Mapping The Domain Architecture And Conformations of Soluble Guanylate Cyclase

描述（由申请人提供）：一氧化氮（NO）信号通路介导多种生理功能，包括血管舒张、神经传递、心肌功能和血小板聚集。可溶性鸟苷酸环化酶（sGC）是NO的主要受体。NO-sGC信号通路功能障碍可导致心脏病、勃起功能障碍、中风和高血压。了解no诱导的sGC活化的分子细节对于开发针对这些疾病状态的治疗方法至关重要。哺乳动物sGC是一种多结构域、异二聚体的血红蛋白。由于全长sGC已被证明难以进行高分辨率结构分析（例如x射线晶体学），因此对其结构和no诱导的构象变化知之甚少。本文提出的具体研究目的是通过平行、互补的蛋白质定位方法来阐明sGC的结构域组织和构象变化。氢-氘交换质谱（HDX-MS）是一种通过测量酰胺质子与氘化溶剂交换速率的变化来绘制蛋白质表面溶剂暴露的强大策略。HDX-MS将用于绘制sGC域截断的相互作用面。将进行丙氨酸扫描，以确定对sGC域间相互作用至关重要的单个残基。为了表征sGC结构域的相对取向，羟基自由基足迹将被采用。在区域间相互作用附近的试剂中产生的羟基自由基可以在近端残基处切割多肽主干。这些解理模式报告了sGC结构域表面之间的接近度。将整合HDX-MS、丙氨酸扫描和羟基自由基足迹的结果，以开发全长sGC的结构域结构模型。然后可以在全长sGC中评估NO刺激引起的sGC结构的变化。HDX-MS将用于绘制no诱导的sGC表面可达性变化，以建立控制sGC环化酶活性的构象变化模型。

DESCRIPTION (provided by applicant): Nitric oxide (NO) signaling pathways mediate diverse physiological functions, including vasodilation, neurotransmission, myocardial function, and platelet aggregation. Soluble guanylate cyclase (sGC) is the primary receptor of NO. Dysfunctions in the NO-sGC signaling pathway can lead to heart disease, erectile dysfunction, stroke, and hypertension. Understanding the molecular details of NO-induced sGC activation is crucial for developing treatments for these disease states. Mammalian sGC is a multi-domain, heterodimeric hemoprotein. Because full-length sGC has proven intractable to high resolution structure analysis (e.g., X-ray crystallography), the domain architecture and NO-induced conformational changes are poorly understood. The specific aims of the research proposed herein are focused on illuminating the domain organization and conformational changes of sGC through parallel, complementary protein mapping approaches. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful strategy for mapping the solvent exposure of protein surfaces by measuring changes in the rates of amide proton exchange with a deuterated solvent. HDX-MS will be used to map the interaction surfaces of sGC domain truncations. Alanine scanning will be performed to define individual residues that are crucial to sGC inter-domain interactions. To characterize the relative orientations of the sGC domains, hydroxyl radical footprinting will be employed. Hydroxyl radicals generated at a reagent tethered near an inter-domain interaction can cleave polypeptide backbones at proximal residues. These cleavage patterns report on the proximities between surfaces of sGC domains. The results of the HDX-MS, alanine scanning, and hydroxyl radical footprinting will be integrated to develop a model of the domain architecture of full-length sGC. Changes in the sGC domain architecture induced by NO stimulus can then be assessed in full-length sGC. HDX-MS will be used to map NO-induced changes in sGC surface accessibility to develop a model of the conformational changes that control sGC cyclase activity. PUBLIC HEALTH RELEVANCE: Nitric oxide (NO) signaling via soluble guanylate cyclase (sGC) mediates diverse physiological processes crucial to circulatory and neurological function. Disruptions in NO/sGC signaling have been linked to heart disease, stroke, erectile dysfunction, and neurodegeneration. The proposed research aims to illuminate the molecular details of NO-induced sGC activation, a prerequisite for developing treatments for diseases related to NO/sGC dysfunction.