Biophysical rescue of Coagulation Factor IXa conformational ensembles from hemophilia B disease mutations

从血友病 B 疾病突变中生物物理拯救凝血因子 IXa 构象整体

• 批准号: 8981574
• 负责人: Michael C. Thompson
• 金额: $ 5.24万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8981574
• 关键词: Active Sites; Affect; Alleles; Allosteric Site; Binding; Binding Sites; Biochemical; Biological Assay; Blood; Blood Coagulation Disorders; Blood coagulation; Calcium; Catalytic Domain; Coagulation Process; Communication; Complex; Computer Simulation; Computing Methodologies; Coupled; Coupling; DNA Sequence Alteration; Data; Data Collection; Dependence; Development; Disease; Distal; Drug Design; Engineering; Enzymes; Equilibrium; Factor IX; Factor IXa; Factor VIIIa; Factor X; Functional disorder; Future; Goals; Hemophilia A; Hemophilia B; Hemostatic function; Hereditary Disease; Heterogeneity; Ions; Knowledge; Maps; Measures; Metals; Methodology; Methods; Modeling; Molecular; Molecular Conformation; Molecular Structure; Mutation; Nature; Other Genetics; Pathway interactions; Patients; Point Mutation; Protein Conformation; Proteins; Regulation; Research; Resolution; Roentgen Rays; Role; Sampling; Series; Site; Structure; Suppressor Mutations; Surface; System; Techniques; Temperature; Testing; Therapeutic Agents; Thrombosis; Variant; Vision; X-Ray Crystallography; base; biophysical techniques; cancer procoagulant; clinically relevant; conformer; disease phenotype; functional restoration; insight; mutant; novel therapeutics; protein complex; protein function; public health relevance; small molecule; therapeutic development

 DESCRIPTION (provided by applicant): The broad goal of the proposed research is to gain a deeper understanding of the molecular mechanisms underlying hemophilia B. The blood coagulation cascade is a complex biochemical system that is regulated extensively in order to achieve hemostasis without inducing thrombosis. A key component in the activation and regulation of sustained coagulation is a protein complex known as the intrinsic Xase. This complex consists of the Factor IXa (fIXa) enzyme, along with its allosteric activator Factor VIIIa (fVIIIa) and several metal ions which also modulate its activity. Hemophilia B is caused by dysfunction of fIXa, the catalytic subunit of the intrinsic Xase. A specific subset of mutations tht cause hemophilia B do not affect the concentration of fIXa in the blood and do not significantly destabilize the protein. However, these mutations do impair the catalytic function of fIXa. We hypothesize that these mutations perturb conformational equilibria that are critical for allosteric communication, substrate binding, and enzymatic turnover. In order to test this hypothesis, we will first dissect allosteric communication pathways in fIXa using multi-temperature X-ray crystallography and computational approaches. These analyses will identify correlated conformational heterogeneity and energetic coupling between distal regions of the enzyme. Next, we will apply this same methodology to fIXa point mutants that are associated with hemophilia B. Combined with functional assays, this information will allow us to quantitatively understand how mutational perturbations to the conformational ensemble effect enzymatic activity. Finally, we will rationally engineer fIXa mutations whose effects suppress the disease phenotype in hemophilia B variants. This strategy of rescuing function by restoring the native fIXa conformational ensemble will inform future development of therapeutics by identifying allosteric networks that can be targeted with small molecules. Looking forward, the methodology of biophysical rescue that we will establish can be applied to other genetic diseases that result from single point mutations.

 描述（由适用提供）：拟议的研究的广泛目标是对血友病B的分子机制有更深入的了解B。血液凝结级联是一个复杂的生化系统，受到广泛调节，以实现没有诱导血栓形成的止血。持续凝结激活和调节的关键成分是一种蛋白质复合物，称为固有Xase。该复合物由IXA因子（FIXA）酶以及其变构激活因子VIIIA（FVIIIA）和几种也调节其活性的金属离子。血友病B是由固有Xase的催化亚基FIXA功能障碍引起的。突变的特定子集导致血友病B不会影响血液中固定剂的浓度，并且不会显着破坏蛋白质的稳定。但是，这些突变确实会损害FIXA的催化功能。假设这些突变扰动构象平衡对于变构至关重要 通信，底物结合和酶促转换。为了检验这一假设，我们将使用多温X射线晶体学和计算方法首先剖析FIXA中的变构通信途径。这些分析将确定酶远端区域之间相关的构象异质性和能量耦合。接下来，我们将对与血友病B相关的FIXA点突变体应用相同的方法。与功能分析相关联，此信息将使我们能够定量地了解突变扰动如何对构象合奏效应酶促活性。最后，我们将合理地设计固定性固定性突变，其影响抑制了血友病B变体中的疾病表型。通过恢复本地固定构象合奏来挽救功能的这种策略将通过识别可以用小分子靶向的变构网络来为理论的未来发展提供信息。展望未来，我们将确定的生物物理救援方法可以应用于单点突变引起的其他遗传疾病。