Probing Protein Dynamics in Neuronal Nitric Oxide Synthase by Quantitative Cross-Linking Mass Spectrometry

Nitric oxide synthase (NOS) is responsible for the biosynthesisof nitric oxide (NO), an important signaling molecule controllingdiverse physiological processes such as neurotransmission and vasodilation.Neuronal NOS (nNOS) is a calmodulin (CaM)-controlled enzyme. In theabsence of CaM, several intrinsic control elements, along with NADP(+) binding, suppress electron transfer across the NOS domains.CaM binding relieves the inhibitory factors to promote the electrontransport required for NO production. The regulatory dynamics of nNOScontrol elements are critical to governing NO signaling, yet mechanisticquestions remain, because the intrinsic dynamics of NOS thwart traditionalstructural biology approaches. Here, we have employed cross-linkingmass spectrometry (XL MS) to probe regulatory dynamics in nNOS, focusingon the CaM-responsive control elements. Quantitative XL MS revealedconformational changes differentiating the nNOS reductase (nNOSred)alone, nNOSred with NADP(+), nNOS-CaM, and nNOS-CaM withNADP(+). We observed distinct effects of CaM vs NADP(+) on cross-linking patterns in nNOSred. CaM induces strikingglobal changes, while the impact of NADP(+) is primarilylocalized to the NADPH-binding subdomain. Moreover, CaM increasesthe abundance of intra-nNOS cross-links that are related to the formationof the inter-CaM-nNOS cross-links. Taken together, these XL MS resultsdemonstrate that CaM and NADP(+) site-specifically alterthe nNOS conformational landscape.