title: Alteration of the C-terminal ligand specificity of the erbin PDZ domain by allosteric mutational effects.
abstract: Modulation of protein binding specificity is important for basic biology and for applied science. Here we explore how binding specificity is conveyed in PDZ (postsynaptic density protein-95/discs large/zonula occludens-1) domains, small interaction modules that recognize various proteins by binding to an extended C terminus. Our goal was to engineer variants of the Erbin PDZ domain with altered specificity for the most C-terminal position (position 0) where a Val is strongly preferred by the wild-type domain. We constructed a library of PDZ domains by randomizing residues in direct contact with position 0 and in a loop that is close to but does not contact position 0. We used phage display to select for PDZ variants that bind to 19 peptide ligands differing only at position 0. To verify that each obtained PDZ domain exhibited the correct binding specificity, we selected peptide ligands for each domain. Despite intensive efforts, we were only able to evolve Erbin PDZ domain variants with selectivity for the aliphatic C-terminal side chains Val, Ile and Leu. Interestingly, many PDZ domains with these three distinct specificities contained identical amino acids at positions that directly contact position 0 but differed in the loop that does not contact position 0. Computational modeling of the selected PDZ domains shows how slight conformational changes in the loop region propagate to the binding site and result in different binding specificities. Our results demonstrate that second-sphere residues could be crucial in determining protein binding specificity.
authors.affiliation: Departamento de Química Física e Instituto de Biotecnología, Facultad de Ciencias, Universidad de Granada, 18071 Granada, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: Murciano-Calles
authors.affiliation: Banting and Best Department of Medical Research and Department of Molecular Genetics, University of Toronto, The Donnelly Centre, 160 College Street, Toronto, Ontario, M5S 3E1, Canada.
authors.forename: Megan E
authors.initials: ME
authors.lastname: McLaughlin
authors.affiliation: Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, Edmond J. Safra Campus Givat Ram, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel.
authors.forename: Ariel
authors.initials: A
authors.lastname: Erijman
authors.affiliation: Banting and Best Department of Medical Research and Department of Molecular Genetics, University of Toronto, The Donnelly Centre, 160 College Street, Toronto, Ontario, M5S 3E1, Canada.
authors.forename: Yogesh
authors.initials: Y
authors.lastname: Hooda
authors.affiliation: Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane QLD 4000, Australia.
authors.forename: Nishant
authors.initials: N
authors.lastname: Chakravorty
authors.affiliation: Departamento de Química Física e Instituto de Biotecnología, Facultad de Ciencias, Universidad de Granada, 18071 Granada, Spain.
authors.forename: Jose C
authors.initials: JC
authors.lastname: Martinez
authors.affiliation: Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, Edmond J. Safra Campus Givat Ram, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel. Electronic address: jshifman@mail.huji.ac.il.
authors.forename: Julia M
authors.initials: JM
authors.lastname: Shifman
authors.affiliation: Banting and Best Department of Medical Research and Department of Molecular Genetics, University of Toronto, The Donnelly Centre, 160 College Street, Toronto, Ontario, M5S 3E1, Canada. Electronic address: sachdev.sidhu@utoronto.ca.
authors.forename: Sachdev S
authors.initials: SS
authors.lastname: Sidhu
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000235:genetics*; D000495:Allosteric Site; D003198:Computer Simulation; D004252:DNA Mutational Analysis; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008024:Ligands; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D012984:Software
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C413364:ERBIN protein, human; D008024:Ligands; D019151:Peptide Library; D010455:Peptides; D005982:Glutathione Transferase
keywords: binding cooperativity; computational modeling; phage display; protein engineering; protein interaction domains