Nitric-oxide synthase forms N-NO-pterin and S-NO-cys: implications for activity, allostery, and regulation.

Inducible nitric-oxide synthase (iNOS) produces biologically stressful levels of nitric oxide (NO) as a potent mediator of cellular cytotoxicity or signaling. Yet, how this nitrosative stress affects iNOS function in vivo is poorly understood. Here we define two specific non-heme iNOS nitrosation sites discovered by combining UV-visible spectroscopy, chemiluminescence, mass spectrometry, and x-ray crystallography. We detected auto-S-nitrosylation during enzymatic turnover by using chemiluminescence. Selective S-nitrosylation of the ZnS(4) site, which bridges the dimer interface, promoted a dimer-destabilizing order-to-disorder transition. The nitrosated iNOS crystal structure revealed an unexpected N-NO modification on the pterin cofactor. Furthermore, the structurally defined N-NO moiety is solvent-exposed and available to transfer NO to a partner. We investigated glutathione (GSH) as a potential transnitrosation partner because the intracellular GSH concentration is high and NOS can form S-nitrosoglutathione. Our computational results predicted a GSH binding site adjacent to the N-NO-pterin. Moreover, we detected GSH binding to iNOS with saturation transfer difference NMR spectroscopy. Collectively, these observations resolve previous paradoxes regarding this uncommon pterin cofactor in NOS and suggest means for regulating iNOS activity via N-NO-pterin and S-NO-Cys modifications. The iNOS self-nitrosation characterized here appears appropriate to help control NO production in response to cellular conditions.

Anchored plasticity opens doors for selective inhibitor design in nitric oxide synthase.

Nitric oxide synthase (NOS) enzymes synthesize nitric oxide, a signal for vasodilatation and neurotransmission at low concentrations and a defensive cytotoxin at higher concentrations. The high active site conservation among all three NOS isozymes hinders the design of selective NOS inhibitors to treat inflammation, arthritis, stroke, septic shock and cancer. Our crystal structures and mutagenesis results identified an isozyme-specific induced-fit binding mode linking a cascade of conformational changes to a new specificity pocket. Plasticity of an isozyme-specific triad of distant second- and third-shell residues modulates conformational changes of invariant first-shell residues to determine inhibitor selectivity. To design potent and selective NOS inhibitors, we developed the anchored plasticity approach: anchor an inhibitor core in a conserved binding pocket, then extend rigid bulky substituents toward remote specificity pockets, which become accessible upon conformational changes of flexible residues. This approach exemplifies general principles for the design of selective enzyme inhibitors that overcome strong active site conservation.

Artificial protein cavities as specific ligand-binding templates: characterization of an engineered heterocyclic cation-binding site that preserves the evolved specificity of the parent protein.

Cavity complementation has been observed in many proteins, where an appropriate small molecule binds to a cavity-forming mutant. Here, the binding of compounds to the W191G cavity mutant of cytochrome c peroxidase is characterized by X-ray crystallography and binding thermodynamics. Unlike cavities created by removal of hydrophobic side-chains, the W191G cavity does not bind neutral or hydrophobic compounds, but displays a strong specificity for heterocyclic cations, consistent with the role of the protein to stabilize a tryptophan radical at this site. Ligand dissociation constants for the protonated cationic state ranged from 6 microM for 2-amino-5-methylthiazole to 1 mM for neutral ligands, and binding was associated with a large enthalpy-entropy compensation. X-ray structures show that each of 18 compounds with binding behavior bind specifically within the artificial cavity and not elsewhere in the protein. The compounds make multiple hydrogen bonds to the cavity walls using a subset of the interactions seen between the protein and solvent in the absence of ligand. For all ligands, every atom that is capable of making a hydrogen bond does so with either protein or solvent. The most often seen interaction is to Asp235, and most compounds bind with a specific orientation that is defined by their ability to interact with this residue. Four of the ligands do not have conventional hydrogen bonding atoms, but were nevertheless observed to orient their most polar CH bond towards Asp235. Two of the larger ligands induce disorder in a surface loop between Pro190 and Asn195 that has been identified as a mobile gate to cavity access. Despite the predominance of hydrogen bonding and electrostatic interactions, the small variation in observed binding free energies were not correlated readily with the strength, type or number of hydrogen bonds or with calculated electrostatic energies alone. Thus, as with naturally occurring binding sites, affinities to W191G are likely to be due to a subtle balance of polar, non-polar, and solvation terms. These studies demonstrate how cavity complementation and judicious choice of site can be used to produce a protein template with an unusual ligand-binding specificity.

