Structure, regulation and dynamics of mammalian nitric oxide synthase

Abstract

Nitric oxide synthase (NOS) enzymes control, by synthesis, the activities of their product nitric oxide (NO) as a signal and defensive cytotoxin. In mammals, three very similar, but differently regulated, NOS isozymes direct NO to distinct roles in neurotransmission, vascular homeostasis and host defense, thereby offering important therapeutic targets. Our mutational, biochemical and biophysical investigations of NOS define structures of its component catalytic oxygenase and electron‐supplying reductase modules, and Ca2+/calmodulin‐bound linker; characterize holo‐enzyme assembly and dynamics; and address current mechanistic questions regarding catalysis, regulation and isozyme‐selective inhibition. Our analyses suggest a large scale swinging motion of the entire FMN‐binding domain of NOS reductase to deliver electrons to NOS oxygenase for catalysis.1 Our inhibitor‐bound NOS oxygenase crystal structures, along with biochemical and mutagenesis results, revealed that plasticity of distant isozyme‐specific second‐ and third‐shell residues modulates conformational changes of invariant first‐shell residues to determine inhibitor selectivity.2 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.1E.D. Garcin et al., (2004) J. Biol. Chem. 279: 37918; 2E.D. Garcin et al. (2008) Nat. Chem. Biol. 4: 700. (Supported by NIH R01 HL58883 and the Skaggs Institute for Chemical Biology).