A sequential binding mechanism in a PDZ domain
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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.
Original language | English |
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Journal | Biochemistry |
Volume | 48 |
Issue number | 30 |
Pages (from-to) | 7089-7097 |
ISSN | 0006-2960 |
DOIs | |
Publication status | Published - 2009 |
Bibliographical note
Keywords: Adaptor Proteins, Signal Transducing; Amino Acid Sequence; Binding Sites; DNA-Binding Proteins; Humans; Ligands; Membrane Proteins; Models, Molecular; Molecular Sequence Data; Oncogene Proteins, Viral; PDZ Domains; Peptides; Protein Binding; Protein Conformation; Protein Denaturation; Protein Folding; Thermodynamics
- Former Faculty of Pharmaceutical Sciences
Research areas
ID: 17365971