Protein Structure

蛋白质结构

• 批准号: 6559232
• 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/6559232
• 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; 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 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射线衍射技术研究蛋白质结构和功能之间的关系。过去一年，我们的工作集中在四个不同领域。 具有抗癌特性的酶 我们一直在研究L-天冬酰胺酶家族的几个成员的晶体结构，其中一些在临床上用作针对儿童淋巴细胞白血病的药物。虽然这些酶的抗癌活性的机制还不清楚，我们集中在他们的酶性质的研究。对大肠杆菌L-天冬酰胺酶的T12 V和T89 V突变体的研究发现，在后一种变体的活性位点中存在一个共价中间体，从而提出了T12可能作为亲核试剂直接参与催化的第一步的假设。其他几种突变体也被结晶化。我们还研究了这些酶与谷氨酸和琥珀酸等配体的复合物，并确定了Wolinella succinogenes相关蛋白质的结构。另一种具有潜在治疗特性的酶是Onconase，一种从青蛙卵中分离的细胞毒性核糖核酸酶。我们一直在重新设计这种酶，以使其适用于人类癌症治疗，并恢复其活性的翻译后修饰的情况下。 细胞因子和细胞因子受体 本课题组对几种细胞因子的晶体结构进行了研究，并在其受体复合物的制备方面取得了一定的进展。我们已经确定，螺旋细胞因子，白细胞介素-10（IL-10），是一个结构域交换的二聚体，其中每个紧凑的一半是由两个相同的分子的片段。EB病毒基因组中编码的相关细胞因子的结构现在已经确定，这提供了病毒用于控制宿主免疫系统的试剂的分子结构的第一个一瞥。我们最近已经纯化和结晶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是一个独特的细胞因子家族的成员，也包括IL-8，以前在本实验室研究。令人惊讶的是，我们发现了MCP-1的四级结构由于晶体堆积力的前所未有的修改。我们还解决了相关趋化因子RANTES的几种修饰的晶体结构。 逆转录病毒酶 由逆转录病毒（如HIV）编码的酶是设计有效药物疗法的主要目标。我们一直在研究与抑制剂复合的天然和耐药HIV-1蛋白酶（PR）的结构，目的是追踪耐药现象的分子基础。我们还确定了猫免疫缺陷病毒（FIV）和马传染性贫血病毒（EIAV）相关酶的结构。大多数HIV-1 PR抑制剂（包括临床使用的抑制剂）对后者PR的抑制作用很差，尽管它们能够切割HIV-1衍生序列。为了研究耐药性的机制，我们解决了与相同抑制剂复合的HIV-1，FIV和EIAV PR的结构，而对FIV PR的无活性突变体与底物的研究有助于描绘催化机制。我们实验室正在研究的另一种逆转录病毒酶是整合酶。我们已经解决了禽肉瘤病毒整合酶的催化结构域的存在和不存在的二价阳离子的原子分辨率，并试图与不同的基板复合物的共结晶。 分子标记 解决了绿色荧光蛋白的许多变体的结构，包括其蓝色突变体，以理解这些分子的光学活性的原理，以及帮助工程设计具有理想光谱特性的新品种。