Suzuki Hironori, Kawasaki Masato, Inuzuka Tatsutoshi, Okumura Mayumi, Kakiuchi Takeshi, Shibata Hideki, Wakatsuki Soichi, Maki Masatoshi
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
Structure. 2008 Oct 8;16(10):1562-73. doi: 10.1016/j.str.2008.07.012.
ALG-2 belongs to the penta-EF-hand (PEF) protein family and interacts with various intracellular proteins, such as Alix and TSG101, that are involved in endosomal sorting and HIV budding. Through X-ray crystallography, we solved the structures of Ca(2+)-free and -bound forms of N-terminally truncated human ALG-2 (des3-20ALG-2), Zn(2+)-bound form of full-length ALG-2, and the structure of the complex between des3-23ALG-2 and the peptide corresponding to Alix799-814 in Zn(2+)-bound form. Binding of Ca(2+) to EF3 enables the side chain of Arg125, present in the loop connecting EF3 and EF4, to move enough to make a primary hydrophobic pocket accessible to the critical PPYP motif, which partially overlaps with the GPP motif for the binding of Cep55 (centrosome protein 55 kDa). Based on these results, together with the results of in vitro binding assay with mutant ALG-2 and Alix proteins, we propose a Ca(2+)/EF3-driven arginine switch mechanism for ALG-2 binding to Alix.
ALG-2属于五聚体EF手型（PEF）蛋白家族，可与多种参与内体分选和HIV出芽的细胞内蛋白相互作用，如Alix和TSG101。通过X射线晶体学，我们解析了N端截短的人ALG-2（des3-20ALG-2）的无钙和结合钙形式的结构、全长ALG-2的结合锌形式的结构，以及des3-23ALG-2与锌结合形式下对应于Alix799-814的肽段形成的复合物的结构。钙离子与EF3结合，使位于连接EF3和EF4的环中的Arg125侧链移动足够距离，形成一个主要的疏水口袋，可容纳关键的PPYP基序，该基序部分与用于结合Cep55（55 kDa中心体蛋白）的GPP基序重叠。基于这些结果，以及与突变型ALG-2和Alix蛋白的体外结合试验结果，我们提出了一种由钙离子/EF3驱动的精氨酸开关机制，用于ALG-2与Alix的结合。
