Protein Structure

蛋白质结构

• 批准号: 8552677
• 负责人: alexander wlodawer
• 金额: $ 157.73万
• 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/8552677
• 关键词: Acquired Immunodeficiency Syndrome; Affinity; Anti-HIV Agents; Antibodies; Antimalarials; Antiviral Agents; Area; Aspartic Endopeptidases; Binding; Binding Sites; Boxing; C-terminal; CCAAT-Enhancer-Binding Protein-beta; CCAAT-Enhancer-Binding Proteins; Carbohydrates; Chimeric Proteins; Complex; Crystallography; Cytokine Receptors; Disaccharides; Disulfides; Docking; Drug resistance; EP300 gene; Enhancers; Enzymes; Family; Family member; Feline Immunodeficiency Virus; Genetic Transcription; HIV Infections; HIV Protease; HIV-1; Histones; Human; Human T-lymphotropic virus 1; Interferons; Interleukin-10; Investigation; Lectin; Ligands; Malignant Neoplasms; Mannose; Modeling; Molecular; Molecular Models; Monosaccharides; Mutation; N-terminal; Oligosaccharides; Pattern; Pepsin A; Peptide Hydrolases; Peptides; Pharmaceutical Preparations; Phosphorylation; Phosphotransferases; Prevention; Proline; Property; Protein Kinase; Proteins; Research; Resolution; Role; STAT1 gene; Site; Source; Structure; Structure-Activity Relationship; Tandem Repeat Sequences; Techniques; Transcriptional Regulation; Transferase; Variant; Work; X ray diffraction analysis; X-Ray Diffraction; Zinc; allergen Bla g 2; base; chromatin remodeling; cockroach allergen; cytokine; design; drug development; human EP300 protein; inhibitor/antagonist; insight; interest; interleukin 20; interleukin-19; interleukin-22; leukemia; member; method development; molecular modeling; monomer; neutralizing antibody; plasmepsin; preclinical study; prevent; promoter; protein complex; protein structure; protein structure function; receptor; synthetic peptide

In the past several years, our work has concentrated in several distinct areas. Crystallographic studies of proteases Crystallographic studies of proteases have been an important area of research of this Section since its establishment. We have been particularly active in the investigation of structure-function relationship in aspartic proteases, including clinically important retroviral enzymes. Our studies of HIV protease, although no longer a major target of active research, are still ongoing and concentrate on the investigation of drug-resistant variants and their complexes with inhibitors. We have investigated retroviral proteases from several other sources such as FIV, RSV, HTLV-1, and, most recently, XMRV. A number of inhibitor complexes of HTLV-1 PR have been analyzed, with the aim of assisting in the development of drugs against HTLV-caused leukemia. XMRV protease was found to share properties of both dimeric retroviral aspartic protease, as well as monomeric pepsin-like enzymes. Complexes of XMRV PR with several inhibitors, including an AIDS drug, have been solved and analyzed. Cockroach allergen Bla g 2 was shown to be an inactive aspartic protease and we solved the structures of two complexes with different specific antibodies. The structures of two plasmepsins, PL-1 and HAP, including their complexes with inhibitors, provided information useful for design of anti-malarial drugs.Lectins and antibodies for the prevention of AIDSWe have been involved in studies of several lectins with antiviral activities, some of them currently being developed in pre-clinical trials as potential drugs preventing HIV infection. We have solved the structure of griffithsin, as free protein and complexed with a number of mono- and disaccharides, explaining the structural basis for its tight binding to branched mannose-rich carbohydrates. We have reengineered