FMN domain (Fig. 1B). On the CaM side, this amino acid makes a salt bridge with Glu 47 and a hydrogen bond with the main chain carbonyl oxygen of Asn 42 for all four molecules in the asymmetric unit. With the FMN domain, Arg 536 makes hydrogen bonds with the main chain carbonyl oxygens of the loop containing Ser 562 , Cys 563 , Ala 564 , and Phe 565 , although the number of hydrogen bonds and their distances are slightly different in the four molecules, suggesting that the hinge is flexible. Arg 536 is highly conserved in all known NOS proteins that have been sequenced thus so far.
Although the individual domains of the complex in the four observed structures are the same, their relative dispositions are different. Fig. 2 shows an overlay of all four structures (superimposing only their FMN domains) that clearly indicates the flexibility of the hinge. There are essentially two distinctive sets of conformations (Mol A (red) and Mol D (purple), and Mol B (blue) and Mol C (green)). The difference between the two most different conformers is a ϳ10°rotation of the CaM-binding peptide (residues Leu 515 -Ser 535 ) along with its bound CaM pivoting on the Arg 536 /Glu 47 (CaM) pair (Figs. 1B and 2, A and B). This rotation results in a large swing of the CaM domain, shifting the Leu 515 C␣ atom by as much as 8 Å. The rotational movement of the CaM-binding peptide along with the bound CaM contributes to the overall movement of the domains that is necessary for the efficient electron transfer among different redox centers in iNOS and most likely in other NOS isozymes as well.
The Two Lobes of CaM Also Flex-Although all four structures of CaM are essentially the same (overall r.m.s. deviation ranges from 0.6 to 0.9 Å), the relative orientation of the two lobes within each CaM molecule is slightly different in all four structures (Fig. 2B). When the four C-lobes of CaM are superimposed, the four N-lobes have r.m.s. deviation values ranging Interactions between the iNOS CaM-binding Peptide and CaM-CaM is bound to the CaM-binding peptide in a classical antiparallel manner as observed in other CaM-peptide complexes, including the complexes of CaM⅐eNOS peptide (38), CaM⅐myosin light-chain kinase peptide (48), and CaM⅐␣II spectrin (42). The CaM-binding peptide of iNOS forms a helix (residues Leu 515 -Ser 535 ), and CaM forms two lobes (the N-terminal lobe (residues Gly 3 -Asp 78 ) and the C-terminal lobe (Thr 79 -Ala 147 )) with the N-terminal side of the helix bound to the C-lobe and the C-terminal half of the peptide wrapped within the N-terminal lobe of CaM (i.e. an antiparallel binding) (Figs. 3 and 4). As in other structures of the CaM⅐peptide complexes, including that of eNOS peptide⅐CaM (38), the interactions between the iNOS peptide and the core of CaM are mainly hydrophobic in nature, satisfying the classical 1-5-8-14 subclass motif of CaM binding (51) (Fig. 3). The 1-5-8-14 residues, Leu/Phe 515 , Val/Ala 519 , Val 522 , and Leu 528 , interact with hydrophobic residues in helix VI, helix VIII, helix V, and helices II/III, respectively, that line the inner cavity of the CaM molecule (Fig. 3, A and B). However, there are a few distinct differences between the structures of CaM bound to the iNOS peptide and CaM bound to the eNOS peptide.
