title: Structural and functional characterization of a ubiquitin variant engineered for tight and specific binding to an alpha-helical ubiquitin interacting motif.
abstract: Ubiquitin interacting motifs (UIMs) are short α-helices found in a number of eukaryotic proteins. UIMs interact weakly but specifically with ubiquitin conjugated to other proteins, and in so doing, mediate specific cellular signals. Here we used phage display to generate ubiquitin variants (UbVs) targeting the N-terminal UIM of the yeast Vps27 protein. Selections yielded UbV.v27.1, which recognized the cognate UIM with high specificity relative to other yeast UIMs and bound with an affinity more than two orders of magnitude higher than that of ubiquitin. Structural and mutational studies of the UbV.v27.1-UIM complex revealed the molecular details for the enhanced affinity and specificity of UbV.v27.1, and underscored the importance of changes at the binding interface as well as at positions that do not contact the UIM. Our study highlights the power of the phage display approach for selecting UbVs with unprecedented affinity and high selectivity for particular α-helical UIM domains within proteomes, and it establishes a general approach for the development of inhibitors targeting interactions of this type.
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, M5G 1X5, Canada.|Department of Biochemistry, University of Toronto, Toronto, Ontario, M5S 1A8, Canada.
authors.forename: Noah
authors.initials: N
authors.lastname: Manczyk
authors.affiliation: Department of Molecular Genetics, University of Toronto, Toronto, Ontario, M5S 1A8, Canada.|Donnelly Centre for Cellular and Biomolecular Research, Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, M5S 3E1, Canada.
authors.forename: Bradley P
authors.initials: BP
authors.lastname: Yates
authors.affiliation: Department of Molecular Genetics, University of Toronto, Toronto, Ontario, M5S 1A8, Canada.|Donnelly Centre for Cellular and Biomolecular Research, Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, M5S 3E1, Canada.
authors.forename: Gianluca
authors.initials: G
authors.lastname: Veggiani
authors.affiliation: Institute of Biochemistry II, Faculty of Medicine, Goethe University, Frankfurt am Main, 60590, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Ernst
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, M5G 1X5, Canada.|Department of Molecular Genetics, University of Toronto, Toronto, Ontario, M5S 1A8, Canada.|Department of Biochemistry, University of Toronto, Toronto, Ontario, M5S 1A8, Canada.
authors.forename: Frank
authors.initials: F
authors.lastname: Sicheri
authors.affiliation: Department of Molecular Genetics, University of Toronto, Toronto, Ontario, M5S 1A8, Canada.|Donnelly Centre for Cellular and Biomolecular Research, Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, M5S 3E1, Canada.
authors.forename: Sachdev S
authors.initials: SS
authors.lastname: Sidhu
mesh_terms: D020816:Amino Acid Motifs; D056827:Endosomal Sorting Complexes Required for Transport / Q000737:chemistry* / Q000235:genetics; D019151:Peptide Library; D000072417:Protein Domains; D015202:Protein Engineering; D020836:Protein Structure, Quaternary; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics; D025801:Ubiquitin / Q000737:chemistry* / Q000235:genetics
chemical_list: D056827:Endosomal Sorting Complexes Required for Transport; D019151:Peptide Library; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; C119627:VPS27 protein, S cerevisiae
keywords: phage display; protein engineering; ubiquitin; ubiquitin interacting motif