Abstract
Nitric oxide synthase (NOS) is responsible for the biosynthesis of nitric oxide (NO), an important signaling molecule controlling diverse physiological processes such as neurotransmission and vasodilation. Neuronal NOS (nNOS) is a calmodulin (CaM)-controlled enzyme. In the absence 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 electron transport required for NO production. The regulatory dynamics of nNOS control elements are critical to governing NO signaling, yet mechanistic questions remain, because the intrinsic dynamics of NOS thwart traditional structural biology approaches. Here, we have employed cross-linking mass spectrometry (XL MS) to probe regulatory dynamics in nNOS, focusing on the CaM-responsive control elements. Quantitative XL MS revealed conformational changes differentiating the nNOS reductase (nNOSred) alone, nNOSred with NADP+, nNOS-CaM, and nNOS-CaM with NADP+. We observed distinct effects of CaM vs NADP+ on cross-linking patterns in nNOSred. CaM induces striking global changes, while the impact of NADP+ is primarily localized to the NADPH-binding subdomain. Moreover, CaM increases the abundance of intra-nNOS cross-links that are related to the formation of the inter-CaM-nNOS cross-links. Taken together, these XL MS results demonstrate that CaM and NADP+ site-specifically alter the nNOS conformational landscape.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.