Jiang Ting, Wan Guanghua, Zhang Haikun, Gyawali Yadav Prasad, Underbakke Eric S, Feng Changjian
College of Pharmacy, University of New Mexico, Albuquerque, New Mexico 87131, United States.
Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, Iowa 50011, United States.
Biochemistry. 2023 Aug 1;62(15):2232-2237. doi: 10.1021/acs.biochem.3c00245. Epub 2023 Jul 17.
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.
一氧化氮合酶（NOS）负责一氧化氮（NO）的生物合成，NO是一种重要的信号分子，控制着多种生理过程，如神经传递和血管舒张。神经元型一氧化氮合酶（nNOS）是一种受钙调蛋白（CaM）调控的酶。在没有CaM的情况下，几个内在控制元件与NADP结合，抑制电子在NOS结构域间传递。CaM结合可解除抑制因子，促进产生NO所需的电子传递。nNOS控制元件的调节动力学对于控制NO信号至关重要，但机制问题仍然存在，因为NOS的内在动力学阻碍了传统结构生物学方法的应用。在这里，我们采用交联质谱（XL MS）来探究nNOS中的调节动力学，重点关注CaM反应性控制元件。定量XL MS揭示了不同构象变化，区分了单独的nNOS还原酶（nNOSred）、带有NADP的nNOSred、nNOS-CaM以及带有NADP的nNOS-CaM。我们观察到CaM与NADP对nNOSred交联模式有不同影响。CaM诱导显著的全局变化，而NADP的影响主要局限于NADPH结合亚结构域。此外，CaM增加了与CaM-nNOS间交联形成相关的nNOS内交联丰度。综上所述，这些XL MS结果表明CaM和NADP位点特异性地改变了nNOS的构象格局。
