Intra- and Intermolecular Dynamics of Dihydrofolate Reductase

描述（由申请人提供）：二氢叶酸还原酶（DHFR）是一种小的，~ 20kd的酶，催化将7,8-二氢叶酸（DHF）还原为5,6,7,8-四氢叶酸（THF），这是DNA生物合成所需的关键代谢物。由于DHFR在代谢中的中心位置和细胞增殖所需的活性，它已成为治疗人类癌症和传染病的一个有吸引力的药物靶点。此外，来自不同生物体的DHFRs序列和结构特性的差异使得许多这些药物具有高特异性。另一方面，对大肠杆菌DHFR的广泛生化和结构研究使DHFR成为蛋白质结构动态波动如何促进酶功能的范例。现在已知DHFR在微秒到毫秒的时间尺度上经历不同构象状态之间的转换，以一种将功能循环中的一个步骤连接到相邻步骤的方式。在这里，大肠杆菌DHFR的研究是鉴于其作为蛋白质-药物相互作用和酶动力学模型的重要性。将采用跨学科的实验方法，结合蛋白质核磁共振弛豫与瞬态和预稳态动力学，研究DHFR对化学变性剂和抗叶酸抑制剂的行为响应，这些亲和剂跨越五个数量级。由于脱落率是结合亲和力的关键决定因素，因此将关注内部动力学和构象变化对配体解离的作用，在天然底物和抗叶酸盐的情况下。皮秒-纳秒波动在稳定束缚三元态和促进协同构象变化中的作用将被评估，特别关注侧链迁移。DHFR系统提供了一个很好的机会来研究内部动力学对不同构象状态下叶酸/抗叶酸排出的影响；相反，这些研究将解决构象背景如何定义配体占用和释放的动力学。鉴于构象变化是集体运动，残基之间的内在连通性将使用核磁共振微扰响应方法进行映射。在整个研究中，重点将放在含有还原性烟酰胺腺嘌呤二核苷酸磷酸辅助因子（NADPH）的DHFR复合物上。总之，本应用旨在获得DHFR中蛋白质-药物相互作用、缓慢构象变化、配体抛射和分子内通信的内部动力学作用的机制见解。

DESCRIPTION (provided by applicant): Dihydrofolate reductase (DHFR) is a small, ~20 kD enzyme that catalyzes the reduction of 7,8- dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate (THF), a key metabolite required for DNA biosynthesis. Because of DHFR's central location in metabolism and required activity for cell proliferation, it has become an attractive drug target for the treatment of human cancers and infectious diseases. Furthermore, differences in sequence and structural properties in DHFRs from different organisms allow many of these drugs to act with high specificity. Under a separate light, extensive biochemical and structural studies on E. coli DHFR have led to DHFR becoming a paradigm for how dynamic fluctuations in protein structure facilitate enzyme function. DHFR is now known to undergo switching between distinct conformational states on a microsecond to millisecond timescale, in a manner that connects one step in the functional cycle to adjacent steps. Here, E. coli DHFR is studied in light of its importance as a model for both protein-drug interactions and enzyme dynamics. An interdisciplinary experimental approach combining protein NMR relaxation with transient and pre-steady-state kinetics will be employed to study the response of DHFR behavior to chemical denaturants and antifolate inhibitors with affinities spanning five orders of magnitude. Since off-rates are a key determinant of binding affinity, attention will be paid to the role of internal dynamics and conformational changes to ligand dissociation, in the cases of both natural substrates and antifolates. The role of picosecond-nanosecond fluctuations in stabilizing bound ternary states and promoting concerted conformational changes will be assessed, with particular focus on side-chain mobility. The DHFR system presents an excellent opportunity to study the influence of internal dynamics on folate/antifolate ejection from different conformational states; conversely, these studies will address how conformational context defines the dynamics of ligand occupancy and release. Given that conformational changes are collective motions, the inherent connectivity between residues will be mapped using an NMR perturbation-response approach. Throughout this research, emphasis will be placed on DHFR complexes containing reduced nicotinamide adenine dinucleotide phosphate cofactor (NADPH). In summary, this application seeks to gain mechanistic insights into the role of internal dynamics in protein-drug interactions, slow conformational changes, ligand ejection, and intramolecular communication in DHFR. Project Narrative Dihydrofolate reductase is the target for drugs used to treat cancer and infectious diseases, and it serves as a model for understanding protein-drug interactions. By using protein NMR spectroscopy and enzyme kinetics to identify mechanisms of protein flexibility that either stabilize or destabilize drug occupancy, a greater understanding of the determinants of drug binding affinity will be obtained. This new knowledge will increase the efficiency of the design of small molecule inhibitors to dihydrofolate reductase and other proteins.