CHRYSTALLIZING DISULFIDE-STABILIZED HIV-1 ENV PROTEINS

描述(由申请人提供)：我们建议制备和纯化足够数量的三聚体HIV-1gp140包膜糖蛋白，以便通过X射线结晶学确定其结构。这项提案中概述的研究将作为康奈尔大学威尔医学院的约翰·摩尔团队和斯克里普斯研究所的兰·威尔逊团队的合作进行；这两个团队分别在包膜糖蛋白生物学和结构研究方面的专业领域是互补的。HIV-1的包膜复合体是中和抗体疫苗的主要焦点。天然的Env复合体是三聚体；它的六个单独的亚基(3gp120‘S，3gp41’S)通过相对较弱的非共价相互作用结合在一起。虽然关于单个gp120和gp41亚基的一些结构信息是可用的，但对三聚体复合体的结构缺乏了解。这尤其是因为稳定的、裂解的三聚体由于其亚基间结合的不稳定性而无法进行分析。我们现在已经制作了一个可溶性的Env蛋白，SOS gp140，它的gp120通过分子间二硫键稳定地连接到gp41的胞外结构域。该蛋白在gp120和gp41之间的蛋白水解点被完全切割，但gp41-gp41相互作用的弱点导致它解离成单体。一个变异的SOS gp140蛋白，命名为SOSIP gp140，在gp41(SOS 1559P或SOSIP gp140)的N-末端七肽重复区域的第559位含有异亮氨酸到脯氨酸的取代。这种蛋白质被完全切割，具有良好的抗原性，主要是三聚体。SOSIP gp140三聚体是非共价缔合的，可以通过凝胶过滤层析进行纯化，在阴离子洗涤剂或微热下分解为单体，但相对耐非离子洗涤剂、高盐或中等酸性pH。它们在37摄氏度的温度下稳定了一周多。我们提出了两个特定的目标：纯化可能更易于结晶的三聚体SOSIP gp140复合体或其变体；结晶Env三聚体，单独或作为CD4-或MAB-复合体，然后分析其结构。从天然Env三聚体的结构中获得的任何信息都可以促进基于Env的疫苗的合理设计。

DESCRIPTION (provided by applicant): We propose to prepare and purify trimeric, HIV-1 gp140 envelope glycoproteins in sufficient quantity for determination of their structure by X-ray crystallography. The research outlined in this proposal will be performed as a collaboration between John Moore's group at the Weill Medical College of Cornell University, and lan Wilson's group at the Scripps Research Institute; the areas of expertise of these groups in, respectively, envelope glycoprotein biology and structural studies are complementary. The Env complex of HIV-1 is the principal focus of neutralizing antibody-based vaccines. The native Env complex is trimeric; its six individual subunits (3 gp120's, 3 gp41's) are held in association by relatively weak, non-covalent interactions. Although some structural information on the individual gp120 and gp41 subunits is available, knowledge of the structure of the trimeric complex is lacking. This is, not least, because stable, cleaved trimers have not been available for analysis, due to the instability of their inter-subunit associations. We have now made a soluble Env protein, SOS gp140, that has gp120 stably linked to the gp41 ectodomain by an intermolecular disulfide bond. This protein is fully cleaved at the proteolysis site between gp120 and gp41 but the weakness of gp41-gp41 interactions cause it to dissociate into monomers. A variant SOS gp140 protein, designated SOSIP gp140, contains an isoleucine-to-proline substitution at position 559 in the N-terminal heptad repeat region of gp41 (SOS 1559P, or SOSIP gp140). This protein is fully cleaved, has favorable antigenic properties and is predominantly trimeric. The SOSIP gp140 trimers are non-covalently associated, can be purified by gel-filtration chromatography, dissociate into monomers after exposure to anionic detergent or mild heat, but are relatively resistant to non-ionic detergents, high salt or exposure to mildly acidic pH. They are stable at 37oC for over a week. We propose two Specific Aims: To purify the trimeric SOSIP gp140 complex or variants thereof that might be more amenable to crystallization; to crystallize the Env trimer, alone or as a CD4- or MAb-complex, then analyze its structure. Any information accruing from the structure of a native Env trimer could facilitate the rational design of Env-based vaccines.