Excision of a proposed electron transfer pathway in cytochrome c peroxidase and its replacement by a ligand-binding channel.

A previously proposed electron transfer (ET) pathway in the heme enzyme cytochrome c peroxidase has been excised from the structure, leaving an open ligand-binding channel in its place. Earlier studies on cavity mutants of this enzyme have revealed structural plasticity in this region of the molecule. Analysis of these structures has allowed the design of a variant in which the specific section of protein backbone representing a previously proposed ET pathway is accurately extracted from the protein. A crystal structure verified the creation of an open channel that overlays the removed segment, extending from the surface of the protein to the heme at the core of the protein. A number of heterocyclic cations were found to bind to the proximal-channel mutant with affinities that can be rationalized based on the structures. It is proposed that small ligands bind more weakly to the proximal-channel mutant than to the W191G cavity due to an increased off rate of the open channel, whereas larger ligands are able to bind to the channel mutant without inducing large conformational changes. The structure of benzimidazole bound to the proximal-channel mutant shows that the ligand accurately overlays the position of the tryptophan radical center that was removed from the wild-type enzyme and displaces four of the eight ordered solvent molecules seen in the empty cavity. Ligand binding also caused a small rearrangement of the redesigned protein loop, perhaps as a result of improved electrostatic interactions with the ligand. The engineered channel offers the potential for introducing synthetic replacements for the removed structure, such as sensitizer-linked substrates. These installed "molecular wires" could be used to rapidly initiate reactions, trap reactive intermediates, or answer unresolved questions about ET pathways.

2-aminopyridines as highly selective inducible nitric oxide synthase inhibitors. Differential binding modes dependent on nitrogen substitution.

4-Methylaminopyridine (4-MAP) (5) is a potent but nonselective nitric oxide synthase (NOS) inhibitor. While simple N-methylation in this series results in poor activity, more elaborate N-substitution such as with 4-piperidine carbamate or amide results in potent and selective inducible NOS inhibition. Evidently, a flipping of the pyridine ring between these new inhibitors allows the piperidine to interact with different residues and confer excellent selectivity.

Interaction of 5-methyltetrahydrofolate and tetrahydrobiopterin on endothelial function.

The present study was designed to investigate the interaction between 5-methyltetrahydrofolate and tetrahydrobiopterin in modulating endothelial function. Tetrahydrobiopterin is a critical cofactor for nitric oxide synthase and maintains this enzyme as a nitric oxide- versus superoxide-producing enzyme. The structure of 5-methyltetrahydrofolate is similar to tetrahydrobiopterin and both agents have been shown to improve endothelium-dependent vasodilatation. We hypothesized that 5-methyltetrahydrofolate interacts with nitric oxide synthase in a fashion analogous, yet independent, of tetrahydrobiopterin to improve endothelial function. We demonstrate that 5-methyltetrahydrofolate binds the active site of nitric oxide synthase and mimics the orientation of tetrahydrobiopterin. Furthermore, 5-methyltetrahydrofolate attenuates superoxide production (induced by inhibition of tetrahydrobiopterin synthesis) and improves endothelial function in aortae isolated from tetrahydrobiopterin-deficient rats. We suggest that 5-methyltetrahydrofolate directly interacts with nitric oxide synthase to promote nitric oxide (vs. superoxide) production and improve endothelial function. 5-Methyltetrahydrofolate may represent an important strategy for intervention aimed at improving tetrahydrobiopterin bioavailability.

Distinct dimer interaction and regulation in nitric-oxide synthase types I, II, and III.

