GYF domain proteomics reveals interaction sites in known and novel target proteins.
GYF domains are conserved eukaryotic adaptor domains that recognize proline-rich sequences. Although the structure and function of the prototypic GYF domain from the human CD2BP2 protein have been characterized in detail, very little is known about GYF domains from other proteins and species. Here we describe the binding properties of four GYF domains of various origins. Phage display in combination with SPOT analysis revealed the PPG(F/I/L/M/V) motif as a general recognition signature. Based on these results, the proteomes of human, yeast, and Arabidopsis thaliana were searched for potential interaction sites. Binding of several candidate proteins was confirmed by pull-down experiments or yeast two-hybrid analysis. The binding epitope of the GYF domain from the yeast SMY2 protein was mapped by NMR spectroscopy and led to a structural model that accounts for the different binding properties of SMY2-type GYF domains and the CD2BP2-GYF domain.
Kofler
Michael
M
Protein Engineering Group, Forschungsinstitut für Molekulare Pharmakologie and Freie Universität Berlin, Robert-Rössle-Str. 10, 13125 Berlin, Germany.
Motzny
Kathrin
K
Freund
Christian
C
D048868:Adaptor Proteins, Signal Transducing; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry; D018604:Epitope Mapping; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D019151:Peptide Library; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D040901:Proteomics; D000072260:RNA Splicing Factors; D016601:RNA-Binding Proteins / Q000032:analysis / Q000737:chemistry; D012097:Repressor Proteins / Q000032:analysis / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis / Q000737:chemistry; D016415:Sequence Alignment; D013379:Substrate Specificity; D020798:Two-Hybrid System Techniques; D033921:Vesicular Transport Proteins
D048868:Adaptor Proteins, Signal Transducing; C120378:CD2BP2 protein, human; D002352:Carrier Proteins; C409040:MSL5 protein, S cerevisiae; D019151:Peptide Library; D011506:Proteins; D000072260:RNA Splicing Factors; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; C083574:SMY2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins