A sequential binding mechanism in a PDZ domain

TY - JOUR

T1 - A sequential binding mechanism in a PDZ domain

AU - Chi, Celestine N

AU - Bach, Anders

AU - Engström, Åke

AU - Wang, Huiqun

AU - Strømgaard, Kristian

AU - Gianni, Stefano

AU - Jemth, Per

N1 - 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

PY - 2009

Y1 - 2009

N2 - 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.

AB - 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.

KW - Former Faculty of Pharmaceutical Sciences

U2 - 10.1021/bi900559k

DO - 10.1021/bi900559k

M3 - Journal article

C2 - 19496620

SN - 0006-2960

VL - 48

SP - 7089

EP - 7097

JO - Biochemistry

JF - Biochemistry

IS - 30

ER -