Homodimer formation activates all nitric-oxide synthases (NOSs). It involves the interaction between two oxygenase domains (NOSoxy) that each bind heme and (6R)-tetrahydrobiopterin (H4B) and catalyze NO synthesis from L-Arg. Here we compared three NOSoxy isozymes regarding dimer strength, interface composition, and the ability of L-Arg and H4B to stabilize the dimer, promote its formation, and protect it from proteolysis. Urea dissociation studies indicated that the relative dimer strengths were NOSIIIoxy >> NOSIoxy > NOSIIoxy (endothelial NOSoxy (eNOSoxy) >> neuronal NOSOXY (nNOSoxy) > inducible NOSoxy (iNOSoxy)). Dimer strengths of the full-length NOSs had the same rank order as judged by their urea-induced loss of NO synthesis activity. NOSoxy dimers containing L-Arg plus H4B exhibited the greatest resistance to urea-induced dissociation followed by those containing either molecule and then by those containing neither. Analysis of crystallographic structures of eNOSoxy and iNOSoxy dimers showed more intersubunit contacts and buried surface area in the dimer interface of eNOSoxy than iNOSoxy, thus revealing a potential basis for their different stabilities. L-Arg plus H4B promoted dimerization of urea-generated iNOSoxy and nNOSoxy monomers, which otherwise was minimal in their absence, and also protected both dimers against trypsin proteolysis. In these respects, L-Arg alone was more effective than H4B alone for nNOSoxy, whereas for iNOSoxy the converse was true. The eNOSoxy dimer was insensitive to proteolysis under all conditions. Our results indicate that the three NOS isozymes, despite their general structural similarity, differ markedly in their strengths, interfaces, and in how L-Arg and H4B influence their formation and stability. These distinguishing features may provide a basis for selective control and likely help to regulate each NOS in its particular biologic milieu.

Automated docking of ligands to an artificial active site: augmenting crystallographic analysis with computer modeling.

The W191G cavity of cytochrome c peroxidase is useful as a model system for introducing small molecule oxidation in an artificially created cavity. A set of small, cyclic, organic cations was previously shown to bind in the buried, solvent-filled pocket created by the W191G mutation. We docked these ligands and a set of non-binders in the W191G cavity using AutoDock 3.0. For the ligands, we compared docking predictions with experimentally determined binding energies and X-ray crystal structure complexes. For the ligands, predicted binding energies differed from measured values by +/- 0.8 kcal/mol. For most ligands, the docking simulation clearly predicted a single binding mode that matched the crystallographic binding mode within 1.0 A RMSD. For 2 ligands, where the docking procedure yielded an ambiguous result, solutions matching the crystallographic result could be obtained by including an additional crystallographically observed water molecule in the protein model. For the remaining 2 ligands, docking indicated multiple binding modes, consistent with the original electron density, suggesting disordered binding of these ligands. Visual inspection of the atomic affinity grid maps used in docking calculations revealed two patches of high affinity for hydrogen bond donating groups. Multiple solutions are predicted as these two sites compete for polar hydrogens in the ligand during the docking simulation. Ligands could be distinguished, to some extent, from non-binders using a combination of two trends: predicted binding energy and level of clustering. In summary, AutoDock 3.0 appears to be useful in predicting key structural and energetic features of ligand binding in the W191G cavity.

Conformational changes in nitric oxide synthases induced by chlorzoxazone and nitroindazoles: crystallographic and computational analyses of inhibitor potency.

Nitric oxide is a key signaling molecule in many biological processes, making regulation of nitric oxide levels highly desirable for human medicine and for advancing our understanding of basic physiology. Designing inhibitors to specifically target one of the three nitric oxide synthase (NOS) isozymes that form nitric oxide from the L-Arg substrate poses a significant challenge due to the overwhelmingly conserved active sites. We report here 10 new X-ray crystallographic structures of inducible and endothelial NOS oxygenase domains cocrystallized with chlorzoxazone and four nitroindazoles: 5-nitroindazole, 6-nitroindazole, 7-nitroindazole, and 3-bromo-7-nitroindazole. Each of these bicyclic aromatic inhibitors has only one hydrogen bond donor and therefore cannot form the bidentate hydrogen bonds that the L-Arg substrate makes with Glu371. Instead, all of these inhibitors induce a conformational change in Glu371, creating an active site with altered molecular recognition properties. The cost of this conformational change is approximately 1-2 kcal, based on our measured constants for inhibitor binding to the wild-type and E371A mutant proteins. These inhibitors derive affinity by pi-stacking above the heme and replacing both intramolecular (Glu371-Met368) and intermolecular (substrate-Trp366) hydrogen bonds to the beta-sheet architecture underlying the active site. When bound to NOS, high-affinity inhibitors in this class are planar, whereas weaker inhibitors are nonplanar. Isozyme differences were observed in the pterin cofactor site, the heme propionate, and inhibitor positions. Computational docking predictions match the crystallographic results, including the Glu371 conformational change and inhibitor-binding orientations, and support a combined crystallographic and computational approach to isozyme-specific NOS inhibitor analysis and design.