Protein Structure

蛋白质结构

• 批准号: 6951658
• 负责人: alexander 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/6951658
• 关键词: Epstein Barr virus; X ray crystallography; antineoplastics; asparaginase; chemokine; cytokine receptors; drug discovery /isolation; drug resistance; drug screening /evaluation; endopeptidases; enzyme activity; enzyme complex; enzyme inhibitors; equine infectious anemia virus; feline immunodeficiency virus; human immunodeficiency virus 1; integrase; interleukin 10; lymphocytic leukemia; neoplasm /cancer pharmacology; protein structure function; subtilisins

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 five 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. 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. Crystal structure of the modified enzyme has been solved. We also solved at atomic resolution the crystal structure of eosinofil-derived neurotoxin (EDN), a related enzyme now being developed as a potential anti-cancer agent. 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 solved the crystal structure of IL-19, a novel chemokine related to IL-10, and are attempting to crystallize its complexes with specific receptors. 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. Proteins with antiviral properties We solved the crystal structure of cyanovirin-N, a protein that prevents binding of HIV to its cellular receptor. Since this is accomplished through interactions with high-mannose sugars, we also solved the structure of two such specific complexes, elucidating the mode of binding. We also obtained crystals of another protein with similar properties, scytovirin. Proteolytic enzymes Our laboratory has been working on crystal structures of a number of proteolytic enzymes. We discovered that sedolisin is a member of the subtilisin family, although it is much larger and contains a Ser-Glu-His catalytic triad. We have recently solved the structure of the proteolytic domain of and ATP-dependent E. coli protease Lon, establishing a new fold and a catalytic dyad Ser-Lys for this very interesting enzyme. We have also solved the structure of the alpha domain of Lon.

我们使用高分辨率X射线衍射技术研究了蛋白质结构和功能之间的关系。在过去的一年中，我们的工作集中在五个不同的领域。 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。尽管这些酶的抗癌活性机制尚不清楚，但我们集中于其酶特性的研究。我们还研究了与配体（例如谷氨酸和琥珀酸）复合的这些酶，并确定了来自wolinella琥珀酸酯的相关蛋白的结构。另一种具有潜在治疗特性的酶是OnConase，conase是一种从青蛙卵中分离出来的细胞毒性核糖核酸酶。为了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，我们一直参与重新工程。修饰酶的晶体结构已解决。我们还以原子分辨率求解了嗜酸性神经毒素（EDN）的晶体结构，这是一种相关的酶，现在正在作为潜在的抗癌剂开发。 细胞因子和细胞因子受体 我们的部分一直在研究几种细胞因子的晶体结构，并在制备其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因子10（IL-10）是一个域折叠的二聚体，其中每个紧凑的一半由两个相同的分子的片段组成。现已确定在爱泼斯坦 - 巴尔病毒基因组中编码的相关细胞因子的结构，这是该病毒用来控制宿主免疫系统的药物的分子结构的第一个瞥见。我们已经解决了与IL-10相关的新型趋化因子IL-19的晶体结构，并试图将其复合物与特定受体结晶。 逆转录病毒酶 由逆转录病毒（例如HIV）编码的酶是设计有效药物疗法的主要靶标。我们一直在研究与抑制剂复合的天然和耐药性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）和马传染病病毒（EIAV）的相关酶的结构。 HIV-1 PR的大多数抑制剂（包括临床用途的抑制剂）抑制后一种PR，尽管它们能够切割HIV-1衍生的序列。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的不活跃突变体则使用底物进行了研究有助于描述催化机制。我们实验室研究的另一种逆转录病毒酶是整合酶。我们已经在存在和不存在与原子分辨率的二价阳离子的情况下解决了禽肉瘤病毒整合酶的催化结构域的结构，并正在尝试对具有不同底物的复合物的共结合化。 具有抗病毒特性的蛋白质 我们解决了Cyanovirin-N的晶体结构，Cyanovirin-n是一种阻止HIV结合其细胞受体的蛋白质。由于这是通过与高臭糖的相互作用来实现的，因此我们还解决了两个此类特定复合物的结构，从而阐明了结合模式。我们还获得了另一种具有相似特性Scytovirin的晶体。 蛋白水解酶我们的实验室一直在研究许多蛋白水解酶的晶体结构。我们发现丝硅丝是枯草菌素家族的成员，尽管它大得多，并且包含ser-glu-His催化三合会。我们最近解决了与ATP依赖性大肠杆菌蛋白酶lon的蛋白水解结构域的结构，为这种非常有趣的酶建立了新的折叠和催化二元组。我们还解决了LON的Alpha域的结构。