Dipak K. Ghosh

Characterization of the reductase domain of rat neuronal nitric oxide synthase generated in the methylotrophic yeast Pichia pastoris. Calmodulin response is complete within the reductase domain itself.

Rat neuronal NO synthase (nNOS) is comprised of a flavin-containing reductase domain and a heme-containing oxygenase domain. Calmodulin binding to nNOS increases the rate of electron transfer from NADPH into its flavins, triggers electron transfer from flavins to the heme, activates NO synthesis, and increases reduction of artificial electron acceptors such as cytochrome c. To investigate what role the reductase domain plays in calmodulins activation of these functions, we overexpressed a form of the nNOS reductase domain (amino acids 724-1429) in the yeast Pichia pastoris that for the first time exhibits a complete calmodulin response. The reductase domain was purified by 2′,5′-ADP affinity chromatography yielding 25 mg of pure protein per liter of culture. It contained 1 FAD and 0.8 FMN per molecule. Most of the protein as isolated contained an air-stable flavin semiquinone radical that was sensitive to FeCN6 oxidation. Anaerobic titration of the FeCN6-oxidized reductase domain with NADPH indicated the flavin semiquinone re-formed after addition of 1-electron equivalent and the flavins could accept up to 3 electrons from NADPH. Calmodulin binding to the recombinant reductase protein increased its rate of NADPH-dependent flavin reduction and its rate of electron transfer to cytochrome c, FeCN6, or dichlorophenolindophenol to fully match the rate increases achieved when calmodulin bound to native full-length nNOS. Calmodulins activation of the reductase protein was associated with an increase in domain tryptophan and flavin fluorescence. We conclude that many of calmodulins actions on native nNOS can be fully accounted for through its interaction with the nNOS reductase domain itself.

N-Terminal Domain Swapping and Metal Ion Binding in Nitric Oxide Synthase Dimerization

Nitric oxide synthase oxygenase domains (NOSox) must bind tetrahydrobiopterin and dimerize to be active. New crystallographic structures of inducible NOSox reveal that conformational changes in a switch region (residues 103–111) preceding a pterin‐binding segment exchange N‐terminal β‐hairpin hooks between subunits of the dimer. N‐terminal hooks interact primarily with their own subunits in the ‘unswapped’ structure, and two switch region cysteines (104 and 109) from each subunit ligate a single zinc ion at the dimer interface. N‐terminal hooks rearrange from intra‐ to intersubunit interactions in the ‘swapped structure’, and Cys109 forms a self‐symmetric disulfide bond across the dimer interface. Subunit association and activity are adversely affected by mutations in the N‐terminal hook that disrupt interactions across the dimer interface only in the swapped structure. Residue conservation and electrostatic potential at the NOSox molecular surface suggest likely interfaces outside the switch region for electron transfer from the NOS reductase domain. The correlation between three‐dimensional domain swapping of the N‐terminal hook and metal ion release with disulfide formation may impact inducible nitric oxide synthase (i)NOS stability and regulation in vivo.

Inducible nitric oxide synthase: role of the N‐terminal β‐hairpin hook and pterin‐binding segment in dimerization and tetrahydrobiopterin interaction

The oxygenase domain of the inducible nitric oxide synthase (iNOSox; residues 1–498) is a dimer that binds heme, L‐arginine and tetrahydrobiopterin (H4B) and is the site for nitric oxide synthesis. We examined an N‐terminal segment that contains a β‐hairpin hook, a zinc ligation center and part of the H4B‐binding site for its role in dimerization, catalysis, and H4B and substrate interactions. Deletion mutagenesis identified the minimum catalytic core and indicated that an intact N‐terminal β‐hairpin hook is essential. Alanine screening mutagenesis of conserved residues in the hook revealed five positions (K82, N83, D92, T93 and H95) where native properties were perturbed. Mutants fell into two classes: (i) incorrigible mutants that disrupt side‐chain hydrogen bonds and packing interactions with the iNOSox C‐terminus (N83, D92 and H95) and cause permanent defects in homodimer formation, H4B binding and activity; and (ii) reformable mutants that destabilize interactions of the residue main chain (K82 and T93) with the C‐terminus and cause similar defects that were reversible with high concentrations of H4B. Heterodimers comprised of a hook‐defective iNOSox mutant subunit and a full‐length iNOS subunit were active in almost all cases. This suggests a mechanism whereby N‐terminal hooks exchange between subunits in solution to stabilize the dimer.

Nitric-oxide synthase output state. Design and properties of nitric-oxide synthase oxygenase/FMN domain constructs.

