Protein Structure

蛋白质结构

• 批准号: 9343603
• 负责人: alexander wlodawer
• 金额: $ 146.21万
• 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/9343603
• 关键词: AIDS prevention; Acquired Immunodeficiency Syndrome; 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; Drug resistance; E1A-associated p300 protein; EP300 gene; Engineering; Enhancers; Enzymes; Family; Family member; Feline Immunodeficiency Virus; HIV Infections; HIV Protease; HIV-1; Histones; Human; Human T-lymphotropic virus 1; Interferons; Interleukin-10; Investigation; Lectin; Ligands; Malignant Neoplasms; Mannose; Monosaccharides; Oligosaccharides; Pepsin A; Peptide Hydrolases; Peptides; Pharmaceutical Preparations; Phosphorylation; Property; Protein Kinase; Proteins; Research; Resolution; STAT1 gene; Serine Proteinase Inhibitors; Source; Structure; Structure-Activity Relationship; Tandem Repeat Sequences; Techniques; Transcription Coactivator; Transcriptional Activation; Transferase; Variant; Work; X ray diffraction analysis; X-Ray Diffraction; Zinc; base; cytokine; design; drug development; inhibitor/antagonist; insight; interest; interleukin 20; interleukin-19; interleukin-22; leukemia; member; method development; monomer; neutralizing antibody; peptide structure; plasmepsin; pre-clinical trial; prevent; promoter; protein complex; protein structure; protein structure function; receptor

In the past several years, our work has concentrated in several distinct areas. Crystallographic studies of proteases and their inhibitors Crystallographic studies of proteases and their inhibitors 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 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. The structures of two plasmepsins, PL-1 and HAP, including their complexes with inhibitors, provided information useful for design of anti-malarial drugs. We have studied the structures of two inhibitors of serine proteases with anticancer properties, EcTI and CrataBL. Lectins and antibodies for the prevention of AIDS We 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. We have solved structures of Fab constructs of antibodies directed against HIV-1 gp41 complexed with different gp41 mimics. These structures allowed us to postulate 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 C/EBP transcriptional activators Transcriptional 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 helical 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 contactinteractions with the peptide binding site on Taz2. We utilized this observation to investigate Taz2 binding to C/EBP proteins. We engineered a chimeric protein in which the entire helical extension of the extended Taz2 domain was substituted by residues comprising the minimal TAD of C/EBPepsilon. As expected, the crystal structure of this peptide (PDB ID:3T92), revealed that 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. 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.

在过去几年中，我们的工作集中在几个不同的领域。蛋白酶及其抑制剂的晶体学研究蛋白酶及其抑制剂的晶体学研究是本部门自成立以来的一个重要研究领域。我们一直特别积极地研究天冬氨酸蛋白酶的结构-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积极研究的主要目标，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。我们已经研究了来自其他几种来源的逆转录病毒蛋白酶，如FIV、RSV、HTLV-1和XMRV。已经分析了许多HTLV-1 PR的抑制剂复合物，目的是帮助开发抗HTLV引起的白血病的药物。发现XMRV蛋白酶共享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性质。XMRV PR与几种抑制剂（包括一种艾滋病药物）的复合物已经得到解决和分析。PL-1和HAP两种纤溶酶及其与抑制剂复合物的结构为抗疟疾药物的设计提供了有用的信息。我们已经研究了两种具有抗癌特性的丝氨酸蛋白酶抑制剂EcTI和TisaBL的结构。我们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HIV感染的潜在药物。我们已经解决了griffithsin的结构，作为游离蛋白质和复杂的一些单糖和二糖，解释其紧密结合的结构基础，支链甘露糖丰富的碳水化合物。我们已经将griffithsin重组为单体形式，并用复杂的寡糖解决了其结构，阐明了其抗病毒特性的基础。制备了许多含有多个单体的串联重复序列的griffithsin构建体，并显示出增加其抗病毒活性。我们还解决了另一种凝集素，scytovirin的原子分辨率结构。我们已经解决了与不同gp 41模拟物复合的针对HIV-1 gp 41的抗体的Fab构建体的结构。这些结构使我们能够假设为什么一些密切相关的抗体是中和性的，而另一些则不是。细胞因子和细胞因子受体本科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，并且正在研究与IL-10相关的几种其他细胞因子的复合物，例如IL-19、IL-20和IL-22。我们已经解决了与其受体复合的干扰素λ-1的结构，发现不同家族成员之间的受体-配体相互作用存在相当大的差异。p300的Taz 2结构域及其与C/EBP转录激活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家族的成员与p300/CBP的Taz 2结构域结合并触发p300/CBP磷酸化。两个短的螺旋序列基序，同源盒A和B，这是保守的C/EBP激活剂之间，并包括其最小的TAD，是必要的p300/CBP结合。为了深入了解C/EBP：p300/CBP相互作用，我们开始了与各种肽配体复合的p300 Taz 2的X射线晶体学研究。我们确定了一段人p300蛋白（残基1723-1836）的晶体结构，对应于扩展的锌结合Taz 2结构域。该构建体的残基1813-1834形成C-末端螺旋的螺旋延伸，并与Taz 2上的肽结合位点进行广泛的晶体接触和相互作用。我们利用这一观察结果来研究Taz 2与C/EBP蛋白的结合。我们设计了一种嵌合蛋白，其中扩展的Taz 2结构域的整个螺旋延伸被包含C/EB肽的最小长度的残基取代。正如预期的那样，该肽（PDB ID：3 T92）的晶体结构显示，对应于C/EB肽的片段形成由短接头连接的两个螺旋，并与来自免疫相关分子的Taz 2的核心结构相互作用。Taz 2上的C/EB肽结合位点与STAT 1的已知结合位点重叠。我们假设C/EBP家族的其他成员以相同的方式与Taz 2结构域相互作用。我们还提出了一个可能的结构框架的C/EBP β依赖性磷酸化的p300的C-末端的蛋白激酶HIPK 2。