Linking the conformational landscape to enzymatic function through functional site distant mutations

一个正在进行的争论涉及构象运动，通常称为动力学，在生物细胞中的作用。 功能。对于酶来说，大尺度畴运动的时间尺度与 表观“$$）。 催化速率（Kr！“#这一观察是主要争论点的发展： "$$.在这方面，拟议的研究 构象运动直接影响催化的真实速率（100%）。“#）或！"# 我将进一步探索构象景观的调制如何确实可以微调“$$没有 快！"# 冲击波！“#和基态结构。这个提议的起源来自我们对人类的研究。 鸟苷酸激酶（hGMPK），一种治疗癌症甚至SARS-CoV-2的潜在治疗靶点， 这促使我们用核磁共振（NMR）光谱来解决hGMPK的第一个结构 (PDB：6NUI）。在解析hGMPK结构的同时，我们表达了一系列的七个功能位点距离（FSD）， “与野生动物相比， hGMPK的非同义单核苷酸变体（nsSNV），其增强了人GMPK的免疫原性。𝑘"# 类型（wt）。有趣的是，wt hGMPK及其nsSNV的2D [1H，15 N]-HSQC NMR光谱表明， 𝑎𝑝𝑝GMP结合）， FSD突变对hGMPK骨架折叠的影响最小，但表观解离速率（表观解离速率） wt和FSD突变体V91 M相差约3000 s-1。我们假设hGMPK的活性可以通过 FSD突变体通过重塑构象景观。利用NMR光谱和等温 我们将在以下两个具体目标中检验这一假设。在目标1中，我们将量化影响 从动力学和热力学角度分析了FSD突变对构象的影响。结果 从这一目标将提供一个全面的图片，在hGMPK催化和结合方案 FSD突变对功能的影响最大。对于目标2，我们将解卷积贡献瞬态 构象景观中的结构通过实验驱动在酶催化中发挥作用， 合奏一代。我们的方案将从无偏分子动力学（MD）中选择hGMPK结构 基于残余偶极耦合和用NMR测量的交叉相关弛豫速率的模拟。的 系综将有助于识别功能上重要的瞬时构象和评估 FSD突变对主链二面角相关运动的影响。据我们所知，这项提议 提供了第一个示例1）。实验驱动的，整体生成的酶跨越 生理相关的时间尺度和2.）热力学和动力学参数的测定 与一系列酶变体上的同一位点结合。这一建议的影响是直接联系 酶的功能。这些成果的直接应用包括药物 发现，其中靶向构象景观内的结构，而不是基态结构 将导致更好的结果，和生物分子设计，其中FSD突变可以实施，以调整 通过操纵构象景观来发挥作用。

An ongoing debate concerns the role conformational motions, often termed dynamics, play in biomolceular funtion. For enzymes, it so happens that the timescales for large-scale domain motions are similar to the apparent "$$). catalytic rate (𝑘!"# This observation is where the major point of contention has developed: do "$$. In this context, the proposed studies conformational motions directly impact the true rate of catalysis (𝑘!"#) or 𝑘!"# will further explore how the modulation of the conformational landscape can indeed fine-tune "$$ without 𝑘!"# impacting 𝑘!"# and the ground state structure. The genesis of this proposal arises from our work with human guanylate kinase (hGMPK), a potential therapeutic target for treating cancer and perhaps even SARS-CoV-2, which motivated us to solve the first structure of hGMPK with nuclear magnetic resonance (NMR) spectroscopy (PDB: 6NUI). While solving the hGMPK structure, we expressed a series of seven functional site distant (FSD), "$$ when compared to the wild- non-synonymous single nucleotide variants (nsSNVs) of hGMPK that enhance 𝑘!"# type (wt). Intriguingly, the 2D [1H,15N]-HSQC NMR spectra of the wt hGMPK and its nsSNVs suggest that the 𝑎𝑝𝑝) for GMP binding to FSD mutations minimally impact hGMPK’s backbone fold, yet the apparent off-rates (𝑘𝑜𝑓𝑓 wt and the FSD mutant V91M differ by ~3000 s-1. We hypothesize that hGMPK’s activity can be modulated with FSD mutants by reshaping the conformational landscape. Utilizing NMR spectroscopy and isothermal calorimetry, we will test this hypothesis in the following two Specific Aims. In Aim 1, we will quantify the impact of the FSD mutations on the conformational landscape from kinetic and thermodynamic perspectives. The results from this Aim will provide a comprehensive picture as to where within the hGMPK catalytic and binding schemes the FSD mutations have the largest impact on function. For Aim 2, we will deconvolute the contribution transient structures within the conformational landscape play in enzymatic catalysis through experimentally driven ensemble generation. Our protocol will select hGMPK structures from unbiased molecular dynamics (MD) simulations based on residual dipolar couplings and cross-correlated relaxation rates measured with NMR. The ensembles will aid in the identification of the functionally important transient conformations and an assessment of the impact the FSD mutations have on backbone dihedral correlated motions. To our knowledge, this proposal provides the first examples 1.) of experimentally driven, ensemble generation for an enzyme spanning physiologically relevant timescales and 2.) of determining thermodynamic and kinetic parameters with ligand binding to the same exact site on a series of enzyme variants. The impact of this proposal is the direct linkage of the conformational landscape to enzymatic function. Immediate applications for these results include drug discovery, where targeting structures within the conformational landscape rather than the ground state structure will lead to better outcomes, and biomolecular design, where FSD mutations can be implemented to adjust function through manipulation of the conformational landscape.