Mammalian nitric-oxide synthases are large modular enzymes that evolved from independently expressed ancestors. Calmodulin-controlled isoforms are signal generators; calmodulin activates electron transfer from NADPH through three reductase domains to an oxygenase domain. Structures of the reductase unit and its homologs show FMN and FAD in contact but too isolated from the protein surface to permit exit of reducing equivalents. To study states in which FMN/heme electron transfer is feasible, we designed and produced constructs including only oxygenase and FMN binding domains, eliminating strong internal reductase complex interactions. Constructs for all mammalian isoforms were expressed and purified as dimers. All synthesize NO with peroxide as the electron donor at rates comparable with corresponding oxygenase constructs. All bind cofactors nearly stoichiometrically and have native catalytic sites by spectroscopic criteria. Modest differences in electrochemistry versus independently expressed heme and FMN binding domains suggest interdomain interactions. These interactions can be convincingly demonstrated via calmodulin-induced shifts in high spin ferriheme EPR spectra and through mutual broadening of heme and FMNH· radical signals in inducible nitricoxide synthase constructs. Blue neutral FMN semiquinone can be readily observed; potentials of one electron couple (in inducible nitric-oxide synthase oxygenase FMN, FMN oxidized/semiquione couple =+70 mV, FMN semiquinone/hydroquinone couple =–180 mV, and heme =–180 mV) indicate that FMN is capable of serving as a one electron heme reductant. The construct will serve as the basis for future studies of the output state for NADPH derived reducing equivalents.

Crystal Structures of Cyanide Complexes of P450Cam and the Oxygenase Domain of Inducible Nitric Oxide Synthase-Structural Models of the Short-Lived Oxygen Complexes

The crystal structure of the ternary cyanide complex of P450cam and camphor was determined to 1.8A resolution and found to be identical with the structure of the active oxygen complex [I. Schlichting et al., 2000, Science 287, 1615]. Notably, cyanide binds in a bent mode and induces the active conformation that is characterized by the presence of two water molecules and a flip of the carbonyl of the conserved Asp251. The structure of the ternary complex of cyanide, L-arginine, and the oxygenase domain of inducible nitric oxide synthase was determined to 2.4A resolution. Cyanide binds essentially linearly, interacts with L-Arg, and induces the binding of a water molecule at the active site. This water is positioned by backbone interactions, located 2.8A from the nitrogen atom of cyanide, and could provide a proton required for O-O bond scission in the hydroxylation reaction of nitric oxide synthase.

Host Response to Infection: the Role of CpG DNA in Induction of Cyclooxygenase 2 and Nitric Oxide Synthase 2 in Murine Macrophages

ABSTRACT Depending on sequence, bacterial and synthetic DNAs can activate the host immune system and influence the host response to infection. The purpose of this study was to determine the abilities of various phosphorothioate oligonucleotides with cytosine-guanosine-containing motifs (CpG DNA) to activate macrophages to produce nitric oxide (NO) and prostaglandin E2 (PGE2) and to induce expression of NO synthase 2 (NOS2) and cyclooxygenase 2 (COX2). As little as 0.3 μg of CpG DNA/ml increased NO and PGE2production in a dose- and time-dependent fashion in cells of the mouse macrophage cell line J774. NO and PGE2 production was noted by 4 to 8 h after initiation of cultures with the CpG DNA, with the kinetics of NO production induced by CpG DNA being comparable to that induced by a combination of lipopolysaccharide and gamma interferon. CpG DNA-treated J774 cells showed enhanced expression of NOS2 and COX2 proteins as determined by immunoblotting, with the relative potencies of the CpG DNAs generally corresponding to those noted for the induction of NO and PGE2 production as well as to those noted for the induction of interleukin-6 (IL-6), IL-12, and tumor necrosis factor. Extracts from CpG DNA-treated cells convertedl-arginine to l-citrulline, but the NOS inhibitorNG-monomethyl-l-arginine (NMMA) inhibited this reaction. The COX2-specific inhibitor NS398 inhibited CpG DNA-induced PGE2 production and inhibited NO production to various degrees. The NOS inhibitors NMMA, 1400W, andN-iminoethyl-l-lysine effectively blocked NO production and increased the production of PGE2 in a dose-dependent fashion. Thus, analogues of microbial DNA (i.e., CpG DNA) activate mouse macrophage lineage cells for the expression of NOS2 and COX2, with the production of NO and that of PGE2occurring in an interdependent manner.

