Molecular Recognition by Clathrin Adaptors

网格蛋白适配器的分子识别

• 批准号: 8553447
• 负责人: James Hurley
• 金额: $ 21.05万
• 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/8553447
• 关键词: Adaptor Signaling Protein; Affinity; Binding; Binding Sites; Biogenesis; Caliber; Clathrin; Clathrin Adaptors; Clathrin-Coated Vesicles; Complex; Endocytosis; Endosomes; Eukaryotic Cell; Goals; Individual; Lysosomes; Measures; Melanosomes; Nervous System Physiology; Organelles; Play; Property; Role; SNAPIN gene; Sorting - Cell Movement; Structure; Tubular formation; Vesicle; flexibility; golgi associated, gamma adaptin homologous, ADP-ribosylation factor interacting protein; molecular recognition; nervous system disorder; protein complex

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 overall 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. BLOC-1 (biogenesis of lysosome-related organelles complex-1) is critical for melanosome biogenesis and has also been implicated in neurological function and disease. BLOC-1 is not a clathrin adaptor, but functions in melanosome biogenesis in concert with the clathrin adaptor complex AP-3. We found that BLOC-1 is an elongated complex that contains one copy each of the eight subunits pallidin, Cappuccino, dysbindin, Snapin, Muted, BLOS1, BLOS2, and BLOS3. The complex appears as a linear chain of eight globular domains, ∼300 Å long and ∼30 Å in diameter. The individual domains are flexibly connected such that the linear chain undergoes bending by as much as 45. Two stable subcomplexes were defined, pallidin-Cappuccino-BLOS1 and dysbindin-Snapin-BLOS2. Both subcomplexes are 1:1:1 heterotrimers that form extended structures as indicated by their hydrodynamic properties. The two subcomplexes appear to constitute flexible units within the larger BLOC-1 chain, an arrangement conducive to simultaneous interactions with multiple BLOC-1 partners in the course of tubular endosome biogenesis and sorting.

网格蛋白适配器的分子识别 网格蛋白外壳在真核细胞内的胞吞作用和内体分选中发挥着普遍存在的基本作用。网格蛋白形成一个笼子，包围着承载货物的囊泡，但网格蛋白本身并不直接与货物结合。通过连接货物和网格蛋白的接头蛋白将货物分类到网格蛋白包被的囊泡中。最著名的通用接头蛋白是异四聚体接头蛋白复合物（AP 复合物）和多模块 GGA 接头蛋白。该项目的总体目标是 1) 识别接头蛋白上货物的结合位点并定量测量它们的亲和力； 2) 确定接头和可溶性货物片段之间的复合物的晶体结构； 3) 使用突变分析关联结构和功能。 BLOC-1（溶酶体相关细胞器复合物的生物发生）对于黑素体生物发生至关重要，并且还与神经功能和疾病有关。 BLOC-1 不是网格蛋白接头，但与网格蛋白接头复合物 AP-3 一起在黑素体生物发生中发挥作用。我们发现 BLOC-1 是一种细长复合物，包含 pallidin、Cappuccino、dysbindin、Snapin、Mated、BLOS1、BLOS2 和 BLOS3 八个亚基各一个副本。该复合物表现为由八个球状结构域组成的线性链，长约 300 Å，直径约 30 Å。各个结构域灵活连接，使得线性链弯曲多达 45°。定义了两个稳定的子复合物：pallidin-Cappuccino-BLOS1 和 Dysbindin-Snapin-BLOS2。两个子复合物都是 1:1:1 异源三聚体，可形成延伸结构，如其流体动力学特性所示。这两个子复合物似乎在较大的 BLOC-1 链中构成了灵活的单元，这种排列有利于在管状内体生物发生和分选过程中与多个 BLOC-1 伴侣同时相互作用。