Conformational acrobatics: The structural basis for isoform-selective inhibition of nitric-oxide synthases

A number of dipeptide amides/peptidomimetics and mechanism-based inactivators act as isoform-selective competitive inhibitors and suicide substrates respectively. X-ray crystallography, site-directed mutagenesis and enzyme kinetics were utilized to understand the structural basis for the observed isoform selective NOS inhibition.; The Pichia pastoris eukaryotic expression system was utilized for recombinant production of bovine endothelial NOS. Following vector construction and transformation, an extensive screening process led to the isolation of a "jackpot clone" which consistently yielded 55--56 mg of purified endothelial NOS holo-enzyme per liter of induced cell culture. Trypsinolysis of the holo-enzyme allowed purification of the endothelial NOS heme domain, suitable in quantity and quality for co-crystallization with dipeptide amides, peptidomimetics and mechanism-based inactivators.; Three nNOS selective competitive inhibitors, Nw-nitroarginine-2,4-L-diaminobutyramide, (4S)-N-(4-amino-5-[aminoethyl]aminopentyl)- N'-nitroguanidine, and L- Nw-nitroarginine-(4R)-amino-L-proline amide bind in a curled conformation in nNOS but bind in an extended conformation in eNOS. Using site-directed mutagenesis it was discovered that a single amino acid difference, D597/N368 (nNOS/eNOS) not only dictates the conformation of the bound inhibitor, but also is responsible for two orders of magnitude of the selectivity towards nNOS. A second variant residue, M336/V106 (nNOS/eNOS) was found to contribute further to the observed selectivity, as L- Nw-nitroarginine-(4R)-amino-L-proline amide is 100 times more potent an inhibitor of N368D/V106M eNOS than of wild-type eNOS.; Additionally the crystal structures were determined of nNOS and eNOS bound to two nNOS selective dipeptide amides built on a Nw-nitro-D-arginine (D-NNA) scaffold, D-Lys-D-NNA-NH2 and D-Phe-D-NNA-NH2. Two isoform-variant residues, M336/L337 and V106/L107 (nNOS, eNOS) lie on a beta-strand that terminates in the mouth of the solvent access channel and contacts the N-terminal D-Lys and D-Phe of these dipeptides. The equivalent beta-strand in iNOS with its corresponding M120/T121 restrict the width of the solvent access channel compared to the other two isoforms. Site-directed mutagenesis and X-ray crystallography confirmed that more important than the identity of these variant residues in the channel is the variation in channel volume among isoforms. In these same D-NNA dipeptide/NOS complexes, isoform variant conformational mobility was observed among a triad of conserved active site residues, S477/Q478/R481 and S248/Q249/R252 (nNOS, eNOS) leading to the binding of mannitol in nNOS but not in eNOS. Lastly, crystallographic studies with the mechanism-based inactivator, No-propyl-L-arginine revealed further variations in active-site volume among isoforms. All of these inter-isoform differences can be exploited for the purposes of isoform selective inhibitor design.