The role of dynamics in enzyme mechanism and allostery

动力学在酶机制和变构中的作用

• 批准号: 9979900
• 负责人: Andrew L Lee
• 金额: $ 32.03万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-01-07 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9979900
• 关键词: Active Sites; Address; Affinity; Allosteric Regulation; Antineoplastic Agents; Basic Science; Behavior; Binding; Binding Sites; Biochemical; Calorimetry; Cells; Chemicals; Colorectal Neoplasms; Communication; Complement; Complex; Coupled; Coupling; Crystallography; Data; Deoxyuridine; Drug Targeting; Engineering; Enzymes; Equilibrium; Escherichia coli; Evaluation; Exhibits; Fluorouracil; Funding; Future; G-Protein-Coupled Receptors; Goals; Growth Factor; Health; Homo; Human; Investigation; Knowledge; Label; Ligand Binding; Link; Metabolic; Metabolism; Methods; Modeling; Molecular; Molecular Conformation; Monitor; Multienzyme Complexes; Mutation; N-terminal; NMR Spectroscopy; NOESY; Nature; Nuclear Hormone Receptors; Nucleotides; Organism; Pharmaceutical Preparations; Phosphotransferases; Problem Solving; Process; Proteins; Protomer; Raltitrexed; Refractory; Regulation; Relaxation; Research; Residual state; Resistance; Resolution; Role; Signal Transduction; Site; Source; Structure; System; Testing; Thermodynamics; Thymidylate Synthase; Titrations; Tumor-Derived; Work; X-Ray Crystallography; analog; antiproliferative drugs; base; cell behavior; chemotherapy; cofactor; cytokine; design; dimer; engineering design; enzyme activity; enzyme mechanism; enzyme structure; expectation; flexibility; genetic regulatory protein; improved; insight; molecular dynamics; mutant; novel; protein complex; protein function; resistance mutation; simulation; structural biology; thymidylate; tool

Abstract – The role of dynamics in enzyme mechanism and allostery Enzymes are complex molecules that perform difficult chemical transformations and regulate biochemical activities that maintain cell health. Although many structures of enzymes are available, numerous aspects of enzyme function remain hidden in their dynamics and transient deformations. NMR spectroscopy, in concert with other methods, has helped to realize how dynamics assists protein function on a variety of timescales. However, such studies have been largely limited to small enzymes and proteins. Typically, enzymes are large with complex features, and they are often oligomeric and symmetric in ways that are intimately tied to their allosteric regulation/function. There is thus a need to increase access to the rich dynamics and other NMR-sensitive parameters that exist in complex enzymes and larger proteins. The work proposed here aims to 1) solve long- standing problems in the general study of allostery in symmetric homodimers and 2) gain crucial information on highly flexible regions important for the function of a (large) metabolic enzyme that is a primary target for chemotherapies. Structural and dynamic processes will be examined in the human (70 kDa) and E. coli (62 kDa) versions of thymidylate synthase (TS), which methylates deoxyuridine monophosphate (dUMP) to produce the dTMP nucleotide. TS is a symmetric homodimer that is “half-the-sites reactive”, which is interesting from the perspective of allostery since the active sites are separated by 35 Å. Although the half-the-sites nature of TS gives an expectation of negative thermodynamic cooperativity between the two subunits, we have showed that substrate binding cooperativity is nonexistent or small in ecTS. By contrast, hTS has pronounced negative binding cooperativity, as well as more conformational changes and additional sequence segments of high flexibility. Comparison of residue-specific behavior in the ecTS and hTS systems will yield insights into mechanisms of allostery in symmetric homodimers. Our previous work on ecTS produced an NMR strategy that enables clean, protomer-selective observation of step-wise ligand binding that is necessary to evaluate intersubunit allosteric mechanisms in homodimers. This work will be extended to further characterize intersubunit communication in ecTS in Aim 1 and applied separately to hTS in Aim 3, along with computational work to supply molecular details of the dynamics. The knowledge gained will advance the delineation of principles of allosteric communication, which are needed to engineer or control it in proteins and improve design of allosteric drugs. In Aim 2, structural and dynamic features of function of hTS will be determined, including the role of the highly flexible and hitherto invisible 29-residue N-terminus. In addition, the means by which a tumor-derived resistance mutation weakens affinity to cancer drug 5-FU will be investigated. In summary, this work will use a combination of NMR, ITC, crystallography, and MD simulations to reveal dynamics-based function and mechanisms of allostery in a complex, symmetric, enzyme homodimer.

