Structural of Biomolecular Systems by X Ray Diffraction

通过 X 射线衍射分析生物分子系统的结构

• 批准号: 6559206
• 负责人: XINHUA JI
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6559206
• 关键词: X ray crystallography; glutathione transferase; guanosinetriphosphatases; phosphotransferases; protein isoforms; protein structure

The primary goals of our Section are to address the structure and function of biomolecular systems with anticancer and antimicrobial significance and to explore the feasibility of drug design targeting such biomolecules. In our efforts to achieve these goals, we have established collaborations within NIH as well as with extramural experts in genetics, molecular biology, protein chemistry, enzymology, carcinogenesis, and medicinal chemistry. These collaborations have greatly extended our range of experiments. Glutathione S-transferase: Structure-based Design of Electrophilic Diazeniumdiolates for Pharmacologic Delivery of Nitric Oxide Many tumors become drug resistant by overexpressing the detoxification enzyme glutathione S-transferase (GST). Of the three major isoforms, alpha, mu, and pi, pi is the predominant form in cancer cells. We are attempting to design agents that will overcome this drug resistance by generating nitric oxide (NO) selectively in the active site of GST-pi, which could increase the effectiveness of anti-cancer therapies. Comparison of the active sites and transition state analogs of the three isozymes revealed a potential strategy for achieving isozyme selectivity. Application of this strategy has resulted in a pi-selective NO donor. If planned cytotoxicity studies show that this donor or subsequent NO donors improve the potency of electrophilic anticancer agents toward GST-pi-overexpressing cells, a means of overcoming drug resistance in some clinically important tumor types may be forthcoming. 6-Hydroxymethyl-7,8-dihydropterin Pyrophosphokinase: Mechanism of Pyrophosphoryl Transfer and Structure-based Design of Novel Antimicrobial Agents 6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is the first enzyme in the folate biosynthetic pathway, catalyzing the transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP). Folate cofactors are essential for life. Mammals derive folates from their diets. In contrast, most microorganisms must synthesize folate de novo. Therefore, HPPK is an ideal target for the development of novel antimicrobial agents, which are urgently needed to fight the worldwide crisis of antibiotic resistance. HPPK contains 158 amino acid residues and is thermostable, which makes it an excellent model system for the study of the pyrophosphoryl transfer mechanism, of which little is known. At atomic resolutions (up to 0.89 Angstrom), we have determined the crystal structures of ligand-free enzyme as well as various well-chosen complexes. Our analysis of these structures has provided essential information on the reaction mechanism of pyrophosphoryl transfer and critical knowledge for the design of novel antimicrobial molecules. Of particular importance is the structure of HPPK in complex with HP and MgAMPCPP at 1.25-Angstrom resolution, which mimics most closely the ternary complex of the enzyme and reveals the atomic details of the catalytic assembly, and therefore has been the basis of our structure-based inhibitor design effort. We have carried out the design, synthesis, biochemical, and crystallographic studies of three bisubstrate-mimicking analogs, each of which consists of a pterin, an adenosine moiety, and a linker composed of 2-4 phosphoryl groups. Era Protein: GTPase-dependent Cell Cycle Regulator Era is an essential GTPase found in every bacterium sequenced to date. Highly conserved Era homologs are also found in eukaryotes, such as mouse and human. The Era homolog may be a candidate for a tumor suppressor, because it is located in a chromosomal region where loss of heterozygosity is often associated with various types of cancer. In bacteria, Era has a regulatory role in cell cycle control by coupling cell growth rate with cytokinesis. Cell division is signaled when a threshold of Era activity is reached. Artificially reducing the expression or impairing the activity of Era results in bacterial cell cycle arrest at a predivisional two-cell stage. The arrest lasts until Era activity accumulates to the threshold level, allowing another cell cycle to start. Because the synthesis of Era itself is positively correlated with growth rate, the cell division rate is thus coordinately maintained. We have determined the crystal structure of Era from Escherichia coli at 2.4-Angstrom resolution, which reveals a two-domain arrangement: an N-terminal domain that resembles p21 Ras and a unique C-terminal domain that contains an RNA-binding motif. The crystal structure determination of Era in complex with GDP and with a GTP analog is in progress. Our analysis of these structures will provide insight into the conformational changes of the protein during GTP hydrolysis, which may be part of the signaling pathway of this cell cycle regulator.

