Molecular Recognition by Clathrin Adaptors

网格蛋白衔接子的分子识别 网格蛋白外壳在真核细胞内的内吞作用和内体分选中起着普遍存在的和基本的作用。网格蛋白形成一个笼，围绕货物轴承囊泡，但网格蛋白本身不直接结合货物。货物通过衔接蛋白被分选到网格蛋白包被的囊泡中，衔接蛋白物理桥接货物和网格蛋白。最著名的通用衔接子是异源四聚体衔接子蛋白复合物（AP复合物）和多模块GGA衔接子蛋白。该项目的总体目标是：1）鉴定衔接蛋白上货物的结合位点并定量测量其亲和力; 2）确定衔接蛋白和可溶性货物片段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。 BLOC-1（溶酶体相关细胞器复合物-1的生物发生）对于黑素体生物发生至关重要，并且也与神经功能和疾病有关。BLOC-1不是网格蛋白衔接子，但在黑素体生物发生中与网格蛋白衔接子复合物AP-3协同起作用。我们发现BLOC-1是一个细长的复合物，包含八个亚基中的每一个的一个拷贝苍白球蛋白，卡布奇诺，dysbindin，Snapin，Muted，BLOS 1，BLOS 2和BLOS 3。该复合物表现为8个球状结构域的线性链，长300，直径30。各个域柔性连接，使得线性链经受多达45 °的弯曲。定义了两个稳定的亚复合物，pallidin-Cappuccino-BL 0 S1和dysbindin-Snapin-BL 0 S2。这两个亚复合物是1：1：1的异源三聚体，形成扩展的结构，如其流体动力学性质所示。这两个亚复合物似乎构成了较大的BLOC-1链内的灵活单元，这种安排有利于在管状内体生物发生和分选过程中与多个BLOC-1伙伴同时相互作用。

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.