PROTEIN STRUCTURE

蛋白质结构

• 批准号: 6422155
• 负责人: ALEX WLODAWER
• 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/6422155
• 关键词: X ray crystallography; antineoplastics; asparaginase; drug resistance; drug screening /evaluation; endoribonucleases; enzyme complex; enzyme inhibitors; equine infectious anemia virus; feline immunodeficiency virus; green fluorescent proteins; human immunodeficiency virus 1; lymphocytic leukemia; monocyte chemoattractant protein 1; mutant; pharmacokinetics; protein structure function

We are studying the relationship between protein structure and function, using the technique of high-resolution X-ray diffraction. In the past year, our work has been concentrated in four distinct areas. Enzymes with Anticancer Properties We have been investigating the crystal structures of several members of the family of L-asparaginases, some of which are used clinically as drugs directed against childhood lymphoblastic leukemia. While the mechanism of anticancer activity of these enzymes is not yet clear, we have concentrated on the studies of their enzymatic properties. Investigations of the T12V and T89V mutants of Escherichia coli L-asparaginase resulted in the discovery of a covalent intermediate in the active site of the latter variant, leading to the postulate that T12 might be directly involved as a nucleophile in the first step of catalysis. Several other mutants were also crystallized. We have also studied these enzymes complexed with ligands such as glutamate and succinic acid, and we determined the structure of a related protein from Wolinella succinogenes. Another enzyme with potential therapeutic properties is Onconase, a cytotoxic ribonuclease isolated from frog eggs. We have been involved in reengineering this enzyme in order to make it applicable to human cancer therapy and to restore its activity in the absence of posttranslational modifications. Cytokines and Cytokine Receptors Our section has been investigating the crystal structures of several cytokines and has made progress in preparing their receptor complexes. We have established that a helical cytokine, interleukin-10 (IL-10), is a domain-swapped dimer in which each compact half is composed of fragments of two identical molecules. The structure of a related cytokine encoded in the genome of Epstein-Barr virus has now been determined, providing the first glimpse of the molecular architecture of an agent used by the virus to control the host's immune system. We have recently purified and crystallized complexes of IL-10 with its specific receptor. We have solved the crystal structure of monocyte chemoattractant protein-1 (MCP-1), one member of a distinct cytokine family that also includes IL-8, previously investigated in this laboratory. Surprisingly, we found an unprecedented modification of the quaternary structure of MCP-1 due to crystal packing forces. We have also solved the crystal structures of several modifications of a related chemokine, RANTES. Retroviral Enzymes encoded by retroviruses such as HIV are prime targets for designing effective drug therapies. We have been studying the structure of native and drug-resistant HIV-1 protease (PR) complexed with inhibitors, with the aim of tracing the molecular basis of the resistance phenomenon. We have also determined the structures of related enzymes from feline immunodeficiency virus (FIV) and equine infectious anemia virus (EIAV). The latter PRs are poorly inhibited by most inhibitors of HIV-1 PR, including those in clinical use, although they are capable of cleaving HIV-1-derived sequences. To study the mechanism of drug resistance, we solved the structures of HIV-1, FIV, and EIAV PRs complexed with an identical inhibitor, while the studies of an inactive mutant of FIV PR with a substrate helped in delineating the catalytic mechanism. Another retroviral enzyme under investigation in our laboratory is integrase. We have solved the structure of the catalytic domain of avian sarcoma virus integrase in the presence and absence of divalent cations to atomic resolution, and are attempting cocrystallization of complexes with different substrates. Molecular Markers The structures of a number of variants of green fluorescent protein, including its blue mutants, were solved in order to understand the principles of the optical activity of these molecules, as well as to help in engineering new varieties with desirable spectral properties.

我们正在利用高分辨率X射线衍射技术来研究蛋白质结构和功能之间的关系。在过去的一年里，我们的工作集中在四个方面。具有抗癌特性的酶我们一直在研究L-天冬酰胺酶家族中几个成员的晶体结构，其中一些被临床用作治疗儿童淋巴细胞白血病的药物。虽然这些酶的抗癌活性机制尚不清楚，但我们主要集中在对它们的酶性质的研究上。对大肠杆菌L-天冬酰胺酶T12V和T89V突变体的研究发现，在后者的活性部位存在一个共价中间体，从而推测T12可能直接作为亲核试剂参与催化反应的第一步。其他几个突变体也被结晶了。我们还研究了这些酶与谷氨酸和琥珀酸等配体的络合作用，并确定了产琥珀酸狼疮相关蛋白的结构。另一种具有潜在治疗作用的酶是Onconase，它是从青蛙卵中分离出来的一种细胞毒性核糖核酸酶。我们已经参与了对这种酶的重新设计，以便使其适用于人类癌症治疗，并在没有翻译后修饰的情况下恢复其活性。细胞因子和细胞因子受体我们的研究组一直在研究几种细胞因子的晶体结构，并在制备它们的受体复合体方面取得了进展。我们已经证实，一种螺旋细胞因子，白介素10(IL-10)，是一种结构域交换的二聚体，其中每个紧凑的一半由两个相同分子的片段组成。爱泼斯坦-巴尔病毒基因组中编码的相关细胞因子的结构现在已经确定，这让人们第一次看到了病毒用来控制宿主免疫系统的试剂的分子结构。我们最近提纯并结晶了IL-10与其特异性受体的复合物。我们已经解决了单核细胞趋化蛋白-1(MCP-1)的晶体结构，MCP-1是本实验室先前研究的一个独特的细胞因子家族的成员，也包括IL-8。令人惊讶的是，我们发现由于晶体堆积力的作用，MCP-1的四元结构发生了前所未有的修饰。我们还解决了相关趋化因子RANTES的几个修饰的晶体结构。艾滋病毒等逆转录病毒编码的逆转录病毒酶是设计有效药物疗法的主要目标。我们一直在研究天然的和耐药的HIV-1蛋白酶(PR)与抑制剂的复合结构，目的是追踪耐药现象的分子基础。我们还测定了猫免疫缺陷病毒(FIV)和马传染性贫血病毒(EIAV)的相关酶的结构。大多数HIV-1 PR的抑制剂，包括临床上使用的那些，对后一种PR的抑制作用很差，尽管它们能够切割HIV-1衍生的序列。为了研究耐药的机制，我们解决了HIV-1、FIV和EIAV PR与相同抑制剂复合的结构，而FIV PR与底物的失活突变体的研究有助于揭示其催化机制。我们实验室正在研究的另一种逆转录病毒酶是整合酶。我们已经解决了在有无二价阳离子存在和不存在的情况下禽肉瘤病毒整合酶催化结构域的结构到原子分辨，并尝试与不同底物的络合物共结晶。分子标记解决了绿色荧光蛋白的一些变体，包括它的蓝色突变体的结构，以便了解这些分子的光学活性原理，以及帮助设计具有理想光谱特性的新品种。