Why Does CaM Bind More Tightly to iNOS than to eNOS or nNOS?-Overlays of the structure of iNOS-CaCaM⅐FMN on that of eNOS peptide⅐CaM or nNOS peptide⅐CaM reveal that the relative orientations of the two lobes of CaM are different in all three structures (Fig. 4). When the two structures of iNOS CaCaM⅐FMN and eNOS peptide⅐CaM are aligned by superimposing the two CaM-binding peptides, the C-lobes align more closely (r.m.s. deviation of 2.0 Å) than their N-lobes (r.m.s. deviation of 7.5 Å), indicating that although the interactions between the peptide and the C-lobe are similar in the two structures, the interactions with the N-lobe are very different. Arg 530 is conserved in all known iNOS sequences and makes salt bridges with Asp 80 , Glu 84 , and Glu 87 , all lying at the inner face of helix V of the CaM C-lobe (Fig. 3, A  and B). The corresponding residue in both nNOS and eNOS is Gly (Fig.  3C), which lacks charged interactions with CaM. Fig. 4 shows a comparison of the structures of iNOS CaCaM⅐FMN with eNOS or nNOS peptide⅐CaM. Leu 523 and Met 527 (Val 527 in rat and Leu 527 in pig) of iNOS, both of which are conserved hydrophobic residues in various iNOSs, lie on the same side of the iNOS peptide helix and are surrounded by the hydrophobic residues of CaM, including Met 71 , Met 72 , and Met 76 of helix IV in the N-lobe and Met 145 in the C-lobe (Fig. 3B). The corresponding residues in eNOS or nNOS are hydrophilic residues (Lys for Leu 523 and Ser or Lys for Met 527 ; Fig. 3C). These residues are exposed to solvent in the structure of CaM when bound to the eNOS peptide (38) or to the nNOS peptide (Protein Data Bank code 2O60). The tight hydrophobic interactions involving Leu 523 and Met 527 of iNOS are largely responsible for pulling back helix IV of CaM, which in turn pulls the entire N-lobe of CaM closer to the N-terminal side of the peptide in the iNOS structure (Fig. 4A). In addition, Lys 520 and Lys 531 of iNOS make salt bridges with Glu 11 and Glu 54 , respectively, of CaM. In the structure of the eNOS peptide⅐CaM complex, the amide nitro-  OCTOBER 30, 2009 • VOLUME 284 • NUMBER 44 gen of the corresponding residue, Asn 501 , makes a hydrogen bond with Glu 11 of CaM, which is a weaker interaction.

Structure of a Complex between Human iNOS and CaM
On the other hand, when the iNOS-CaCaM⅐FMN structure is compared with the nNOS peptide structure, the r.m.s. differences between the C␣ atoms of the two structures are 3.6 Å for the N-lobe and 2.4 Å for the C-lobe. The CaM molecule in the nNOS peptide⅐CaM structure adopts a more subtle strategy for its Lys 744 and Lys 748 (human nNOS numbering, homologs of Leu 523 and Met 527 of iNOS, respectively) to avoid contact with the hydrophobic residues of CaM (Met 71 , Met 72 , Met 76 , and Met 145 ) by unwinding one turn of helix V, pushing helix IV away from the nNOS peptide (Fig. 4B).
The stronger interaction between the iNOS peptide and CaM is consistent with the results of various biochemical studies of CaM binding to the NOS isozymes. Venema and co-workers (52,53) showed that the CaM-binding peptides of bovine eNOS and murine iNOS have dissociation constants for CaM binding of 4.0 and 1.5 nM, respectively. These authors also showed that residues 524 -532 of iNOS are necessary for high affinity to CaM, which is consistent with our observations regarding the residues Leu 523 and Met 527 . Censarek et al. (54) studied the thermodynamic parameters for the binding of CaM to the CaM-binding peptide of nNOS in the presence and absence of Ca 2ϩ . Their studies show that the murine nNOS peptide E736K mutant (equivalent position to Lys 520 of iNOS) binds to CaM in the presence of EGTA with a K d of 1.4 M (20-fold higher than the wild type peptide in the presence of Ca 2ϩ ). They also observed that a replacement of the hydrophobic residues of the iNOS CaM-binding peptide with charged residues significantly decreased the affinity of the peptide to CaM (55). Spratt et al. (56) showed that, for iNOS, the N-lobe of CaM alone stimulated ϳ75% of wild type CaM in NO production, whereas the C-lobe alone had 38% of the wild type activity. Thus, the tighter binding of CaM to iNOS comes largely from the N-lobe binding to iNOS.
