GYF Domain Proteomics Reveals Interaction Sites in Known and Novel Target Proteins

Abstract

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. 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. The GYF domain is a protein interaction domain ubiquitously expressed in eukaryotic species (1Nishizawa K. Freund C. Li J. Wagner G. Reinherz E.L. Identification of a proline-binding motif regulating CD2-triggered T lymphocyte activation.Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 14897-14902Google Scholar, 2Freund C. Dotsch V. Nishizawa K. Reinherz E.L. Wagner G. The GYF domain is a novel structural fold that is involved in lymphoid signaling through proline-rich sequences.Nat. Struct. Biol. 1999; 6: 656-660Google Scholar). It belongs to the functional class of proline-rich sequence (PRS) 1The abbreviations used are: PRS, proline-rich sequence; EVH1, Ena-Vasp homology 1; GFP, green fluorescent protein; GYN4, GYF domain-containing protein binding to Not4; GYN4-PR, GYN4-GYF, comprising the C-terminal proline-rich motif; NPWBP, Npw38-binding protein; PERQ1, PERQ amino acid rich with GYF domain protein 1; PERQ2, PERQ amino acid rich with GYF domain protein 2; PR-SYH1, SYH1-GYF, comprising the N-terminal proline-rich motif; SYH1, SMY2 homolog 1; SH3, Src homology 3; snRNP, small nuclear ribonucleoprotein; SWAN, SH3/WW domain anchor protein in the nucleus; UEV, ubiquitin E2 (ubiquitin carrier protein) variant; HA, hemagglutinin; SGD, Saccharomyces Genome Database; eIF, eukaryotic initiation factor; E3, ubiquitin-protein isopeptide ligase; TAIR, The Arabidopsis Information Resource. recognition domains such as SH3 (3Mayer B.J. Hamaguchi M. Hanafusa H. A novel viral oncogene with structural similarity to phospholipase C.Nature. 1988; 332: 272-275Google Scholar, 4Stahl M.L. Ferenz C.R. Kelleher K.L. Kriz R.W. Knopf J.L. Sequence similarity of phospholipase C with the non-catalytic region of src.Nature. 1988; 332: 269-272Google Scholar), WW (5Bork P. Sudol M. The WW domain: a signalling site in dystrophin?.Trends Biochem. Sci. 1994; 19: 531-533Google Scholar), EVH1 (6Niebuhr K. Ebel F. Frank R. Reinhard M. Domann E. Carl U.D. Walter U. Gertler F.B. Wehland J. Chakraborty T. A novel proline-rich motif present in ActA of Listeria monocytogenes and cytoskeletal protei