Intra- and Intermolecular Dynamics of Dihydrofolate Reductase

描述（由申请人提供）：二氢叶酸还原酶（DHFR）是一种约20 kD的小酶，可催化7，8-二氢叶酸（DHF）还原为5，6，7，8-四氢叶酸（THF），后者是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感染性疾病的有吸引力的药物靶标。此外，来自不同生物体的DHFR的序列和结构特性的差异允许这些药物中的许多以高特异性起作用。在单独的灯光下，对E.大肠杆菌DHFR已经导致DHFR成为蛋白质结构的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，其方式将功能循环中的一个步骤连接到相邻步骤。给你E coli DHFR作为蛋白质-药物相互作用和酶动力学模型的重要性。一个跨学科的实验方法相结合的蛋白质NMR弛豫与瞬态和预稳态动力学将被用来研究DHFR行为的化学变性剂和抗叶酸剂抑制剂的亲和力跨越五个数量级的响应。由于解离速率是结合亲和力的关键决定因素，因此在天然底物和抗叶酸剂的情况下，将注意内部动力学和构象变化对配体解离的作用。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，特别关注侧链的流动性。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的叶酸/抗叶酸剂排出的影响;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，残基之间的固有连接将使用NMR扰动响应方法进行映射。在整个研究过程中，重点将放在含有还原型烟酰胺腺嘌呤二核苷酸磷酸辅因子（NADPH）的DHFR复合物上。总之，该应用程序旨在获得机制的见解，在蛋白质-药物相互作用，缓慢的构象变化，配体排出，和分子内通信DHFR的内部动力学的作用。 项目叙述 二氢叶酸还原酶是用于治疗癌症和感染性疾病的药物的靶标，并且它作为理解蛋白质-药物相互作用的模型。通过使用蛋白质NMR光谱和酶动力学，以确定蛋白质的灵活性，无论是稳定或不稳定的药物占用的机制，更好地了解药物结合亲和力的决定因素将获得。这一新知识将提高二氢叶酸还原酶和其他蛋白质的小分子抑制剂的设计效率。

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.