Protein Structure

蛋白质结构

• 批准号: 10014357
• 负责人: alexander wlodawer
• 金额: $ 183.59万
• 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/10014357
• 关键词: AIDS prevention; Acquired Immunodeficiency Syndrome; Anti-HIV Agents; Antibodies; Antimalarials; Antiviral Agents; Area; Aspartic Endopeptidases; Binding; Binding Sites; C-terminal; CCAAT-Enhancer-Binding Protein-beta; CCAAT-Enhancer-Binding Proteins; Carbohydrates; Chimeric Proteins; Clinical; Complex; Cryoelectron Microscopy; Crystallization; 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; Investigational Drugs; JAK1 gene; Lectin; Ligands; Malignant Neoplasms; Mannose; Oligosaccharides; Pepsin A; Peptide Hydrolases; Peptides; Pharmaceutical Preparations; Phosphorylation; Property; Protein Kinase; Proteins; Research; Resolution; Roentgen Rays; STAT1 gene; Serine Proteinase Inhibitors; Source; Structural Protein; Structure; Structure-Activity Relationship; Subcellular structure; Tandem Repeat Sequences; Techniques; Transcription Coactivator; Transcriptional Activation; Transferase; Variant; Work; X ray diffraction analysis; Zinc; anti-cancer; cytokine; design; dimer; drug development; endopeptidase La; inhibitor/antagonist; insight; interest; interleukin 20; interleukin-19; interleukin-22; leukemia; member; method development; monomer; neutralizing antibody; peptide structure; plasmepsin; preclinical trial; prevent; promoter; protein complex; protein structure; protein structure function; receptor; recruit

In the past several years, our work has concentrated in several distinct areas. 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. The structure of Lon protease was investigated by a combination of crystallography and cryo-EM. 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, IL-22, and IL-24. 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. We also solved the structure of the intracellular domain of the interferon lambda receptor complexed with fragment of JAK1. 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 contact interactions 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自该科成立以来一直是该科的一个重要研究领域。我们一直在研究天冬氨酸蛋白酶的结构-功能关系，包括临床重要的逆转录酶。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，并集中在耐药变体及其抑制剂复合物的研究上。我们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。许多HTLV-1 PR的抑制剂复合物已经被分析，目的是帮助开发针对htlv引起的白血病的药物。发现XMRV蛋白酶具有二聚体逆转录病毒天冬氨酸蛋白酶和单体胃蛋白酶样酶的特性。XMRV PR与几种抑制剂（包括一种艾滋病药物）的配合物已经得到解决和分析。两种血浆蛋白酶PL-1和HAP的结构及其与抑制剂的配合物为抗疟疾药物的设计提供了有用的信息。采用晶体学和低温电镜相结合的方法研究了Lon蛋白酶的结构。我们研究了两种具有抗癌特性的丝氨酸蛋白酶抑制剂EcTI和CrataBL的结构。我们已经参与了几种具有抗病毒活性的凝集素的研究，其中一些目前正在进行临床前试验，作为预防艾滋病毒感染的潜在药物。我们已经解决了griffithsin的结构，作为游离蛋白质，并与许多单糖和双糖络合，解释了它与富含甘露糖的支链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，并用一个复杂的低聚糖解决了它的结构，阐明了它抗病毒特性的基础。制备了许多含有多个单体串联重复序列的griffithsin构建物，并证明其抗病毒活性增加。我们还解决了另一种凝集素，细胞毒蛋白的原子分辨率结构。我们已经解决了针对HIV-1 gp41的抗体与不同gp41模拟物络合的Fab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可以中和，而另一些则不能。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。我们纯化并结晶了IL-10及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，如IL-19、IL-20、IL-22和IL-24。我们已经解决了干扰素lambda-1与其受体络合的结构，发现不同家族成员之间的受体-配体相互作用存在相当大的差异。我们还解决了与JAK1片段络合的干扰素受体胞内结构域的结构。C/EBP转录激活蛋白的转录激活依赖于组蛋白乙酰转移酶（HATs） CBP/p300在启动子/增强子区域的募集。C/EBP家族成员结合p300/CBP的Taz2结构域并触发p300/CBP磷酸化。两个短的螺旋序列基序，同源盒A和B，在C/EBP激活剂之间保守，包含它们的最小tad，是p300/CBP结合所必需的。为了深入了解C/EBP:p300/CBP相互作用，我们开始了p300 Taz2与各种肽配体络合的x射线晶体学研究。我们确定了人类p300蛋白片段（残基1723-1836）与扩展的锌结合Taz2结构域相对应的晶体结构。该结构的残基1813-1834形成c端螺旋的螺旋延伸，并与Taz2上的肽结合位点进行广泛的晶体接触相互作用。我们利用这一观察结果来研究Taz2与C/EBP蛋白的结合。我们设计了一种嵌合蛋白，其中扩展的Taz2结构域的整个螺旋延伸被包含C/EBPepsilon最小TAD的残基取代。正如预期的那样，该肽（PDB ID:3T92）的晶体结构显示，C/EBPepsilon TAD对应的片段形成两个螺旋，由一个短连接体连接，并与来自对称相关分子的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。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。我们还提出了一种可能的结构框架，用于蛋白激酶HIPK2对p300 C-末端的C/ ebpbeta依赖性磷酸化。