Mapping calmodulin-induced conformational during activation of neuronal nitric oxide synthase by H/D exchange mass spectrometry

Neuronal nitric oxide synthase (nNOS) produces nitric oxide (NO), a unique signaling gas molecule involved in retrograde neuronal signaling. Homodimeric nNOS is composed of a reductase and oxidase subdomain, connected by a mobile FMN subdomain. The reductase domain delivers reducing equivalents to the heme of the oxidase domain, where NO is generated from arginine. Delivery of reducing equivalents is tightly regulated by Ca2+ via the intermediary Ca2+-sensor calmodulin (CaM). We examined conformational changes induced by CaM-binding that encourage the inter-domain delivery using hydrogen-deuterium exchange mass spectrometry (HDX-MS). HDX-MS revealed critical regulatory surfaces on the reductase domain and FMN subdomain. Changes in the reductase domain were most pronounced in a regulatory beta-finger, a structural element unique to the CaM-regulated isoforms of NOS. Interestingly, the CaM-perturbed regions encompass two serine residues that are subject to phosphorylation, a modification reported to tune nNOS activity. Changes in the mobile FMN subdomain were more distributed, largely localized to the distal surface opposite the FMN cofactor. This surface includes an auto-inhibitory insert involved in docking the FMN subdomain to the reductase domain. Taken together, the HDX-MS results define several inter-domain interaction surfaces that are released upon Ca2+/CaM-induced activation