Molecular Recognition by Clathrin Adaptors

网格蛋白适配器的分子识别

• 批准号: 7734084
• 负责人: James Hurley
• 金额: $ 34.46万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7734084
• 关键词: Adaptor Signaling Protein; Address; Affinity; Binding; Binding Sites; C-terminal; Cations; Clathrin; Clathrin Adaptors; Clathrin-Coated Vesicles; Complex; Consensus; Cytoplasmic Tail; Down-Regulation; Ear; Endocytosis; Eukaryotic Cell; Goals; HIV-1; IGF Type 2 Receptor; Integral Membrane Protein; Laboratories; Ligands; Measures; Mediating; Monophenol Monooxygenase; Mutation; National Institute of Child Health and Human Development; Peptides; Personal Satisfaction; Play; Proteins; Purpose; Recording of previous events; Role; Signal Transduction; Sorting - Cell Movement; Structure; System; TFAP2A gene; Transcription Factor AP-1; Tyrosine; Variant; Vesicle; Viral Proteins; Work; base; golgi associated, gamma adaptin homologous, ADP-ribosylation factor interacting protein; molecular recognition; novel; receptor

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.

网格蛋白适配器的分子识别 网格蛋白涂层在内吞作用和真核生物细胞内的内体分选中起普遍存在的基本作用。网状蛋白形成围绕载有货物的囊泡的笼子，但网状蛋白本身并未直接与货物结合。货物通过物理桥接货物和网格蛋白的衔接蛋白将货物分类。最著名的通用适配器是异晶衔接蛋白复合物（AP复合物）和多模块化GGA适配器蛋白。该项目的目标是1）确定适配器蛋白上货物的结合位点并定量测量其亲和力； 2）确定适配器和可溶性货物碎片之间复合物的晶体结构； 3）使用突变分析将结构和功能联系起来。 特定的分类信号将跨膜蛋白直接到内体溶菌体系统的隔室。基于酪氨酸的排序信号与AP复合物（AP -1，-2，-3和4）的大型亚基的EAR结构域结合，并且已经表现得很好。分类受体的细胞质尾巴中存在的DXXLL形式的酸性簇 - 二氨酸信号，例如与GGA适配器蛋白的VHS结构域的VHS结构域认识到阳离子独立于阳离子依赖性和阳离子依赖性甘露糖6-磷酸受体。在先前的工作中，我们的实验室确定了与DXXLL-MOTIF肽结合的GGA VHS结构域的结构。我们继续表征与相关蛋白或肽配体复合物中GGA蛋白的所有结构域。 DXXXLL基序存在于医学上重要的宿主蛋白中，例如CD4和病毒蛋白，例如HIV-1 NEF。与DXXLL基序不同，它与AP复合物而不是GGAS结合。尽管具有多个实验室的重要性和实质性的历史以及多年的努力，但DXXXLL图案的结构基础尚不清楚。为了解决这个问题，我们的实验室生成了一种有能力与DXXXLL基序肽结合的变体AP-2复合物。 CD4下调取决于在C末端的NEF中保守的（D/E）XXXL（L/I） - 型二李酮基序，Nef的柔性环将介导与clathrin辅助络合物的结合，与clathrin appter ap-1，ap-2和ap-3介导。在2007 - 8年，我们与Bonifacino Lab（NICHD）合作，以确定该循环中的共识（D/E）D序列是NEF与AP-2相互作用的第二个保守的决定因素，尽管不是AP-1和AP-3。我们小组的主要作用是净化竞争与NEF结合的AP-2核心的变体形式。该二肽基序中的突变消除了AP-2结合和CD4下调。酪氨酸酶的二利氨酸基序在其天然环境和NEF的背景下都可以独立于二氧化基序与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AP-2是NEF下调CD4的关键网状蛋白适配器，并揭示了dilecine分类信号中先前未知的多样性。