title: Deciphering the kinetic binding mechanism of dimeric ligands using a potent plasma-stable dimeric inhibitor of postsynaptic density protein-95 as an example.
abstract: Dimeric ligands can be potent inhibitors of protein-protein or enzyme-substrate interactions. They have increased affinity and specificity toward their targets due to their ability to bind two binding sites simultaneously and are therefore attractive in drug design. However, few studies have addressed the kinetic mechanism of interaction of such bivalent ligands. We have investigated the binding interaction of a recently identified potent plasma-stable dimeric pentapeptide and PDZ1-2 of postsynaptic density protein-95 (PSD-95) using protein engineering in combination with fluorescence polarization, isothermal titration calorimetry, and stopped-flow fluorimetry. We demonstrate that binding occurs via a two-step process, where an initial binding to either one of the two PDZ domains is followed by an intramolecular step, which produces the bidentate complex. We have determined all rate constants involved in the binding reaction and found evidence for a conformational transition of the complex. Our data demonstrate the importance of a slow dissociation for a successful dimeric ligand but also highlight the possibility of optimizing the intramolecular association rate. The results may therefore aid the design of dimeric inhibitors in general.
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: Marie
authors.initials: M
authors.lastname: Gottschalk
authors.forename: Anders S
authors.initials: AS
authors.lastname: Kristensen
authors.forename: Kristian
authors.initials: K
authors.lastname: Strømgaard
authors.forename: Per
authors.initials: P
authors.lastname: Jemth
mesh_terms: D000595:Amino Acid Sequence; D019281:Dimerization; D007700:Kinetics; D008024:Ligands; D008565:Membrane Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D009842:Oligopeptides / Q000097:blood / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D054731:PDZ Domains; D011485:Protein Binding; D055550:Protein Stability
chemical_list: D008024:Ligands; D008565:Membrane Proteins; D009842:Oligopeptides