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

 描述(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。血液凝固级联是一个复杂的生化系统，受到广泛的调节，以实现止血而不会导致血栓形成。激活和调节持续凝血的一个关键成分是一种被称为内在Xase的蛋白质复合体。该复合体由因子IXa(FixA)酶及其变构激活剂因子VIIIa(FVIIIa)和几个也调节其活性的金属离子组成。血友病B是由固有Xase的催化亚单位FixA功能障碍引起的。导致血友病B的特定突变子集不会影响FixA在血液中的浓度，也不会显着破坏蛋白质的稳定性。然而，这些突变确实削弱了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、底物结合和酶的周转。为了验证这一假说，我们将首先使用多温度X射线结晶学和计算方法剖析FixA中的变构通讯途径。这些分析将确定相关的构象异质性和酶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相关的固定点突变。结合功能分析，这些信息将使我们能够定量地了解构象集成的突变扰动如何影响酶活性。最后，我们将合理地设计FixA突变，其作用是抑制血友病B变种的疾病表型。这种通过恢复天然FixA构象集合来挽救功能的策略将通过识别可以被小分子靶向的变构网络来指导未来的治疗学发展。展望未来，我们将建立的生物物理拯救方法学可以应用于其他由单点突变引起的遗传病。

 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.