Molecular Recognition by Clathrin Adaptors

特异的分选信号将跨膜蛋白引导到内核溶酶体系统的隔室。基于酪氨酸的分类信号与AP复合物大亚基（AP1、2、3和4）的穗结构域结合，并已被很好地表征。DXXLL形式的酸簇二苯胺信号存在于分选受体的细胞质尾部，如阳离子依赖型和阳离子依赖型甘露糖6-磷酸受体，可被GGA接头蛋白的VHS结构域识别。在之前的工作中，我们的实验室确定了与dxxll基序肽结合的GGA VHS结构域的结构。我们接着描述了GGA蛋白的所有结构域与相关蛋白质或肽配体的复合物。DXXXLL基序存在于医学上重要的宿主蛋白如CD4和病毒蛋白如HIV-1 Nef中。与DXXLL基序不同，它与AP复合物结合，而不是与GGAs结合。尽管它的重要性和丰富的历史，以及多个实验室多年的努力，DXXXLL基序的结构基础是未知的。为了解决这个问题，我们的实验室设计并表达了一种能够结合DXXXLL基序肽的AP2复合物变体。

Specific sorting signals direct transmembrane proteins to the compartments of the endosomal-lysosomal system. The tyrosine-based sorting signal binds to the ear domains of the large subunits of AP complexes (AP1, 2, 3, and 4), and has been well characterized. Acidic-cluster-dileucine signals of the form DXXLL present within the cytoplasmic tails of sorting receptors, such as the cation-independent and cation-dependent mannose 6-phosphate receptors, are recognized by the VHS domain of the GGA adaptor proteins. In previous work, our laboratory determined the structure of the GGA VHS domain bound to DXXLL-motif peptides. We went on to characterize all of the domains of the GGA proteins in complex with relevant protein or peptide ligands. The DXXXLL motif exists in medically important host proteins such as CD4 and viral proteins such as HIV-1 Nef. Unlike the DXXLL motif, it binds to AP complexes, not GGAs. Despite its importance and substantial history, and years of effort by multiple laboratories, the structural basis for the DXXXLL motif is unknown. To address this problem, our lab engineered and expressed a variant AP2 complex that is competent to bind to DXXXLL motif peptides. Nef is an accessory protein of human and simian immunodeficiency viruses. It is a critical determinant of pathogenesis that promotes the progression from infection to AIDS. The pathogenic effects of Nef are in large part dependent on its ability to downregulate the macrophage and T-cell coreceptor, CD4. It has been proposed that Nef induces downregulation by linking the cytosolic tail of CD4 to components of the host-cell protein trafficking machinery. To identify these components, Juan Bonifacinos laboratory, with whom we collaborate, developed a novel Nef-CD4 downregulation system in Drosophila melanogaster S2 cells. They found that human immunodeficiency virus type 1 (HIV-1) Nef downregulates human CD4 in S2 cells and that this process is subject to the same sequence requirements as in human cells. An RNA interference screen targeting protein trafficking genes in S2 cells revealed a requirement for clathrin and the clathrin-associated, plasma membrane-localized AP2 complex in the downregulation of CD4. The requirement for AP2 was confirmed in the human cell line HeLa. A yeast three-hybrid system and glutathione S-transferase pull-down analyses using the recombinant AP2 complex variant produced in our laboratory were used to demonstrate a robust, direct interaction between HIV-1 Nef and AP2. This interaction requires a dileucine motif in Nef that is also essential for downregulation of CD4.