griffithsin into a monomeric form and solved its structure with a complex oligosaccharide, elucidating the basis of its antiviral properties. A number of constructs of griffithsin containing tandem repeats of multiple monomers were prepared and shown to increase their antiviral activity. We have also solved atomic-resolution structure of another lectin, scytovirin, and conducted extensive studies aimed at elucidating its disulfide patterns. We have solved structures of Fab constructs of antibodies directed against HIV-1 gp41 complexed with different gp41 mimics. These structures are being interpreted in order to find why some closely related antibodies are neutralizing, while others are not.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 purified and crystallized complexes of IL-10 with its specific receptor and are studying complexes of several other cytokines related to IL-10, such as IL-19, IL-20, and IL-22. We have solved the structure of interferon lambda-1 complexed with its receptor, finding considerable differences in the receptor-ligand interactions between different family members.Crystallographic studies of the Taz2 domain of p300 and its interactions with transcription factorsTranscriptional activation by C/EBP proteins relies on recruitment of the histone acetyl transferases (HATs) CBP/p300 to the promoter/enhancer region. Members of the C/EBP family bind to the Taz2 domain of p300/CBP and trigger p300/CBP phosphorylation. Two short sequence motifs, homology boxes A and B, which are conserved between C/EBP activators and comprise their minimal TADs, are necessary for p300/CBP binding. In order to gain insights into C/EBP:p300/CBP interactions we initiated x-ray crystallographic studies of the p300 Taz2 complexed to various peptide ligands. We determined the crystal structure of a segment of the human p300 protein (residues 1723-1836) corresponding to the extended zinc-binding Taz2 domain. Residues 1813-1834 from this construct form a helical extension of the C-terminal helix and make extensive crystal contact interactions with the peptide binding site on Taz2. Based on the analysis of crystal contacts and molecular modeling, we proposed a hypothetical model of the binding of phosphorylated p53 to Taz2. Subsequently, the interactions between the core TAD of C/EBPepsilon and human p300 Taz2 were determined from the crystal structure (PDB ID:3T92) of a chimeric protein composed of p300 Taz2 (residues 1723-1818) and C/EBPepsilon (residues 37-61). The segment corresponding to the C/EBPepsilon TAD forms two helices connected by a short linker and interacts with the core structure of Taz2 from the symmetry-related molecule. The C/EBPepsilon binding site on Taz2 overlaps with the known binding site of STAT1, suggesting competition between the members of the two families. To confirm that we found the specific binding site, we showed that structure-guided mutations in Taz2 diminished tenfold the affinity of interaction with synthetic peptide encompassing C/EBP/epsilon minimal TAD. We postulate that other members of the C/EBP family interact with the Taz2 domain in the same manner. We also propose a possible structural framework for C/EBPbeta-dependent phosphorylation of the p300 C-terminus by protein kinase HIPK2 and suggest that phosphorylation of the p300/CBP C terminus may be executed by other proline-directed kinases which dock to sites N-terminal to homology box A of C/EBP proteins. This study thus provides structural insights important for understanding the molecular mechanisms underlying the role of C/EBP proteins in chromatin remodeling and transcriptional regulation.