摘要-动力学在酶机制和变构中的作用 酶是复杂的分子，执行困难的化学转化并调节生物化学反应。 维持细胞健康的活动。虽然酶的许多结构是可用的，但是酶的许多方面是不可用的。 酶的功能仍然隐藏在它们的动力学和瞬时变形中。核磁共振光谱，与 其他方法，有助于认识到动力学如何帮助蛋白质在各种时间尺度上发挥作用。然而，在这方面， 这些研究主要限于小的酶和蛋白质。通常，酶是大的，具有复杂的 特征，并且它们通常是低聚的和对称的，与它们的变构密切相关。 调节/功能。因此，有必要增加对丰富的动态和其他NMR敏感信息的访问。 存在于复杂酶和较大蛋白质中的参数。本文的主要目的是：（1）解决长期的... 在对称同二聚体变构的一般研究中存在的问题，2）获得关于 高度灵活的区域对（大）代谢酶的功能很重要，代谢酶是主要靶点， 化疗结构和动力学过程将在人类（70 kDa）和E。大肠杆菌（62 kDa） 胸苷酸合酶（TS）的形式，其甲基化脱氧尿苷一磷酸（dUMP）以产生 dTMP核苷酸。TS是对称的同二聚体，其是“半位点反应性的”，这是有趣的，因为TS是对称的。 由于活性位点相隔35 μ m，因此从变构的角度来看。尽管TS的一半站点性质 给出了两个亚基之间的负热力学协同性的预期，我们已经表明， 底物结合协同性在ecTS中不存在或很小。相比之下，HTS具有明显的负向 结合协同性，以及更多的构象变化和额外的序列片段的高 灵活性.比较ecTS和hTS系统中的残留物特异性行为将深入了解 对称同二聚体的变构机制。我们先前关于ecTS的工作产生了一种NMR策略， 能够清楚地、选择性地观察逐步配体结合， 同二聚体中的亚基间变构机制。这项工作将被扩展到进一步表征intersubunit 目标1中的ecTS中的通信，并单独应用于目标3中的hTS，沿着计算工作，以提供 动力学的分子细节。所获得的知识将推进对变构原理的描述 这是工程师或控制蛋白质中的它和改进变构药物设计所需要的。在 目的2、确定高温超导体功能的结构特征和动力学特征，包括高温超导体中高分子的作用。 柔性和迄今不可见的29个残基的N-末端。此外，肿瘤来源的耐药性的方式 将研究突变减弱对癌症药物5-FU的亲和力。总之，这项工作将使用一个组合 NMR，ITC，晶体学和MD模拟，以揭示基于动力学的功能和机制， 在复杂的、对称的、酶同二聚体中的变构。

项目成果

Backbone and ILV methyl resonance assignments of E. coli thymidylate synthase bound to cofactor and a nucleotide analogue.

• DOI: 10.1007/s12104-013-9482-6
• 发表时间: 2014-04
• 期刊: Biomolecular NMR assignments
• 影响因子: 0.9
• 作者: Sapienza PJ;Lee AL
• 通讯作者: Lee AL

Contrasting roles of dynamics in protein allostery: NMR and structural studies of CheY and the third PDZ domain from PSD-95.

• DOI: 10.1007/s12551-015-0169-3
• 发表时间: 2015-06-01
• 期刊: Biophysical reviews
• 作者: Lee, Andrew L

Thermodynamic and NMR Assessment of Ligand Cooperativity and Intersubunit Communication in Symmetric Dimers: Application to Thymidylate Synthase.

• DOI: 10.3389/fmolb.2018.00047
• 发表时间: 2018
• 期刊: Frontiers in molecular biosciences
• 影响因子: 5
• 作者: Lee AL;Sapienza PJ
• 通讯作者: Sapienza PJ

Structure and dynamics of the G121V dihydrofolate reductase mutant: lessons from a transition-state inhibitor complex.

• DOI: 10.1371/journal.pone.0033252
• 发表时间: 2012
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Mauldin RV;Sapienza PJ;Petit CM;Lee AL
• 通讯作者: Lee AL

Evidence for dynamics in proteins as a mechanism for ligand dissociation.

• DOI: 10.1038/nchembio.769
• 发表时间: 2012-01-15
• 期刊: NATURE CHEMICAL BIOLOGY
• 影响因子: 14.8
• 作者: Carroll, Mary J.;Mauldin, Randall V.;Gromova, Anna V.;Singleton, Scott F.;Collins, Edward J.;Lee, Andrew L.
• 通讯作者: Lee, Andrew L.