The Structure of the iNOS Reductase Domain in the Closed and Open Conformations- Fig. 5 shows a modeled structure of the iNOS reductase domain, including the bound CaM, in the closed and open conformations. iNOS does not have the AR (a ϳ40-residue insertion in the FMN domain) that is present in the constitutive NOSs and has a shorter C-terminal tail compared with the other two NOSs (tail lengths of 21, 33, and 42 residues for iNOS, nNOS, and eNOS, respectively). iNOS also has a shorter loop in place of the long ␤-finger (or SI, for small insertion found in the connecting domain of all NOSs compared with CYPOR) that exists in nNOS and eNOS. Therefore, the structure of iNOS reductase domain should be more similar to the CYPOR structure (Fig. 5A) than to the nNOS reductase domain. When the iNOS loop was modeled after the CYPOR loop, the short iNOS loop did not interfere with CaM binding (Fig. 5B, left). As in the CYPOR structure, when the iNOS reductase domain is in the closed conformation (Fig. 5B, left), the two flavin cofactors are juxtaposed at their respective xylene ring side of the flavin ring (C7-methyl to C8-methyl distance ϳ4 Å). However, in the cases of eNOS and nNOS, the SI has to move slightly when CaM binds to the enzyme, and/or the CaM-binding helix together with the bound CaM must bend back slightly at the Arg hinge (Arg 517 in human eNOS and Arg 757 in human nNOS), so that there is no steric hindrance between the bound CaM and SI. It is possible that this bending of the SI triggers movements of the AR and the long C-terminal tail, both of which are present in eNOS and nNOS, releasing the locked conformation of the reductase domain, as previously suggested (13,31,36,57).
In this unlocked conformation, AR and the long C-terminal tail of nNOS (and eNOS) are removed from their inhibitory (locked) positions, as seen in the structure of the nNOS reductase domain without the bound CaM (37) (also see Fig. 5A). It has been shown that in CYPOR, the indole ring of the penultimate residue Trp 676 has to move away from the re-face of the FAD isoalloxazine ring, when the nicotinamide ring of NADPH binds to CYPOR (58). Similarly, it is most likely that the aromatic side chain of the Phe that is found at the corresponding positions in all NOSs would have to move away when NADPH binds the FAD in NOSs and then move back when NADP ϩ is released. The extra tail found in NOS hampers this movement, which in turn decreases the rate of hydride transfer from NADPH to FAD and electron transfer from FAD to FMN, as has been shown in the tail-truncated NOS mutants (25,59) and CYPOR mutants with the NOS C-terminal tail (60). In fact, the 42 residue-long eNOS C-terminal tail most likely contributes to eNOS having the weakest activity among three NOSs. One other factor contributing to the slow activity of eNOS has been shown to be the linker between the two flavin domains (61).
For the open conformation, we modeled the entire iNOS reductase structure with the bound CaM, based on the structure of a mutant CYPOR, in which four residues of the hinge between the FMN domain and the FAD domain were deleted (15) (Protein Data Bank code 3ES9). The resulting structure is shown in Fig. 5B (right). There are very few contact surfaces among the three domains, CaM, the FMN domain, and the FAD domain, suggesting that all three domains are flexing from each other. The oxygenase domain dimer also forms a discreet module separated by the 10-residue linker from the CaM-binding domain (Fig. 5B). Thus, each of the four domains of the iNOS molecule forms a discreet module, and the four modules are connected to each other by flexible linkers. The linkers between the heme domain and CaM-binding region and between the FMN and FAD domains in iNOS are 10 and 24 residues, respectively. The connection between the bound CaM and the FMN domain is the hinge, which pivots at Arg 536 (Arg 517 in human eNOS and Arg 757 in human nNOS).
In the constitutive NOSs, when CaM is not bound to the CaM-binding region, the CaM-binding helix becomes unstructured and forms part of the linker between the two halves (the oxygenase domain and reductase domain), making this linker extremely long (Ͼ40 residues). Then the interaction between the two domains becomes inefficient, behaving almost bimolecular rather than unimolecular and resulting in inefficient electron transfer from FMN to heme and, consequently, poor/ negligible NO production in the absence of CaM. From our current composite structure of the iNOS reductase domain, including the bound CaM, it is unlikely that there would be any direct interactions between CaM and the C-terminal tail in iNOS. Roman and Masters (13) suggested that in nNOS, there would be direct interactions between the AR and C-terminal tail and between AR and CaM (13). Considering that the C-terminal tail is much longer in nNOS, it is entirely possible to have a direct interaction between the AR and the tail in nNOS in the closed conformation. However, it is not as obvious that there would be any direct interaction between CaM and AR in the open conformation of nNOS. A definitive answer must await the structure of a CaM-bound form of the nNOS or the eNOS reductase domain.
