PROTEIN STRUCTURE

我们正在利用高分辨率x射线衍射技术研究蛋白质结构与功能之间的关系。一年来，我们的工作主要集中在四个方面。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，其中一些在临床上被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，但我们已经集中研究了它们的酶性质。对大肠杆菌l -天门汀酶T12V和T89V突变体的研究发现，后一种突变体的活性位点存在共价中间体，从而推测T12可能作为亲核试剂直接参与了催化的第一步。其他几个突变体也结晶了。我们还研究了这些酶与谷氨酸和琥珀酸等配体的络合，并确定了琥珀酸Wolinella相关蛋白的结构。另一种具有潜在治疗特性的酶是Onconase，一种从青蛙卵中分离出来的细胞毒性核糖核酸酶。我们参与了这种酶的重新设计，以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本部门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物方面取得了进展。我们已经确定，螺旋细胞因子，白细胞介素-10 (IL-10)，是一个区域交换二聚体，其中每个紧凑的一半是由两个相同分子的片段组成。爱泼斯坦-巴尔病毒（Epstein-Barr virus）基因组中编码的一种相关细胞因子的结构现已确定，这使人们首次了解了该病毒用来控制宿主免疫系统的一种物质的分子结构。我们最近纯化和结晶了IL-10及其特定受体的复合物。我们已经解决了单核细胞化学吸引蛋白-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，之前在这个实验室研究过。令人惊讶的是，我们发现由于晶体堆积力，MCP-1的四元结构发生了前所未有的改变。我们还解决了相关趋化因子RANTES的几种修饰的晶体结构。由逆转录病毒（如HIV）编码的逆转录酶是设计有效药物治疗的主要目标。我们一直在研究天然和耐药HIV-1蛋白酶（PR）与抑制剂络合的结构，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。后一种PR被大多数HIV-1 PR抑制剂（包括临床使用的抑制剂）抑制较差，尽管它们能够切割HIV-1衍生序列。为了研究耐药机制，我们解决了HIV-1， FIV和EIAV PR与相同抑制剂络合的结构，而对FIV PR与底物的失活突变体的研究有助于描述催化机制。我们实验室正在研究的另一种逆转录酶是整合酶。我们已经解决了禽肉瘤病毒整合酶在存在和不存在二价阳离子的催化结构域的原子分辨率，并正在尝试与不同底物的配合物共结晶。为了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，研究人员解决了许多绿色荧光蛋白变体（包括其蓝色突变体）的结构。

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.