Three nitric-oxide synthase (NOS) isozymes play crucial, but distinct, roles in neurotransmission, vascular homeostasis, and host defense, by catalyzing Ca²⁺/calmodulin-triggered NO synthesis. Here, we address current questions regarding NOS activity and regulation by combining mutagenesis and biochemistry with crystal structure determination of a fully assembled, electron-supplying, neuronal NOS reductase dimer. By integrating these results, we structurally elucidate the unique mechanisms for isozyme-specific regulation of electron transfer in NOS. Our discovery of the autoinhibitory helix, its placement between domains, and striking similarities with canonical calmodulin-binding motifs, support new mechanisms for NOS inhibition. NADPH, isozyme-specific residue Arg¹⁴⁰⁰, and the C-terminal tail synergistically repress NOS activity by locking the FMN binding domain in an electron-accepting position. Our analyses suggest that calmodulin binding or C-terminal tail phosphorylation frees a large scale swinging motion of the entire FMN domain to deliver electrons to the catalytic module in the holoenzyme.

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.

Postsynaptic nitric oxide (NO) production affects synaptic plasticity and neuronal cell death. Ca²⁺fluxes through the NMDA receptor (NMDAR) stimulate the production of NO by neuronal nitric oxide synthase (nNOS). However, the mechanisms by which nNOS activity is regulated are poorly understood. We evaluated the effect of neuronal stimulation with glutamate on the phosphorylation of nNOS. We show that, in cortical neurons, a low glutamate concentration (30 μm) induces rapid and transient NMDAR-dependent phosphorylation of S1412 by Akt, followed by sustained phosphorylation of S847 by CaMKII (calcium-calmodulin-dependent kinase II). We demonstrate that phosphorylation of S1412 by Akt is necessary for activation of nNOS by the NMDAR. nNOS mutagenesis confirms that these phosphorylations respectively activate and inhibit nNOS and, thus, transiently activate NO production. A constitutively active (S1412D), but not a constitutively repressed (S847D) nNOS mutant elevated surface glutamate receptor 2 levels, demonstrating that these phosphorylations can control AMPA receptor trafficking via NO. Notably, an excitotoxic stimulus (150 μmglutamate) induced S1412, but not S847 phosphorylation, leading to deregulated nNOS activation. S1412D did not kill neurons; however, it enhanced the excitotoxicity of a concomitant glutamate stimulus. We propose a swinging domain model for the regulation of nNOS: S1412 phosphorylation facilitates electron flow within the reductase module of nNOS, increasing nNOS sensitivity to Ca²⁺-calmodulin. These findings suggest a critical role for a kinetically complex and novel series of regulatory nNOS phosphorylations induced by the NMDA receptor for thein vivocontrol of nNOS.