Model of the Entire Holo iNOS Dimer in the Conformation Enabling the Transfer of Electrons from FMN to Heme-The entire iNOS holo dimer was constructed by docking the oxygenase domain dimer onto the open conformation of the reductase domain. From the in silico docking using GRAMM-X, the only solution that satisfies the two conditions (that there be a 2-fold symmetry for the entire dimeric molecule and that the distance for the heme-CaM linker in each monomer be Ͻ35 Å) is shown in Fig. 5C. The final solution has a linker distance of 19 Å, a reasonable distance for an 11-residue linker between the oxygenase and CaM domains. Interestingly, the FMN domain of one monomer interacts with the oxygenase domain of the other monomer, although this was not imposed as a docking condition. This intersubunit electron transfer arrangement is consistent with previously observed biochemical studies revealing the role of CaM in electron transfer between the two subunits in the dimeric NOS molecule (11,62).
The contact area between the FMN domain and the oxygenase domain of the other monomer is 510 Å 2 . The contact area between the FMN domain and the oxygenase domain in the same monomer is 215 Å 2 . Together, these areas are about 10% of the total surface of the FMN domain of 7400 Å 2 , considerably less than the corresponding contact area with the FAD domain found in the closed form (960 Å 2 , 13% of the total). Ilagan et al. (16) have proposed four docking models for nNOS in which the contact areas between the two domains (the oxygenase and FMN domains) range between 400 and 900 Å 2 . Whether any one of the Ilagan models is similar to our model is not clear. In our current model, which is the only NOS docking model so far based on the entire iNOS structure (albeit a model structure) rather than based on only the oxygenase and FMN domains, there are no appreciable contact areas between the oxygenase domain and other parts of the reductase domain. In our model, as in the Ilagan models, the interactions between the two domains are largely electrostatic, with the oxygenase domain being positive and the FMN domain negative (Fig. 5D). In this docked model, the closest distance between FMN and heme (the C7 methyl group of FMN and the vinyl group of the heme B ring) is ϳ12 Å, similar to the distance found between FMN and heme in the docked structure of the CYPOR-P450 2B4 complex (15). Interestingly, Trp 372 of the oxygenase domain lies between the two cofactors, suggesting that it acts as an electron conduit between FMN and heme.
In conclusion, the data presented here provide new structural information on NOS, namely the linkage between the oxygenase domain and the reductase domain, supporting the modular nature and flexibility between different modules of NOS isozymes. The data also provide insight into how different modules interact with each other during catalysis. By combining the structure of the human iNOS FMN domain in complex with CaM with known structures of the rat nNOS reductase domain (36,37) and the open form structure of rat CYPOR (15), we have constructed a model of the entire iNOS reductase domain together with bound CaM for both open and closed states. The composite structure together with known biochemical information allows us to propose a mechanism that is accompanied by structural changes during the catalytic cycle. First, the hydride transfer mechanism from NADPH to FAD should be very similar to that of CYPOR. As observed in rat CYPOR (58), the conserved phenylalanine (Phe 1125 in human iNOS) at the C terminus of all three NOSs must move during catalysis to give the nicotinamide ring access to the re-face of the FAD ring. This movement most likely requires a conformational change at the entire C-terminal tail in all three NOSs. Second, electron transfer between the two flavins also requires fine tuning of the alignment of the two flavin rings. The conformation change of the C-terminal tail must be responsible for the movement of the FMN domain in iNOS, in which CaM is always bound, and there is no direct interaction between the C-terminal tail and CaM. However, in constitutive NOS, when CaM binds, it is most likely that the interaction between CaM and SI (found only in constitutive NOS) triggers large conformational changes that move both the tail and AR from their locked conformations (between the FMN and FAD domains) to unlocked positions, freeing the FMN domain to coordinate its movement with the binding of NADPH and release of NADP ϩ . The bound CaM in constitutive NOS may directly interact with AR to stabilize the unlocked conformation, as suggested by Roman and Masters (13). Third, our open conformation, which is necessary for the electron transfer between FMN and heme, requires a drastic movement of the FMN domain together with bound CaM and is accomplished by two linkers (one between the FMN and FAD domains, as observed in the linker-shortened CYPOR mutant (15), and the other between the oxygenase domain and the CaM domain) and the hinge between the FMN and CaM domains. The hinge between the two CaM lobes contributes to the fine tuning of both FMN-heme and FMN-FAD alignments for optimal electron transfer between these cofactors.