NMR distinction of single- and multiple-mode binding of small-molecule protein ligands.
Identifying and characterizing small-molecule inhibitors of protein-protein interactions is of high interest for drug discovery and for chemical genetics studies of biological pathways. Very often, initial hits or first-generation compounds have low micromolar dissociation constants and cause line broadening in NMR spectra. It is very important for subsequent structure-based compound optimization to know if this line broadening is caused by intermediate exchange of the dissociation kinetics only or in addition by multiple binding modes. Here, we present an approach of how to distinguish these two situations and demonstrate its experimental application. Two very similar small-molecule ligands of Bcl-xL are considered that cause both severe line broadening of interface residues. We show that one compound exhibits single-mode binding, and broadening is just due to dissociation kinetics in the intermediate exchange regime, and the line broadening can be overcome by providing excess ligand. In the other case, line broadening is due to dissociation kinetics and exchange between multiple bound conformations, and broadening cannot be overcome by providing excess ligand. The procedures used are very general and can also be applied to characterizing protein-protein and protein-nucleic acid interactions.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Mikhail
M
Reibarkh
Thomas J
TJ
Malia
Gerhard
G
Wagner
D001665:Binding Sites; D008024:Ligands; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D009842:Oligopeptides; D011485:Protein Binding; D051020:bcl-X Protein / Q000737:chemistry* / Q000378:metabolism
D008024:Ligands; D009842:Oligopeptides; D051020:bcl-X Protein; C048812:kinetensin