Binding and activation of nitric oxide synthase isozymes by calmodulin EF hand pairs

Calmodulin (CaM) is a cytosolic Ca2+ signal‐transducing protein that binds and activates many different cellular enzymes with physiological relevance, including the nitric oxide synthase (NOS) isozymes. CaM consists of two globular domains joined by a central linker; each domain contains an EF hand pair. Four different mutant CaM proteins were used to investigate the role of the two CaM EF hand pairs in the binding and activation of the mammalian inducible NOS (iNOS) and the constitutive NOS (cNOS) enzymes, endothelial NOS (eNOS) and neuronal NOS (nNOS). The role of the CaM EF hand pairs in different aspects of NOS enzymatic function was monitored using three assays that monitor electron transfer within a NOS homodimer. Gel filtration studies were used to determine the effect of Ca2+ on the dimerization of iNOS when coexpressed with CaM and the mutant CaM proteins. Gel mobility shift assays were performed to determine binding stoichiometries of CaM proteins to synthetic NOS CaM‐binding domain peptides. Our results show that the N‐terminal EF hand pair of CaM contains important binding and activating elements for iNOS, whereas the N‐terminal EF hand pair in conjunction with the central linker region is required for cNOS enzyme binding and activation. The iNOS enzyme must be coexpressed with wild‐type CaM in vitro because of its propensity to aggregate when residues of the highly hydrophobic CaM‐binding domain are exposed to an aqueous environment. A possible role for iNOS aggregation in vivo is also discussed.

FMN fluorescence in inducible NOS constructs reveals a series of conformational states involved in the reductase catalytic cycle.

Nitric oxide synthases (NOSs) produce NO as a molecular signal in the nervous and cardiovascular systems and as a cytotoxin in the immune response. NO production in the constitutive isoforms is controlled by calmodulin regulation of electron transfer. In the tethered shuttle model for NOS reductase function, the FMN domain moves between NADPH dehydrogenase and oxygenase catalytic centers. Crystal structures of neuronal NOS reductase domain and homologs correspond to an ‘input state’, with FMN in close contact with FAD. We recently produced two domain ‘output state’ (oxyFMN) constructs showing calmodulin dependent FMN domain association with the oxygenase domain. FMN fluorescence is sensitive to enzyme conformation and calmodulin binding. The inducible NOS (iNOS) oxyFMN construct is more fluorescent than iNOS holoenzyme. The difference in steady state fluorescence is rationalized by the observation of a series of characteristic states in the two constructs, which we assign to FMN in different environments. OxyFMN and holoenzyme share open conformations with an average lifetime of ∼ 4.3 ns. The majority state in holoenzyme has a short lifetime of ∼ 90 ps, probably because of FAD–FMN interactions. In oxyFMN about 25–30% of the FMN is in a state with a lifetime of 0.9 ns, which we attribute to quenching by heme in the output state. Occupancy of the output state together with our previous kinetic results yields a heme edge to FMN distance estimate of 12–15 Å. These results indicate that FMN fluorescence is a valuable tool to study conformational states involved in the NOS reductase catalytic cycle.

Deletion of the autoregulatory insert modulates intraprotein electron transfer in rat neuronal nitric oxide synthase

Comparative CO photolysis kinetics studies on wild‐type and autoregulatory (AR) insert‐deletion mutant of rat nNOS holoenzyme were conducted to directly investigate the role of the unique AR insert in the catalytically significant FMN–heme intraprotein electron transfer (IET). Although the amplitude of the IET kinetic traces was decreased two‐ to three‐fold, the AR deletion did not change the rate constant for the calmodulin‐controlled IET. This suggests that the rate‐limiting conversion of the electron‐accepting state to a new electron‐donating (output) state does not involve interactions with the AR insert, but that AR may stabilize the output state once it is formed.

Mutations in the FMN Domain Modulate MCD Spectra of the Heme Site in the Oxygenase Domain of Inducible Nitric Oxide Synthase

The nitric oxide synthase (NOS) output state for NO production is a complex of the flavin mononucleotide (FMN)-binding domain and the heme domain, and thereby it facilitates the interdomain electron transfer from the FMN to the catalytic heme site. Emerging evidence suggests that interdomain FMN-heme interactions are important in the formation of the output state because they guide the docking of the FMN domain to the heme domain. In this study, notable effects of mutations in the adjacent FMN domain on the heme structure in a human iNOS bidomain oxygenase/FMN construct have been observed by using low-temperature magnetic circular dichroism (MCD) spectroscopy. The comparative MCD study of wild-type and mutant proteins clearly indicates that a properly docked FMN domain contributes to the observed L-Arg perturbation of the heme MCD spectrum in the wild-type protein and that the conserved surface residues in the FMN domain (E546 and E603) play key roles in facilitating a productive alignment of the FMN and heme domains in iNOS.