Small-molecule binding to an intrinsically disordered protein revealed by experimental NMR 19F transverse spin-relaxation

Intrinsically disordered proteins are highly dynamic biomolecules that rapidly interconvert between many structural conformations. Traditionally, these proteins have been considered un-druggable because of their lack of classical long-lived binding pockets. Recent evidence suggests that intrinsically disordered proteins can bind small, drug-like molecules, however, there are limited approaches to characterize these interactions experimentally. Here we demonstrate that ligand-detected 19F transverse relaxation rates (R2) obtained from Nuclear Magnetic Resonance spectroscopy are highly sensitive to the interaction between a small-molecule and an intrinsically disordered protein, in contrast to chemical shift perturbations which are minimally sensitive for this interaction. With this method, we show that the small molecule, 5-fluoroindole, interacts with the disordered domains of non-structural protein 5A from hepatitis C virus with a Kd of 260 {+/-} 110 M. We also demonstrate that 5-fluoroindole remains highly dynamic in the bound form. Our findings suggest that ligand-detected 19F transverse relaxation measurements could represent a highly effective screening strategy to identify molecules capable of interacting with these traditionally elusive, dynamic biomolecules.