我们的主要目标是解决抗癌和抗菌意义的生物分子系统的结构和功能，并探索针对这些生物分子的药物设计的可行性。在我们努力实现这些目标的过程中，我们在NIH内部以及与遗传学、分子生物学、蛋白质化学、酶学、致癌作用和药物化学方面的校外专家建立了合作关系。这些合作极大地扩展了我们的实验范围。 谷胱甘肽S-转移酶：基于结构的亲电二氮鎓二醇盐药物释放一氧化氮的设计 许多肿瘤通过过度表达解毒酶谷胱甘肽S-转移酶（GST）而产生耐药性。在三种主要亚型α、μ和pi中，pi是癌细胞中的主要形式。我们正试图设计药物，通过在GST-π的活性位点选择性地产生一氧化氮（NO）来克服这种耐药性，这可以提高抗癌疗法的有效性。三种同工酶的活性位点和过渡态类似物的比较揭示了实现同工酶选择性的潜在策略。这种策略的应用导致了pi-选择性NO供体。如果计划的细胞毒性研究表明，该供体或随后的NO供体提高了亲电子抗癌剂对GST-π过表达细胞的效力，则克服某些临床重要肿瘤类型中的耐药性的手段可能即将到来。 6-羟甲基-7，8-二氢蝶呤焦磷酸激酶：焦磷酸转移机理及基于结构的新型抗菌剂设计 6-羟甲基-7，8-二氢蝶呤焦磷酸激酶（6-Hydroxymethyl-7，8-dihydropterin pyrophosphokinase，HPPK）是叶酸生物合成途径中的第一个酶，催化焦磷酸从ATP转化为6-羟甲基-7，8-二氢蝶呤（6-hydroxymethyl-7，8-dihydropterin，HP）。叶酸辅助因子是生命所必需的。哺乳动物从它们的饮食中获取叶酸。相反，大多数微生物必须从头合成叶酸。因此，HPPK是开发新型抗菌药物的理想靶标，这是对抗全球抗生素耐药性危机的迫切需要。HPPK含有158个氨基酸残基，是热稳定的，这使得它成为一个很好的模型系统的焦磷酸转移机制的研究，其中知之甚少。在原子分辨率（高达0.89埃），我们已经确定了无配体酶以及各种精心选择的复合物的晶体结构。我们对这些结构的分析为焦磷酰基转移的反应机理和新型抗菌分子的设计提供了必要的信息。特别重要的是1.25埃分辨率下HPPK与HP和MgAMPCPP复合物的结构，它最接近地模拟了酶的三元复合物，并揭示了催化组装的原子细节，因此一直是我们的基础基于结构的抑制剂设计工作。我们已经进行了设计，合成，生物化学，和晶体学研究的三个双底物模拟类似物，其中每一个组成的蝶呤，腺苷部分，和连接器组成的2 - 4磷酰基。 Era蛋白：GTP酶依赖性细胞周期调节因子 Era是迄今为止在测序的每种细菌中发现的必需的GTdR。高度保守的Era同源物也发现于真核生物中，例如小鼠和人类。Era同源物可能是肿瘤抑制因子的候选者，因为它位于杂合性丢失通常与各种类型的癌症相关的染色体区域。在细菌中，Era通过偶联细胞生长速率与胞质分裂在细胞周期控制中具有调节作用。当达到Era活性的阈值时，细胞分裂发出信号。牺牲性地减少Era的表达或损害Era的活性导致细菌细胞周期停滞在分裂前的两细胞阶段。这种停滞持续到Era活性积累到阈值水平，允许另一个细胞周期开始。由于Era本身的合成与生长速率正相关，因此细胞分裂速率得以协调维持。我们已经确定了Era的晶体结构，从大肠杆菌在2.4埃分辨率，这揭示了一个两个结构域的安排：一个N-末端结构域，类似于p21 Ras和一个独特的C-末端结构域，包含一个RNA结合基序。与GDP和GTP类似物复合的Era的晶体结构测定正在进行中。我们对这些结构的分析将提供深入了解GTP水解过程中蛋白质的构象变化，这可能是这种细胞周期调节剂的信号通路的一部分。