title: A sequential binding mechanism in a PDZ domain.
abstract: Conformational selection and induced fit are two well-known mechanisms of allosteric protein-ligand interaction. Some proteins, like ubiquitin, have recently been found to exist in multiple conformations at equilibrium, suggesting that the conformational selection may be a general mechanism of interaction, in particular for single-domain proteins. Here, we found that the PDZ2 domain of SAP97 binds its ligand via a sequential (induced fit) mechanism. We performed binding experiments using SAP97 PDZ2 and peptide ligands and observed biphasic kinetics with the stopped-flow technique, indicating that ligand binding involves at least a two-step process. By using an ultrarapid continuous-flow mixer, we then detected a hyperbolic dependence of binding rate constants on peptide concentration, corroborating the two-step binding mechanism. Furthermore, we found a similar dependence of the rate constants on both PDZ and peptide concentration, demonstrating that the PDZ2-peptide interaction involves a precomplex, which then undergoes a conformational change, and thereby follows an induced fit mechanism.
authors.affiliation: Department of Medical Biochemistry and Microbiology, Uppsala University, BMC Box 582, SE-75123 Uppsala, Sweden.
authors.forename: Celestine N
authors.initials: CN
authors.lastname: Chi
authors.forename: Anders
authors.initials: A
authors.lastname: Bach
authors.forename: Ake
authors.initials: A
authors.lastname: Engström
authors.forename: Huiqun
authors.initials: H
authors.lastname: Wang
authors.forename: Kristian
authors.initials: K
authors.lastname: Strømgaard
authors.forename: Stefano
authors.initials: S
authors.lastname: Gianni
authors.forename: Per
authors.initials: P
authors.lastname: Jemth
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000235:genetics; D000074288:Discs Large Homolog 1 Protein; D006801:Humans; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000235:genetics; D054731:PDZ Domains; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013816:Thermodynamics
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C513314:DLG1 protein, human; D004268:DNA-Binding Proteins; D000074288:Discs Large Homolog 1 Protein; C052603:E6 protein, Human papillomavirus type 18; D008024:Ligands; D008565:Membrane Proteins; D009856:Oncogene Proteins, Viral; D010455:Peptides