Structure/Function of Complex II Oxidoreductase

描述（由申请人提供）：复合物II酶是具有两个空间上分离的活性位点的整合膜异源四聚体，所述活性位点将琥珀酸盐和富马酸盐的氧化还原偶联至醌和醌醇的氧化还原。在人类中，复合物II是一种线粒体酶，通过参与TCA循环（通过琥珀酸氧化）和氧化磷酸化（通过醌还原）来产生能量，其突变与肿瘤形成和神经变性有关。该研究计划的长期目标是揭示催化，组装和生理功能的机制。在拟议的研究中，我们将确定复合物II组装因子的作用，揭示复合物II如何有助于体内信号传导，并展示疾病相关突变如何改变复合物II功能。 目的1：直到最近，复合物II组装被认为是自催化进行的，但现在已经在人类中确定了两个组装因子。两者都被提出来帮助辅因子插入，但支持这一点的已发表证据是脆弱的，也没有组装的共识机制。了解这些组装因子的作用，将提供深入了解这一重要蛋白质复合物组装的基本机制。我们将使用大肠杆菌复合物II来揭示每个组装因子的作用，确定它们是否直接或间接起作用，并使用基因缺失和互补、诱变、体内位点特异性交联、结合测定和晶体学来确定是否有额外的组装因子。 目的2：在人类和细菌中，复合物II的活性是生物能量学之外的功能所必需的。例如，在大肠杆菌中，鞭毛的组装和对富马酸盐的趋化反应需要复合物II同系物QFR的活性。由于未来的抗菌剂可能会直接破坏细菌的运动，这将是重要的是要揭示QFR如何支持鞭毛组装。我们将结合联合收割机位点特异性交联、结合测定、运动测定、晶体学和计算建模来确定QFR如何影响鞭毛扭矩环的构象以促进组装和影响趋化性。 目的3：编码人类复合物II的基因突变与多效性临床表现相关。与这些突变相关的细胞变化已在文献中报道，但几乎没有人知道这些突变对酶活性的生化后果。确定与复合体II突变相关的生化变化对于开发线粒体疾病的未来疗法是重要的。因此，我们将开发一个人类复合物II的表达系统，并评估疾病相关突变对酶组装，动力学和结构的影响。

DESCRIPTION (provided by applicant): Complex II enzymes are integral-membrane heterotetramers with two spatially separated active sites that couple the oxidoreduction of succinate and fumarate to the oxidoreduction of quinone and quinol. In humans, Complex II is a mitochondrial enzyme that contributes to energy generation by participating in both the TCA cycle (via succinate oxidation) and oxidative phosphorylation (via quinone reduction), and its mutation is associated with tumor formation and neurodegeneration. The long-term objective of this research program is to reveal mechanisms of catalysis, assembly, and physiological function. In the proposed research, we will identify roles of Complex II assembly factors, reveal how Complex II contributes to in vivo signaling, and demonstrate how disease-associated mutations alter Complex II function. Aim 1: Until very recently, Complex II assembly was thought to proceed autocatalytically, but two assembly factors have now been identified in humans. Both are proposed to assist in cofactor insertion, but the published evidence supporting this is tenuous and there is no consensus mechanism of assembly. Understanding the roles of these assembly factors will provide insight into fundamental mechanisms of assembly of this essential protein complex. We will use Escherichia coli Complex II to reveal the role of each assembly factor, identify if they work directly or indirectly, and identify whether thre are additional assembly factors using gene deletion and complementation, mutagenesis, in vivo site-specific cross-linking, binding assays, and crystallography. Aim 2: In both humans and bacteria, Complex II activity is required for functions outside of bioenergetics. For example, in E coli, assembly of the flagellum and the chemotactic response to fumarate requires activity of the Complex II homolog QFR. Since future antimicrobials could be directed toward disruption of bacterial motility, it will be important to reveal how QFR supports flagellar assembly. We will combine site-specific cross- linking, binding assays, motility assays, crystallography, and computational modeling to identify how QFR influences the conformation of the flagellar torque ring to promote assembly and influence chemotaxis. Aim 3: Mutations in the genes encoding human Complex II are associated with pleiotropic clinical presentations. Cellular changes associated with these mutations have been reported in the literature, but almost nothing is known about the biochemical consequences of these mutations on enzyme activity. Identifying the biochemical changes associated with Complex II mutation is important for developing future therapies for mitochondrial disease. We will therefore develop an expression system for human Complex II and assess the consequences of disease-associated mutations on enzyme assembly, kinetics, and structure.