Protein Structure

在过去的几年里，我们的工作集中在几个不同的领域。自该科成立以来，对蛋白酶及其抑制剂的结晶学研究一直是该科的一个重要研究领域。我们在天冬氨酸蛋白酶的结构-功能关系的研究中尤其活跃，包括临床上重要的逆转录病毒酶。我们对HIV蛋白水解酶的研究虽然不再是活跃研究的主要目标，但仍在进行中，并集中在耐药变异体及其与抑制剂的复合体的研究上。我们已经研究了来自其他几个来源的逆转录病毒蛋白酶，如FIV，RSV，HTLV-1和XMRV。分析了HTLV-1PR的一些抑制剂复合体，目的是帮助开发抗HTLV引起的白血病的药物。XMRV蛋白水解酶既具有二聚体逆转录病毒天冬氨酸蛋白水解酶的性质，又具有单体胃蛋白酶样酶的性质。XMRV PR与包括一种艾滋病药物在内的几种抑制剂的络合物已经被解析和分析。两种血浆蛋白PL-1和HAP的结构，包括它们与抑制剂的络合物，为抗疟疾药物的设计提供了有用的信息。用结晶学和低温电子显微镜相结合的方法对Lon蛋白酶的结构进行了研究。我们研究了两种具有抗癌活性的丝氨酸蛋白酶抑制剂ECTI和CrataBL的结构。预防艾滋病的凝集素和抗体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，并与一些单糖和双糖络合，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。我们已经将格里菲辛重新设计成单体形式，并用复杂的寡糖解决了它的结构，阐明了它的抗病毒特性的基础。制备了许多含有多个单体串联重复序列的Griffithsin结构，并证明它们具有更强的抗病毒活性。我们还解决了另一种凝集素--镰刀菌素的原子分辨结构。我们已经解决了针对HIV-1gp41的抗体与不同的gp41模拟物复合的Fab结构。这些结构使我们能够推测为什么一些密切相关的抗体是中和的，而另一些则不是。细胞因子和细胞因子受体我们研究组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体方面取得了进展。我们已经提纯和结晶了IL-10与其特异性受体的复合物，并正在研究其他几种与IL-10相关的细胞因子的复合物，如IL-19、IL-20、IL-22和IL-24。我们已经解决了干扰素lambda-1与其受体的复合体结构，发现不同家族成员之间的受体-配体相互作用存在很大差异。我们还解决了与JAK1片段络合的干扰素lambda受体胞内区的结构。结晶学研究p300的Taz2结构域及其与C/EBP转录激活因子的相互作用依赖于组蛋白乙酰转移酶(HATS)CBP/p300在启动子/增强子区的募集。C/EBP家族成员与p300/CBP的Taz2结构域结合并触发p300/CBP的磷酸化。两个短螺旋序列基序A和B是p300/CBP结合所必需的，它们在C/EBP激活子之间保守，并包含它们的最小TADS。为了深入了解C/EBP：P300/CBP的相互作用，我们启动了p300Taz2与不同多肽配体络合的X射线结晶学研究。我们确定了人类p300蛋白(残基1723-1836)的晶体结构，对应于扩展的锌结合Taz2结构域。来自该结构的残基1813-1834形成C末端螺旋的螺旋延伸，并与Taz2上的肽结合部位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组成最小TAD的C/EBPepsilon残基所取代。不出所料，该多肽(PDB ID：3T92)的晶体结构表明，与C/EBPepsilon TAD对应的片段形成两个由短连接子连接的螺旋，并从对称性相关分子与Taz2的核心结构相互作用。Taz2上的C/EBPepsilon结合位点与STAT1的已知结合位点重叠。我们假设C/EBP家族的其他成员以同样的方式与Taz2结构域相互作用。我们还提出了一种可能的结构框架，通过蛋白激酶HIPK2依赖C/EBPβ对p300 C末端进行磷酸化。

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.