Structural of Biomolecular Systems by X Ray Diffraction

本研究组的主要目标是研究具有抗癌和抗微生物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进行药物设计的可行性。为了实现这些目标，我们在NIH内部以及与遗传学、分子生物学、蛋白质化学、酶学、癌症发生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：用于一氧化氮药物递送的亲电重氮二醇酸盐的结构设计 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。在三种主要的亚型，即α、u和pi中，pi是癌细胞中的主要亚型。我们正试图设计一种药物，通过在GST-pi的活性部位选择性地产生一氧化氮(NO)来克服这种耐药性，这可能会增加抗癌治疗的有效性。三种同工酶的活性部位和过渡态类似物的比较揭示了实现同工酶选择性的一种潜在策略。这一策略的应用导致了pi-选择性的NO供体。如果计划中的细胞毒性研究表明，这个供体或随后的NO供体提高了亲电抗癌剂对GST-pi过表达细胞的效力，那么克服某些临床重要肿瘤类型的耐药性的方法可能就会出现。 6-羟甲基-7，8-二氢蝶呤焦磷酸激酶：焦磷酸转移机理及新型抗菌剂的结构设计 6-羟甲基-7，8-二氢蝶呤焦磷酸激酶(HPPK)是叶酸生物合成途径中的第一个酶，催化焦磷酸从ATP转移到6-羟甲基-7，8-二氢蝶呤(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得叶酸。相反，大多数微生物必须从头合成叶酸。因此，HPPK是开发新型抗菌剂的理想靶点，而新型抗菌剂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，具有热稳定性，是研究焦磷酰化转移机理的良好模型体系。在原子分辨率(高达0.89埃)下，我们测定了无配体酶和各种精选的配合物的晶体结构。我们对这些结构的分析为焦磷酰基转移的反应机理提供了重要的信息，并为新型抗菌分子的设计提供了关键知识。特别重要的是HPPK在1.25埃分辨率下与HP和MgAMPCPP络合物的结构，它最接近于酶的三元络合物，并揭示了催化组装的原子细节，因此一直是我们基于结构的抑制剂设计工作的基础。我们进行了三个双底物类似物的设计、合成、生化和结晶学研究，每个类似物都由一个蝶呤、一个腺苷部分和一个由2-4个磷酰基组成的连接基组成。 Era蛋白：GTPase依赖的细胞周期调节因子 Era是迄今为止在所有已测序的细菌中都发现的一种重要的GTP酶。在真核生物中也发现了高度保守的Era同源物，如小鼠和人类。Era同源基因可能是肿瘤抑制基因的候选基因，因为它位于染色体区域，杂合性缺失通常与各种类型的癌症相关。在细菌中，ERA通过将细胞生长速度与胞质分裂相结合，在细胞周期控制中发挥调节作用。当达到ERA活动的阈值时，细胞分裂就被发出信号。人为地减少ERA的表达或削弱ERA的活性会导致细菌细胞周期停滞在分裂前的两个细胞阶段。这种抑制持续到ERA活性累积到阈值水平，允许另一个细胞周期开始。由于Era本身的合成与生长速率呈正相关，因此细胞分裂速率被协调地维持。我们在2.4埃分辨率下测定了大肠杆菌Era的晶体结构，揭示了一个两个结构域的排列：一个类似于p21Ras的N-末端结构域和一个独特的包含RNA结合基序的C-末端结构域。Era与GDP络合物和GTP类似物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对GTP水解过程中蛋白质构象变化的洞察，这可能是该细胞周期调节因子信号通路的一部分。

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.