Protein Structure

在过去几年中，我们的工作集中在几个不同的领域。蛋白酶的晶体学研究自该科成立以来，蛋白酶的晶体学研究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，以及最近的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。蟑螂变应原blag2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决了两个复合物的结构。我们还研究了一种细菌atp依赖性蛋白酶Lon，发现其蛋白水解结构域具有独特的折叠，从而建立了一个具有Ser-Lys催化双偶体的新蛋白酶家族。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。具有抗病毒活性的凝集素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。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模式的广泛研究。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和IL-22。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。p300的Taz2结构域及其与p53和C/EBP转录因子相互作用的晶体学研究。CBP及其同源物p300是组蛋白乙酰转移酶，通过特殊结构域与多种转录因子相互作用来调节基因表达。我们确定了人类p300蛋白片段（残基1723-1836）的晶体结构与扩展的锌结合Taz2结构域相对应。利用束缚Zn离子的异常衍射信号求解了晶体结构。该结构包括由三个锌离子稳定的非典型螺旋束，与先前确定的较短肽的溶液结构非常相似。该结构的残基1813-1834形成了c端螺旋的螺旋延伸，并与Taz2的肽结合位点进行了广泛的晶体接触相互作用，为Taz2识别不同的transactivation domains （TADs）的机制提供了额外的见解。基于对这些接触的分析和分子模型，我们提出了磷酸化p53与Taz2结合的假设模型。目前，我们利用晶体接触相互作用来研究Taz2与C/EBP蛋白的结合。MCL蛋白纯化核心制备了嵌合体蛋白Taz2(1723-1818)C/EBPbeta(37-61)，其中Taz2初始结构的25个C末端残基被C/EBPbeta转录因子最小TAD对应的氨基酸取代。在Argonne的同步加速器SER-CAT光束线上收集了P6(5)组蛋白结晶和1.5分辨率的x射线衍射数据。人类C/EBPbeta蛋白片段（残基37-60）采用螺旋-螺旋-螺旋构象，与对称相关分子Taz2的核心结构相互作用。这个推测的C/EBPbeta结合位点与已知的STAT1在Taz2上的结合位点重叠。

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.