Protein Structure

蛋白质结构

基本信息

批准号：
8348987
负责人：
alexander wlodawer
金额：
$ 159.33万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
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/8348987
关键词：
ATP-Dependent Proteases Acquired Immunodeficiency Syndrome Adopted Amino Acids 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 Complex Crystallography Cytokine Receptors Data Disaccharides Disulfides Drug resistance EP300 gene Enzymes Family Family member Feline Immunodeficiency Virus Gene Expression HIV Infections HIV Protease Helix-Turn-Helix Motifs Histones Human Human T-lymphotropic virus 1 Interferons Interleukin-10 Investigation Ions Lectin Ligands Malignant Neoplasms Mannose Modeling Molecular Conformation Molecular Models Monosaccharides Oligosaccharides Pattern Pepsin A Peptide Hydrolases Peptides Pharmaceutical Preparations Property Protease Domain Proteins Research Resolution STAT1 gene Signal Transduction Solutions Source Structure Structure-Activity Relationship Synchrotrons Techniques Transactivation Transferase Variant Work X ray diffraction analysis X-Ray Diffraction Zinc allergen Bla g 2 base beamline cockroach allergen cytokine design drug development human EP300 protein inhibitor/antagonist insight interest interleukin 20 interleukin-19 interleukin-22 leukemia method development molecular modeling plasmepsin preclinical study prevent protein complex protein purification protein structure protein structure function receptor transcription factor

项目摘要

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. We have also been investigating a bacterial ATP-dependent protease Lon, finding that is proteolytic domain has a unique fold and thus establishes a new family of proteases with a Ser-Lys catalytic dyad. The structures of two plasmepsins, PL-1 and HAP, including their complexes with inhibitors, provided information useful for design of anti-malarial drugs. Lectins with antiviral activity 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. We have also solved atomic-resolution structure of another lectin, scytovirin, and conducted extensive studies aimed at elucidating its disulfide patterns. 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 p53 and C/EBP transcription factors. CBP and its paralogue p300 are histone acetyl transferases that regulate gene expression by interacting with multiple transcription factors via specialized domains. We determined the crystal structure of a segment of human p300 protein (residues 1723-1836) corresponding to the extended zinc-binding Taz2 domain. The crystal structure was solved utilizing an anomalous diffraction signal of the bound Zn ions. The structure comprises an atypical helical bundle stabilized by three Zn ions and closely resembles the solution structures determined previously for shorter peptides. 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 of Taz2, providing additional insights into the mechanism of recognition of diverse transactivation domains (TADs) by Taz2. Based on the analysis of these contacts and molecular modeling we proposed a hypothetical model of the binding of phosphorylated p53 to Taz2. Currently, we use the crystal contact interactions to investigate Taz2 binding to C/EBP proteins. A chimera protein, Taz2(1723-1818)C/EBPbeta(37-61), where 25 C-terminal residues from the initial Taz2 construct were replaced by amino acids corresponding to the minimal TAD from the C/EBPbeta transcription factor was produced by the Protein Purification Core of MCL. The protein crystallized in the P6(5) group and X-Ray diffraction data extending to 1.5 resolution were collected at synchrotron SER-CAT beamline at Argonne. The segment of the human C/EBPbeta protein (residues 37-60) adopts helix-turn-helix conformation and interacts with the core structure of Taz2 from the symmetry related molecule. This putative C/EBPbeta binding site overlaps with the known binding site of STAT1 on Taz2.

在过去的几年中，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一直是自建立以来本节的重要领域。我们在天冬氨酸蛋白酶（包括临床上重要的逆转录病毒酶）中对结构功能关系的研究特别活跃。我们对HIV蛋白酶的研究虽然不再是积极研究的主要靶标，但仍在进行中，并集中于对抗药性变体及其与抑制剂的复合物进行研究。我们已经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。已经分析了许多HTLV-1 PR的抑制剂复合物，目的是协助开发针对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蛋白酶以及单体胃蛋白酶样酶的特性。 XMRV PR的复合物与包括AIDS药物在内的多种抑制剂的复合物已得到解决和分析。蟑螂过敏原BLA G 2被证明是一种非活性天冬氨酸蛋白酶，我们解决了具有不同特异性抗体的两个配合物的结构。我们还一直在研究一个细菌依赖ATP的蛋白酶lon，发现蛋白水解结构域具有独特的折叠，因此建立了具有Ser-Lys催化dyad的新蛋白酶家族。两种纤溶酶PL-1和HAP的结构（包括其与抑制剂的复合物）提供了有关抗疟疾药物设计有用的信息。具有抗病毒活性的凝集素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，并与许多单糖和二糖构成复合，从而解释了其与富含甘露糖的富含甘露糖的碳水化合物紧密结合的结构基础。我们已经将Griffithsin重新设计为单体形式，并用复杂的寡糖解决了其结构，从而阐明了其抗病毒特性的基础。我们还解决了另一种凝集素Scytovirin的原子分辨率结构，并进行了旨在阐明其二硫化模式的广泛研究。细胞因子和细胞因子受体我们的截面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。我们已经纯化和结晶的IL-10及其特定受体的复合物，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。我们已经解决了干扰素lambda-1与其受体复合的结构，发现了不同家族成员之间的受体 - 配体相互作用的差异。 P300 TAZ2结构域的晶体学研究及其与p53和C/EBP转录因子的相互作用。 CBP及其旁系同源物p300是组蛋白乙酰转移酶，通过通过专用域与多个转录因子相互作用来调节基因表达。我们确定了与扩展的锌结合TAZ2结构域相对应的人p300蛋白段（残基1723-1836）的晶体结构。使用结合Zn离子的异常衍射信号来解决晶体结构。该结构包括由三个Zn离子稳定的非典型螺旋束，并与先前针对较短肽确定的溶液结构非常相似。该构造中的残基1813-1834形成了C末端螺旋的螺旋扩展，并与TAZ2的肽结合位点进行了广泛的晶体接触相互作用，从而提供了对识别多种反式反式激活结构域（TADS）识别的机制的更多见解。基于对这些接触和分子建模的分析，我们提出了磷酸化p53与TAZ2结合的假设模型。当前，我们使用晶体接触相互作用来研究TAZ2与C/EBP蛋白的结合。嵌合蛋白TAZ2（1723-1818）C/EBPBETA（37-61），其中最初TAZ2构建体的25个C末端残基被MCL的蛋白质纯化核心产生了与C/EBPBETA转录因子的最小TAD相对应的氨基酸。在Argonne的同步加速器ser-cat梁线上收集了在P6（5）组中结晶至1.5分辨率的X射线衍射数据的蛋白质。人C/EBPBETA蛋白（残基37-60）的片段采用螺旋 - 旋转螺旋构象，并与与对称性相关分子的TAZ2的核心结构相互作用。这个推定的C/EBPBETA结合位点与TAZ2上的STAT1的已知结合位点重叠。