PRODUCTION OF HIV AND HIV RELATED PROTEINS FOR STRUCTURAL STUDIES

背景：HIV和HIV相关蛋白是通过重组DNA方法产生的，用于高分辨率结构分析。选择的蛋白质对病毒的生命周期和结构完整性很重要，因此代表了基于合理结构的药物设计的潜在靶点。协作小组(在下面的括号中指出)进行了实际的结构确定。用X射线衍射或多维核磁共振确定蛋白质的三维结构需要生产大量高纯度和物理上均一的蛋白质。此外，通过核磁共振确定蛋白质结构需要用稳定同位素H-2、C-13和N-15的组合生物合成标记蛋白质。结果：(1)Nef是一种23 kDa的蛋白质，与HIV的致病特性密切相关。最近完成了Nef的溶液结构。基于这种结构，我们进行了几次缺失和定点突变，以改善蛋白质的物理性质。我们现在正在研究涉及Nef的一些特定的蛋白质-蛋白质相互作用。一个重要的相互作用是与CD4的细胞质尾巴。Lck SCR酪氨酸激酶的N-末端结构域也与该区域结合。因此，NEF可能通过置换lck SCR酪氨酸激酶而发挥其生物学效应。为了进一步了解这些相互作用，我们在大肠杆菌中表达了lck的N端部分和CD4的细胞质尾巴。我们为核磁共振研究生产和配制这些蛋白质的努力仍在继续。(2)HIV Rev是HIV表达所必需的重要调控因子。这种RNA结合蛋白有很强的自我缔合形成超高分子量纤维聚合物的倾向。使用这些纤维，光谱方法，冷冻电子显微镜和图像分析被用来获得低分辨率的蛋白质模型。我们继续努力通过关注蛋白质的较小区域，特别是介导与其目标RNA(RRE)结合的N末端一半，来获得更多的结构信息。融合蛋白已在细菌中表达，其特性还在继续。(3)在我们对二聚体HIV蛋白酶的持续研究中，我们研究了第95位残基的可逆氧化，并认为这调节了HIV I型和II型酶的活性。残基95是半胱氨酸(类型I)或蛋氨酸(类型II)，这些位于二聚体界面的含硫氨基酸的氧化以可逆的方式抑制活性。这些研究可以作为HIV-蛋白酶的氧化还原调节的模型，其功能可能在氧化应激(Davis)下的细胞中被调节。还开展了HIV-1蛋白酶前体的蛋白分解加工工作。在这些研究中，与天然转码区相连的HIV-1蛋白酶在大肠杆菌中表达(TFR-PR)。利用TFR-PR结构，研究了自催化成熟的机理。蛋白酶N端的裂解导致二聚体结构的稳定(Louis)。(4)MAP30是一种具有抗HIV和抗肿瘤活性的植物蛋白。建立了用于核磁共振结构测定的30 kDa蛋白质的纯化方法和生物合成标记方法。(Torchia，Bax，Wang和S.Lee-Huang)。高分辨结构表明，MAP30与蓖麻毒素A链和其他核糖体失活蛋白中观察到的二级结构元素相似，与蓖麻毒素A链折叠相适应。随后的生化分析表明，MAP30是一种DNA糖苷酶/AP裂解酶。这种活性可以解释MAP30抑制HIV-1整合酶以及解释其抗HIV/抗肿瘤活性。(5)HIV的组装是由Gag基因产物介导的(P55)。壳。HIV-1蛋白被HIV-1酶切成四个亚基：p17(基质蛋白)、p24(衣壳蛋白)、p7(NC)和C端p6。裂解产物形成成熟的病毒衣壳。成熟的病毒具有锥形或杆状外观。艾滋病毒核衣壳的组织和整体形态尚未明确确定。我们已经生产了P55，并打算通过生物物理和高分辨率电子显微镜监测组装的衣壳结构，研究这种蛋白质的体外成熟过程。我们已经开始了这项工作，在细菌中表达了几个单独的成分：p24，p17和p7。在一项相关的研究中，我们正在研究涉及p17的N末端结构域和gp41的细胞质尾部的特定蛋白质-蛋白质相互作用。在这项工作中，我们表达了SIV和HIV gp41的~150个残基细胞质区域。虽然p17可以相对容易地表达和纯化，但gp41部分已被证明是非常有问题的。我们已经获得了这些蛋白质作为GST融合蛋白，并继续检测它们的化学和物理性质，试图将它们用于结构工作。摘要：免疫缺陷病毒(HIV)由许多具有调节和结构作用的蛋白质组成。在细菌中使用重组DNA方法表达对病毒生命周期重要的HIV蛋白质和具有抗HIV活性的蛋白质。这些蛋白质被提纯，然后进行研究，以确定它们的化学和物理性质。研究这些蛋白质分子结构的NIH研究人员可以获得特征良好的蛋白质。这种结构信息可能会为靶向药物的设计和发现提供动力。

Background:HIV and HIV-related proteins are produced by recombinant DNA methods for high-resolution structural analyses. The proteins are selected which are important for the life cycle of the virus and for its structural integrity and, thus, represent potential targets for rational structure-based drug design. Collaborating groups (indicated below in parentheses) performed the actual structure determinations. The determination of the 3-D structure of proteins by X-ray diffraction or multidimensional NMR required the production of large quantities of highly purified and physically homogeneous protein. In addition, protein structure determination by NMR requires protein biosynthetically labeled with combinations of the stable isotopes: H-2, C-13 and N-15. Results:(1) Nef is a 23 kDa protein essential for the pathogenic properties of HIV. The solution structure of Nef was completed recently. Based on the structure, we have made several deletion and site-directed mutations that improve the physical properties of the protein. We are now investigating some of the specific protein-protein interactions involving Nef. One important interaction is with the cytoplasmic tail of CD4. The N-terminal domain of the lck Scr tyrosine kinase also binds to this region. Nef may, thus, exert its biological effect by displacement of the lck Scr tyrosine kinase. To gain further insights into these interactions, we have expressed the N-terminal portion of the lck and the cytoplasmic tail of CD4 in E.coli. Our attempts to produce and formulate these proteins for NMR studies continue. (2) HIV Rev is an important regulatory factor required for HIV expression. This RNA binding protein has a strong tendency to self-associate into very high molecular weight fibrous polymers. Using these fibers, spectroscopic methods and cryo-electron microscopy and image analysis were used to obtain a low-resolution model of the protein. We have continued our efforts to gain more structural information by focusing on smaller regions of the protein, especially the N-terminal half that mediates binding to its target RNA (RRE). Fusion proteins have been expressed in bacteria and their characterization continues.