Structural and Functional Basis of the Vitamin K Cycle

维生素K循环支持血液凝固、骨矿化和血管钙稳态。一把钥匙 这个循环中的酶，维生素K环氧化物还原酶(VKOR)，是维生素K拮抗剂(VKAs)的靶标。尽管 其广泛的临床应用，VKA的剂量(例如，华法林)很难控制，过量可导致死亡 在流血。改善剂量调节需要了解VKA如何抑制VKOR，VKOR是一种膜- 很难用结构和生物化学研究来表征的嵌入酶。我们的长期目标是 阐明整个维生素K循环的生理过程及其与VKA的相互作用。这个周期 从γ-羧化开始，这是维生素K依赖蛋白活性所必需的修饰，包括 几种凝血因子。羧基酶的活性需要维生素K对苯二酚的环氧化。VKOR 通过还原环氧化物来再生这种辅因子，并通过电子转移来保持这种还原酶的活性。 小路。VKOR还有一个Paralog，VKORL，它具有相同的活动，但对华法林相对不敏感 抑制力。由于组织分布的不同，VKOR主要支持凝血和VKORL 可能支持非凝血过程。本申请的目的是阐明其机制。 VKOR和VKORL催化和维生素K拮抗利用我们在膜结构生物学方面的专业知识。我们的 假设是：(1)华法林治疗窗口狭窄的部分原因是它是一种紧密结合的抑制剂。 VKOR的剂量范围受VKOR水平的限制；(2)VKOR的细胞活性通过替代方案维持 电子转移途径；和(3)共同的结构机制控制华法林不敏感的VKORL 以及VKOR中的华法林耐药突变。为了支持这些假设，我们实现了一个长期目标 用几种VKA和底物维生素K环氧化物测定人VKOR的晶体结构， 并确定了VKORL同系物在华法林结合和无配体状态下的结构。我们发现 VKOR中不同的华法林耐药突变组，并确定了控制华法林的关键残基 VKORL的灵敏度。我们还证明了华法林在体外是一种紧密结合的抑制剂。我们将检验我们的假设 具体有三个目标：(1)我们将展示华法林在细胞环境中的紧密结合，了解其 与VKOR的氧化还原状态相关，并测试降低VKOR是否可以释放结合的华法林；(2)我们将 确定VKA如何抑制VKOR催化，阐明VKOR的还原步骤和反应中间体， 并描述维持VKOR活性的电子转移途径；以及3)我们将定义结构 并调查VKORL变异是否导致骨质疏松症。配备着我们的 最近开发的结构工具，我们将演示关于VKA抑制范围的创新概念， VKOR的催化途径，以及干扰凝血和骨健康的突变。因此，拟议的 研究将显著提高我们对VKOR功能及其与VKA相互作用的知识，从而导致 改进了华法林管理。

The vitamin K cycle supports blood coagulation, bone mineralization, and vascular calcium homeostasis. A key enzyme in this cycle, vitamin K epoxide reductase (VKOR), is the target of vitamin K antagonists (VKAs). Despite their extensive clinical use, the dose of VKAs (e.g., warfarin) is hard to regulate and overdose can lead to fatal bleeding. Improving the dose regulation requires understanding how VKAs inhibit VKOR, which is a membrane- embedded enzyme that is difficult to characterize with structural and biochemical studies. Our long-term goal is to elucidate the physiological process of the entire vitamin K cycle and its interaction with VKAs. This cycle begins with γ-carboxylation, a modification required for the activity of vitamin-K-dependent proteins, including several coagulation factors. The carboxylase activity requires the epoxidation of vitamin K hydroquinone. VKOR regenerates this cofactor by reducing the epoxide, and this reductase activity is maintained by electron-transfer pathways. VKOR also has a paralog, VKORL, which has the same activity but is relatively insensitive to warfarin inhibition. Owing to differences in tissue distribution, VKOR primarily supports blood coagulation and VKORL likely supports non-coagulation processes. The objective of this application is to elucidate the mechanisms of VKOR and VKORL catalysis and vitamin K antagonism using our expertise in membrane structure biology. Our hypotheses are: (1) the narrow therapeutic window of warfarin is in part because it is a tight-binding inhibitor whose dose range is limited by VKOR levels; (2) the cellular activity of VKOR is maintained by alternative electron-transfer pathways; and (3) a common structural mechanism governs the warfarin insensitivity of VKORL and warfarin-resistant mutations in VKOR. To support these hypotheses, we have achieved a long-standing goal of determining the crystal structures of human VKOR with several VKAs and with the substrate, vitamin K epoxide, and have determined the structures of a VKORL homolog in its warfarin-bound and ligand-free states. We found distinct groups of warfarin-resistant mutations in VKOR, and identified key residues that control the warfarin sensitivity of VKORL. We also showed that warfarin is a tight-binding inhibitor in vitro. We will test our hypotheses with three specific aims: (1) we will show the tight binding of warfarin in a cellular environment, understand its correlation with VKOR's redox status, and test whether reducing VKOR can release bound warfarin; (2) we will determine how VKAs inhibit VKOR catalysis, elucidate the reduction steps and reaction intermediate of VKOR, and characterize the electron-transfer pathways that maintain VKOR activity; and 3) we will define the structural basis of warfarin resistance and investigate whether VKORL variations lead to osteoporosis. Armed with our recently developed structural tools, we will demonstrate innovative concepts about the inhibition range of VKAs, the catalytic pathway of VKOR, and mutations interfering with coagulation and bone health. Thus, the proposed studies will significantly advance our knowledge of VKOR function and its interaction with VKAs, leading to improved warfarin management.