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: 9330246
• 负责人: Michael C. Thompson
• 金额: $ 5.92万
• 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/9330246
• 关键词: Active Sites; Affect; Alleles; Allosteric Site; Binding; Binding Sites; Biochemical; Biological Assay; Biophysics; Blood; Blood Coagulation Disorders; Blood coagulation; Calcium; Catalytic Domain; Coagulation Process; Communication; Complex; Computer Simulation; Computing Methodologies; Coupled; Coupling; Crystallization; DNA Sequence Alteration; Data; Data Collection; Dependence; Disease; Distal; Drug Design; Engineering; Enzymes; Factor IX; Factor IXa; Factor VIIIa; Factor X; Functional disorder; Future; Goals; Hemophilia A; Hemophilia B; Hemostatic function; Hereditary Disease; Heterogeneity; Impairment; Ions; Knowledge; Maps; Measures; Metals; Methodology; Methods; Modeling; Modernization; Molecular; Molecular Conformation; Molecular Structure; Mutation; Nature; Other Genetics; Pathologic; Pathway interactions; Patients; Point Mutation; Protein Conformation; Proteins; Regulation; Research; Resolution; Resuscitation; Roentgen Rays; Role; Sampling; Series; Site; Structure; Suppressor Mutations; Surface; System; Techniques; Temperature; Testing; Thrombosis; Variant; Vision; X-Ray Crystallography; biophysical techniques; cancer procoagulant; clinically relevant; conformer; disease phenotype; functional restoration; hexachlorocyclohexane x-factor; insight; mutant; novel therapeutics; protein complex; protein function; public health relevance; small molecule; synergism; 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（由申请人提供）：拟议研究的广泛目标是更深入地了解血友病B的分子机制。凝血级联是一个复杂的生化系统，其被广泛调节以实现止血而不诱导血栓形成。激活和调节持续凝血的关键组分是称为内源性Xase的蛋白质复合物。该复合物由因子IXa（fIXa）酶、沿着其变构激活剂因子VIIIa（fVIIIa）和也调节其活性的几种金属离子组成。血友病B是由fIXa功能障碍引起的，fIXa是内源性Xase的催化亚基。引起血友病B的特定突变亚组不影响血液中fIXa的浓度，也不显著破坏蛋白质的稳定性。然而，这些突变确实损害了fIXa的催化功能。我们假设这些突变扰乱了对变构至关重要的构象平衡。 通讯、底物结合和酶周转。为了验证这一假设，我们将首先使用多温X射线晶体学和计算方法解剖fIXa中的变构通信途径。这些分析将确定相关的构象异质性和酶的远端区域之间的能量耦合。接下来，我们将把同样的方法应用于与血友病B相关的fIXa点突变体。结合功能测定，这些信息将使我们能够定量地了解突变扰动的构象合奏效果酶的活性。最后，我们将合理地设计fIXa突变，其作用抑制血友病B变体中的疾病表型。这种通过恢复天然fIXa构象整体来挽救功能的策略将通过鉴定可以用小分子靶向的变构网络来告知治疗剂的未来发展。展望未来，我们将建立的生物物理拯救方法可以应用于由单点突变引起的其他遗传疾病。