1 research outputs found
Probing Protein Dynamics in Neuronal Nitric Oxide Synthase by Quantitative Cross-Linking Mass Spectrometry
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