Molecular Recognition by Clathrin Adaptors

笼状蛋白适配器的分子识别作用 在真核细胞的内吞作用和胞体分选中，网状蛋白被层起着普遍而基本的作用。网状蛋白形成一个笼子，包裹着载货的小泡，但网状蛋白本身并不直接与货物结合。货物通过连接货物和笼蛋白的接头蛋白被分类到覆盖着笼状蛋白的小泡中。最著名的通用接头是异四聚体接头蛋白复合体(AP复合体)和多模块GGA接头蛋白。该项目的目标是1)确定接头蛋白上货物的结合位点，并定量测量它们的亲和力；2)确定接头与可溶性货物片段之间的复合体的晶体结构；3)利用突变分析将结构和功能联系起来。 特定的分选信号将跨膜蛋白定向到内体-溶酶体系统的隔室。基于酪氨酸的分选信号与AP复合体(AP-1、-2、-3和4)的大亚基的EAR结构域结合，并得到了很好的表征。存在于分选受体细胞质尾部的DXXLL形式的酸性簇二亮氨酸信号，如阳离子非依赖性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。在以前的工作中，我们实验室确定了与DXXLL基序多肽结合的GGA VHS结构域的结构。我们继续研究了GGA蛋白与相关蛋白质或多肽配体形成的复合体中的所有结构域。DXXXLL基序存在于重要的医学宿主蛋白中，如CD4和病毒蛋白，如HIV-1 Nef。与DXXLL基序不同，它与AP复合体结合，而不是GGA。尽管它的重要性和悠久的历史，以及多个实验室多年来的努力，DXXXLL基序的结构基础尚不清楚。为了解决这个问题，我们的实验室产生了一个变异的AP-2复合体，它能够与DXXXLL基序多肽结合。 CD_4的下调依赖于Nef C-末端的一个保守的(D/E)XXXL(L/I)型二亮氨酸基序，该基序介导与笼蛋白适配器复合体AP-1、AP-2和AP-3的结合。2007-2008年，我们与Bonifacino实验室(NICHD)合作，在该环中确定了一个共识(D/E)D基序，作为Nef与AP-2相互作用的第二个保守决定因素，尽管不与AP-1和AP-3相互作用。我们小组的主要作用是提纯AP-2核心蛋白的变种形式，使其能够与Nef结合。这个二酸基序的突变使AP-2结合和CD4下调都失效。酪氨酸酶的二亮氨酸基序，在其自然环境和Nef环境中，都可以独立于二酸基序与AP-2结合。这些结果确定了一种新型的AP-2相互作用决定因素，支持了AP-2是Nef下调CD4的关键网状蛋白适配器的概念，并揭示了二亮氨酸分选信号之间以前未被认识到的多样性。

Molecular Recognition by Clathrin Adaptors The clathrin coat plays a ubiquitous and fundamental role in endocytosis and in endosomal sorting within the eukaryotic cell. Clathrin forms a cage that surrounds cargo-bearing vesicles, but clathrin itself does not directly bind to cargo. Cargo is sorted into clathrin-coated vesicles by adaptor proteins that physically bridge cargo and clathrin. The best-known general purpose adaptors are the heterotetrameric adaptor protein complexes (AP complexes) and the multimodular GGA adaptor proteins. The goals of this project are 1) to identify the binding sites for cargo on adaptor proteins and measure their affinities quantitatively; 2) to determine the crystal structures of complexes between adaptors and soluble cargo fragments; and 3) to relate structure and function using mutational analysis. 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 (AP-1, -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 generated a variant AP-2 complex that is competent to bind to DXXXLL motif peptides. CD4 downregulation depends on a conserved (D/E)XXXL(L/I)-type dileucine motif in the C-terminal, flexible loop of Nef, which mediates binding to the clathrin adaptor complexes AP-1, AP-2, and AP-3. In 2007-8, we collaborated with the Bonifacino lab (NICHD) to identify a consensus (D/E)D motif within this loop as a second, conserved determinant of interaction of Nef with AP-2, though not with AP-1 and AP-3. The principal role of our group was to purify the variant form of the AP-2 core competent to bind to Nef. Mutations in this diacidic motif abrogate both AP-2 binding and CD4 downregulation. A dileucine motif from tyrosinase, both in its native context and in the context of Nef, can bind to AP-2 independently of the diacidic motif. These results identified a novel type of AP-2 interaction determinant, supporting the notion that AP-2 is the key clathrin adaptor for the downregulation of CD4 by Nef, and revealed a previously unrecognized diversity among dileucine sorting signals.