Department of Pharmaceutical Sciences, 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. 2024 Jun 4;63(11):1395-1411. doi: 10.1021/acs.biochem.4c00157. Epub 2024 May 15.
Nitric oxide synthase (NOS) in mammals is a family of multidomain proteins in which interdomain electron transfer (IET) is controlled by domain-domain interactions. Calmodulin (CaM) binds to the canonical CaM-binding site in the linker region between the FMN and heme domains of NOS and allows tethered FMN domain motions, enabling an intersubunit FMN-heme IET in the output state for NO production. Our previous cross-linking mass spectrometric (XL MS) results demonstrated site-specific protein dynamics in the CaM-responsive regions of rat neuronal NOS (nNOS) reductase construct, a monomeric protein [Jiang et al., , 2023, 62, 2232-2237]. In this work, we have extended our combined approach of XL MS structural mapping and AlphaFold structural prediction to examine the homodimeric nNOS oxygenase/FMN (oxyFMN) construct, an established model of the NOS output state. We employed parallel reaction monitoring (PRM) based quantitative XL MS (qXL MS) to assess the CaM-induced changes in interdomain dynamics and interactions. Intersubunit cross-links were identified by mapping the cross-links onto top AlphaFold structural models, which was complemented by comparing their relative abundances in the cross-linked dimeric and monomeric bands. Furthermore, contrasting the CaM-free and CaM-bound nNOS samples shows that CaM enables the formation of the intersubunit FMN-heme docking complex and that CaM binding induces extensive, allosteric conformational changes across the NOS regions. Moreover, the observed cross-links sites specifically respond to changes in ionic strength. This indicates that interdomain salt bridges are responsible for stabilizing and orienting the output state for efficient FMN-heme IET. Taken together, our targeted qXL MS results have revealed that CaM and ionic strength modulate specific dynamic changes in the CaM/FMN/heme complexes, particularly in the context of intersubunit interdomain FMN-heme interactions.
哺乳动物中的一氧化氮合酶 (NOS) 是一组多结构域蛋白，其中结构域间电子转移 (IET) 受结构域间相互作用控制。钙调蛋白 (CaM) 与 NOS 的 FMN 和血红素结构域之间的连接区中的典型 CaM 结合位点结合，并允许连接的 FMN 结构域运动，从而在产生 NO 的输出状态下实现亚基间的 FMN-血红素 IET。我们之前的交联质谱 (XL MS) 结果表明，在大鼠神经元 NOS (nNOS) 还原酶构建体的 CaM 反应区域存在特定于位点的蛋白质动力学，该构建体是一种单体蛋白 [Jiang 等人，2023 年，62，2232-2237]。在这项工作中，我们扩展了我们的 XL MS 结构映射和 AlphaFold 结构预测相结合的方法，以检查NOS 输出状态的建立模型，即同二聚体 nNOS 加氧酶/FMN (oxyFMN) 构建体。我们采用平行反应监测 (PRM) 基于定量 XL MS (qXL MS) 来评估 CaM 诱导的结构域间动力学和相互作用的变化。通过将交联映射到顶级 AlphaFold 结构模型上来鉴定亚基间交联，并用它们在交联二聚体和单体带中的相对丰度进行补充。此外，对比 CaM 游离和 CaM 结合的 nNOS 样品表明，CaM 使亚基间 FMN-血红素对接复合物的形成成为可能，并且 CaM 结合诱导 NOS 区域的广泛变构构象变化。此外，观察到的交联位点特异性响应离子强度的变化。这表明结构域间盐桥负责稳定和定向输出状态，以实现有效的 FMN-血红素 IET。总之，我们的靶向 qXL MS 结果表明，CaM 和离子强度调节 CaM/FMN/血红素复合物中特定的动态变化，特别是在亚基间结构域间 FMN-血红素相互作用的背景下。
