剪接因子Puf60的U2AF同源基序的二聚化及蛋白质结合特异性
Dimerization and protein binding specificity of the U2AF homology motif of the splicing factor Puf60.
作者信息
Corsini Lorenzo, Hothorn Michael, Stier Gunter, Rybin Vladimir, Scheffzek Klaus, Gibson Toby J, Sattler Michael
机构信息
Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany, the Institute of Structural Biology, Helmholtz Zentrum Mu¨nchen, Ingolsta¨dter Landstrasse 1, 85764 Neuherberg, Germany, and the Munich Center for Integrated Protein Science and Chair Biomolecular NMR, Department Chemie, Technische Universita¨t Mu¨nchen, Lichtenbergstrasse 4, 85747 Garching, Germany.
Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany, the Institute of Structural Biology, Helmholtz Zentrum Mu¨nchen, Ingolsta¨dter Landstrasse 1, 85764 Neuherberg, Germany, and the Munich Center for Integrated Protein Science and Chair Biomolecular NMR, Department Chemie, Technische Universita¨t Mu¨nchen, Lichtenbergstrasse 4, 85747 Garching, Germany; Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany, the Institute of Structural Biology, Helmholtz Zentrum Mu¨nchen, Ingolsta¨dter Landstrasse 1, 85764 Neuherberg, Germany, and the Munich Center for Integrated Protein Science and Chair Biomolecular NMR, Department Chemie, Technische Universita¨t Mu¨nchen, Lichtenbergstrasse 4, 85747 Garching, Germany; Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany, the Institute of Structural Biology, Helmholtz Zentrum Mu¨nchen, Ingolsta¨dter Landstrasse 1, 85764 Neuherberg, Germany, and the Munich Center for Integrated Protein Science and Chair Biomolecular NMR, Department Chemie, Technische Universita¨t Mu¨nchen, Lichtenbergstrasse 4, 85747 Garching, Germany.
出版信息
J Biol Chem. 2009 Jan 2;284(1):630-639. doi: 10.1074/jbc.M805395200. Epub 2008 Oct 29.
PUF60 is an essential splicing factor functionally related and homologous to U2AF(65). Its C-terminal domain belongs to the family of U2AF (U2 auxiliary factor) homology motifs (UHM), a subgroup of RNA recognition motifs that bind to tryptophan-containing linear peptide motifs (UHM ligand motifs, ULMs) in several nuclear proteins. Here, we show that the Puf60 UHM is mainly monomeric in physiological buffer, whereas its dimerization is induced upon the addition of SDS. The crystal structure of PUF60-UHM at 2.2 angstroms resolution, NMR data, and mutational analysis reveal that the dimer interface is mediated by electrostatic interactions involving a flexible loop. Using glutathione S-transferase pulldown experiments, isothermal titration calorimetry, and NMR titrations, we find that Puf60-UHM binds to ULM sequences in the splicing factors SF1, U2AF65, and SF3b155. Compared with U2AF65-UHM, Puf60-UHM has distinct binding preferences to ULMs in the N terminus of SF3b155. Our data suggest that the functional cooperativity between U2AF65 and Puf60 may involve simultaneous interactions of the two proteins with SF3b155.
PUF60是一种与U2AF(65)功能相关且同源的必需剪接因子。其C端结构域属于U2AF（U2辅助因子）同源基序家族（UHM），是RNA识别基序的一个亚组，可与几种核蛋白中含色氨酸的线性肽基序（UHM配体基序，ULM）结合。在此，我们表明Puf60 UHM在生理缓冲液中主要为单体，而加入SDS后会诱导其二聚化。分辨率为2.2埃的PUF60-UHM晶体结构、核磁共振数据和突变分析表明，二聚体界面由涉及一个柔性环的静电相互作用介导。通过谷胱甘肽S-转移酶下拉实验、等温滴定量热法和核磁共振滴定，我们发现Puf60-UHM与剪接因子SF1、U2AF65和SF3b155中的ULM序列结合。与U2AF65-UHM相比，Puf60-UHM对SF3b155 N端的ULM具有不同的结合偏好。我们的数据表明，U2AF65和Puf60之间的功能协同作用可能涉及这两种蛋白质与SF3b155的同时相互作用。
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