TY - JOUR
T1 - Deciphering the kinetic binding mechanism of dimeric ligands using a potent plasma-stable dimeric inhibitor of postsynaptic density protein-95 as an example
AU - Chi, Celestine N
AU - Bach, Anders
AU - Gottschalk, Marie
AU - Kristensen, Anders Skov
AU - Strømgaard, Kristian
AU - Jemth, Per
PY - 2010/9/3
Y1 - 2010/9/3
N2 - 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.
AB - 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.
KW - Former Faculty of Pharmaceutical Sciences
U2 - 10.1074/jbc.M110.124040
DO - 10.1074/jbc.M110.124040
M3 - Journal article
C2 - 20576616
SN - 0021-9258
VL - 285
SP - 28252
EP - 28260
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 36
ER -