CHRYSTALLIZING DISULFIDE-STABILIZED HIV-1 ENV PROTEINS

描述（由申请人提供）：我们准备制备和纯化三聚体HIV-1 gp140包膜糖蛋白，以获得足够的量，以便通过x射线晶体学测定其结构。本提案中概述的研究将由康奈尔大学威尔医学院的约翰·摩尔小组和斯克里普斯研究所的伊恩·威尔逊小组合作进行；这些小组的专业领域，分别在包膜糖蛋白生物学和结构研究是互补的。HIV-1的Env复合体是中和基于抗体的疫苗的主要焦点。天然的Env复合物是三聚体；它的6个亚基（3个gp120’s， 3个gp41’s）通过相对弱的非共价相互作用结合在一起。虽然一些关于gp120和gp41亚基的结构信息是可用的，但缺乏对三聚体复合物结构的了解。这不仅是因为稳定的、断裂的三聚体由于其亚基间关联的不稳定性而无法用于分析。我们现在已经制造了一种可溶性的Env蛋白，SOS gp140，其中gp120通过分子间二硫键稳定地连接到gp41外结构域。该蛋白在gp120和gp41之间的蛋白水解位点被完全切割，但gp41-gp41相互作用的弱点导致其解离成单体。一个SOS gp140蛋白的变体，被命名为SOSIP gp140，在gp41 （SOS 1559P，或SOSIP gp140）的n端七核苷酸重复区559位含有异亮氨酸到脯氨酸的取代。该蛋白是完全切割的，具有良好的抗原性，主要是三聚体。SOSIP gp140三聚体是非共价结合的，可以通过凝胶过滤层析纯化，暴露于阴离子洗涤剂或温和的热量后解离成单体，但相对耐非离子洗涤剂，高盐或暴露于温和的酸性ph。它们在37℃下稳定一周以上。我们提出了两个具体目标：纯化三聚体SOSIP gp140复合物或其变体，使其更易于结晶；将Env三聚体单独或作为CD4-或mab -复合物结晶，然后分析其结构。从天然Env三聚体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.