title: Cys-Gly specific dipeptidase Dug1p from S. cerevisiae binds promiscuously to di-, tri-, and tetra-peptides: Peptide-protein interaction, homology modeling, and activity studies reveal a latent promiscuity in substrate recognition.
abstract: Dug1p is a recently identified novel dipeptidase and plays an important role in glutathione (GSH) degradation. To understand the mechanism of its substrate recognition and specificity towards Cys-Gly dipeptides, we characterized the solution properties of Dug1p and studied the thermodynamics of Dug1p-peptide interactions. In addition, we used homology modeling and ligand docking approaches to get structural insights into Dug1p-peptide interaction. Dug1p exists as dimer and the stoichiometry of peptide-Dug1p complex is 2:1 indicating each monomer in the dimer binds to one peptide. Thermodynamic studies indicate that the free energy change for Dug1p-peptide complex formation is similar (▵G(bind) ∼ -7.0 kcal/mol) for a variety of peptides of different composition and length (22 peptides). Three-dimensional model of Dug1p is constructed and docking of peptides to the modeled structure suggests that hydrogen bonding to active site residues (E172, E171, and D137) lock the N-terminal of the peptide into the binding site. Dug1p recognizes peptides in a metal independent manner and peptide binding is not sensitive to salts (dlogK/dlog[salt] ∼ 0) over a range of [NaCl] (0.02-0.5 M), [ZnCl(2)], and [MnCl(2)] (0-0.5 mM). Our results indicate that promiscuity in peptide binding results from the locking of peptide N-terminus into the active site. These observations were supported by our competitive inhibition activity assays. Dug1p activity towards Cys-Gly peptide is significantly reduced (∼ 70%) in the presence of Glu-Cys-Gly. Therefore, Dug1p can recognize a variety of oligopeptides, but has evolved with post-binding screening potential to hydrolyze Cys-Gly peptides selectively.
authors.affiliation: Institute of Microbial Technology, Sector 39A, Chandigarh 160036, India.
authors.forename: Hardeep
authors.initials: H
authors.lastname: Kaur
authors.forename: Manish
authors.initials: M
authors.lastname: Datt
authors.forename: Mary Krishna
authors.initials: MK
authors.lastname: Ekka
authors.forename: Monica
authors.initials: M
authors.lastname: Mittal
authors.forename: Appu Kumar
authors.initials: AK
authors.lastname: Singh
authors.forename: Sangaralingam
authors.initials: S
authors.lastname: Kumaran
mesh_terms: D000494:Allosteric Regulation; D001667:Binding, Competitive; D004150:Dipeptidases / Q000737:chemistry* / Q000378:metabolism*; D004151:Dipeptides / Q000737:chemistry / Q000378:metabolism*; D004795:Enzyme Stability; D008024:Ligands; D008345:Manganese / Q000494:pharmacology; D008958:Models, Molecular; D009994:Osmolar Concentration; D011485:Protein Binding; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013057:Spectrum Analysis; D013379:Substrate Specificity; D013816:Thermodynamics; D015032:Zinc / Q000494:pharmacology
chemical_list: D004151:Dipeptides; D008024:Ligands; D029701:Saccharomyces cerevisiae Proteins; D008345:Manganese; C022291:cystinylglycine; C523458:DUG1 protein, S cerevisiae; D004150:Dipeptidases; D015032:Zinc