在过去的几年里，我们的工作集中在几个不同的领域。蛋白酶的晶体学研究 蛋白酶的晶体学研究自本室成立以来一直是一个重要的研究领域。我们特别积极地研究天冬氨酸蛋白酶的结构与功能关系，包括临床上重要的逆转录病毒酶。我们对 HIV 蛋白酶的研究虽然不再是活跃研究的主要目标，但仍在进行中，并集中于耐药变体及其与抑制剂的复合物的研究。我们研究了来自其他几个来源的逆转录病毒蛋白酶，例如 FIV、RSV、HTLV-1 以及最近的 XMRV。一些 HTLV-1 PR 抑制剂复合物已被分析，目的是协助开发抗 HTLV 引起的白血病的药物。 XMRV 蛋白酶被发现具有二聚体逆转录病毒天冬氨酸蛋白酶以及单体胃蛋白酶样酶的特性。 XMRV PR 与多种抑制剂（包括艾滋病药物）的复合物已得到解析和分析。蟑螂过敏原 Bla g 2 被证明是一种无活性的天冬氨酸蛋白酶，我们用不同的特异性抗体解析了两种复合物的结构。两种血浆蛋白酶PL-1和HAP的结构，包括它们与抑制剂的复合物，为抗疟疾药物的设计提供了有用的信息。预防艾滋病的凝集素和抗体我们已经参与了几种具有抗病毒活性的凝集素的研究，其中一些目前正在临床前试验中开发，作为预防艾滋病毒感染的潜在药物。我们已经解析了格里菲辛的结构，作为游离蛋白质并与许多单糖和二糖复合，解释了其与富含支链甘露糖的碳水化合物紧密结合的结构基础。我们将 griffithsin 重新设计成单体形式，并用复杂的寡糖解析了其结构，阐明了其抗病毒特性的基础。制备了许多含有多个单体串联重复的格里菲辛构建体，并显示其可增强其抗病毒活性。我们还解决了另一种凝集素 scytovirin 的原子分辨率结构，并进行了广泛的研究，旨在阐明其二硫键模式。我们已经解决了针对 HIV-1 gp41 与不同 gp41 模拟物复合的抗体 Fab 构建体的结构。对这些结构进行解释是为了找出为什么一些密切相关的抗体具有中和作用，而另一些则不能。 细胞因子和细胞因子受体 我们部门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。我们已经纯化并结晶了IL-10与其特异性受体的复合物，并正在研究与IL-10相关的其他几种细胞因子的复合物，例如IL-19、IL-20和IL-22。我们解析了干扰素 lambda-1 与其受体复合的结构，发现不同家族成员之间的受体-配体相互作用存在显着差异。 p300 的 Taz2 结构域及其与转录因子相互作用的晶体学研究 C/EBP 蛋白的转录激活依赖于组蛋白乙酰转移酶 (HAT) CBP/p300 向启动子/增强子区域的募集。 C/EBP 家族成员与 p300/CBP 的 Taz2 结构域结合并触发 p300/CBP 磷酸化。两个短序列基序，即同源盒 A 和 B，在 C/EBP 激活剂之间保守并包含其最小 TAD，是 p300/CBP 结合所必需的。为了深入了解 C/EBP:p300/CBP 相互作用，我们启动了与各种肽配体复合的 p300 Taz2 的 X 射线晶体学研究。我们确定了与延伸的锌结合 Taz2 结构域相对应的人类 p300 蛋白片段（残基 1723-1836）的晶体结构。该构建体的残基 1813-1834 形成 C 端螺旋的螺旋延伸，并与 Taz2 上的肽结合位点产生广泛的晶体接触相互作用。基于晶体接触分析和分子建模，我们提出了磷酸化 p53 与 Taz2 结合的假设模型。随后，根据由 p300 Taz2（残基 1723-1818）和 C/EBPepsilon（残基 37-61）组成的嵌合蛋白的晶体结构（PDB ID：3T92）确定了 C/EBPepsilon 核心 TAD 与人 p300 Taz2 之间的相互作用。对应于 C/EBPepsilon TAD 的片段形成两个由短接头连接的螺旋，并与对称相关分子的 Taz2 核心结构相互作用。 Taz2 上的 C/EBPepsilon 结合位点与已知的 STAT1 结合位点重叠，表明两个家族成员之间存在竞争。为了证实我们找到了特异性结合位点，我们发现 Taz2 中的结构引导突变使与包含 C/EBP/epsilon 最小 TAD 的合成肽相互作用的亲和力降低了十倍。我们假设 C/EBP 家族的其他成员以相同的方式与 Taz2 结构域相互作用。我们还提出了蛋白激酶 HIPK2 对 p300 C 末端进行 C/EBPβ 依赖性磷酸化的可能结构框架，并表明 p300/CBP C 末端的磷酸化可能由其他脯氨酸定向激酶执行，这些激酶对接至 C/EBP 蛋白同源框 A 的 N 末端位点。因此，这项研究为理解 C/EBP 蛋白在染色质重塑和转录调控中作用的分子机制提供了重要的结构见解。