Microsecond Timescale Conformational Dynamics of a Small-Molecule Ligand within the Active Site of a Protein

The possible internal dynamics of non-isotope-labeled small-molecule ligands inside a target protein is inherently difficult to capture. Whereas high crystallographic temperature factors can denote either static disorder or motion, even moieties with very low B-factors can be subject to vivid motion between symmetry-related sites. Here we report the experimental identification of internal mu s timescale dynamics of a high-affinity, natural-abundance ligand tightly bound to the enzyme human carbonic anhydrase II (hCAII) even within a crystalline lattice. The rotamer jumps of the ligand's benzene group manifest themselves both, in solution and fast magic-angle spinning solid-state NMR 1H R1 rho relaxation dispersion, for which we obtain further mechanistic insights from molecular-dynamics (MD) simulations. The experimental confirmation of rotameric jumps in bound ligands within proteins in solution or the crystalline state may improve understanding of host-guest interactions in biology and supra-molecular chemistry and may facilitate medicinal chemistry for future drug campaigns. Symmetric motions of bound ligands contribute to the entropic changes upon binding but are difficult to detect via conventional methods. Here, Kotschy, Soldner et al. develop solid-state NMR approaches focusing on the protons of the ligand to reveal an aromatic ring flip of the benzylic group of the inhibitor.#image

Automated summary

This study reports the experimental identification of internal microsecond-scale dynamics of a high-affinity ligand tightly bound to the enzyme human carbonic anhydrase II (hCAII), even within a crystalline lattice. The authors demonstrate the existence of ligand's rotamer jumps through both solution and solid-state NMR experiments, and gain further insights through molecular dynamics simulations. This experimental confirmation of rotameric jumps in bound ligands within proteins in solution or the crystalline state is significant for understanding host-guest interactions in biology and supra-molecular chemistry, and for facilitating medicinal chemistry in drug research.