Dimerization and protein binding specificity of the U2AF homology motif of the splicing factor Puf60.
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.
Corsini
Lorenzo
L
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.
Hothorn
Michael
M
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.
Stier
Gunter
G
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.
Rybin
Vladimir
V
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.
Scheffzek
Klaus
K
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.
Gibson
Toby J
TJ
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.
Sattler
Michael
M
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. Electronic address: sattler@helmholtz-muenchen.de.
D020816:Amino Acid Motifs / Q000502:physiology; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D006801:Humans; D019906:Nuclear Magnetic Resonance, Biomolecular; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D020836:Protein Structure, Quaternary / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D000072260:RNA Splicing Factors; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000737:chemistry* / Q000378:metabolism; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D000072668:Splicing Factor U2AF; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D000072260:RNA Splicing Factors; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; D017413:Ribonucleoprotein, U2 Small Nuclear; D012261:Ribonucleoproteins; C107467:SF1 protein, human; C112491:SF3B1 protein, human; D000072668:Splicing Factor U2AF; D014157:Transcription Factors; C000607494:U2AF2 protein, human; C410595:poly-U binding splicing factor 60KDa; D012967:Sodium Dodecyl Sulfate