(3) In our continuing studies on the dimeric HIV protease, we have studied the reversible oxidation of residue 95 and suggest this regulates the activity of both HIV type I and type II enzyme. Residue 95 is cysteine (type I) or methionine (type II) and oxidation of these sulphur containing amino acids, located at the dimer interface, inhibits activity in an reversible manner. These studies may be useful as a model of redox regulation of the HIV-protease whose function may be regulated in cells under oxidative stress (Davis). Work has also been carried out on the proteolytic processing of the HIV-1 protease precursor. For these studies the HIV-1 protease linked to the native transframe region was expressed in E.coli (TFR-PR). Using the TFR-PR construct, the mechanism of the autocatalytic maturation was studied. Cleavage at the N-terminus of the protease leads to stabilization of the dimeric structure (Louis). (4) MAP30 is a plant protein with anti-HIV and anti-tumor activities. Purification methods and biosynthetic labeling protocols for this 30 kDa protein were developed for NMR structure determination. (Torchia, Bax, Wang and S.Lee-Huang). The high-resolution structure revealed that MAP30 adapts the ricin A chain fold with secondary structure elements similar to those observed in ricin A chain and other ribosome inactivating proteins. Subsequent biochemical assays (Pommier) showed that MAP30 acts as a DNA glycosidase/ ap lyase. This activity could account for MAP30 inhibition of HIV-1 integrase as well as explaining its anti HIV/anti-tumor activities. (5) The assembly of HIV is mediated by the gag gene product (p55). Shell. The gag polyprotein is cleaved by HIV-1 protease into four subunits: p17 (matrix protein), p24 (capsid protein), p7 (NC) and C-terminal p6. The cleaved product forms mature viral capsids. The mature viruses have a cone- or rod-shaped appearance. The organization and overall morphology of the HIV nucleocapsid has not yet been clearly established. We have produced p55 and intend to study the maturation of this protein in-vitro, monitoring the assembled capsid structures by biophysical and high-resolution electron microscopy. We have initiated this work by expressing in bacteria several of the individual components: p24, p17 and p7. In a related study we are investigating specific protein-protein interactions involving the N-terminal domain of p17 and the cytoplasmic tail of gp41. For this work we have expressed the ~150 residue cytoplasmic regions of both SIV and HIV gp41. Although the p17 can be expressed and purified with relative ease, the gp41 moieties have proved very problematic. We have obtained these proteins as GST-fusion proteins and continue to examine their chemical and physical properties in an attempt to formulate them for structural work. Summary:The immunodeficiency virus (HIV) comprises a number of proteins with regulatory and structural roles. HIV proteins important for the virus life cycle, and proteins which have anti-HIV activity, are expressed in bacteria using recombinant DNA methods. The proteins are purified then studied to establish their chemical and physical properties. Well-characterized proteins are made available to NIH investigators who study the molecular structure of these proteins. This structural information may provide impetus for targeted drug design and discovery.