Measurement of multiple torsional angles from one-dimensional solid-state NMR spectra: application to the conformational analysis of a ligand in its biological receptor site

Abstract

Knowledge of the three-dimensional structure of a ligand in the binding site of its biological receptor is a valuable asset that can assist disease research and guide drug discovery. Solid-state nuclear magnetic resonance (SSNMR) is a useful high-resolution technique for the structural analysis of small molecule or peptide ligands when bound to receptors. SSNMR-derived constraints on the molecular conformations of isotopically (e.g., ¹³C and ¹⁵N) enriched ligands usually take the form of through-space distances between atomic nuclei that are separated by three or more bonds. It is advantageous to supplement such distance measurements with independent geometric constraints to resolve structural ambiguities arising from molecular symmetry. Here it is demonstrated that multiple torsional angle constraints can be measured directly for a uniformly labelled biological ligand at a realistically low concentration (150 nmoles) in a practicable experiment time. A simple adaptation of a standard one-dimensional ¹³C double-quantum filtered SSNMR experiment is used to measure the relative orientations of C–H bonds in CH₂–CH and CH₂–CH₂ groups, which influence ¹³C double quantum signal amplitudes in a predictable way. The methodology is applied to uniformly ¹³C and ¹⁵N labelled glutamate ([U-¹³C,¹⁵N]Glu) bound to the ligand binding domain of the ionotropic glutamate receptor 2 (GluR2) in a microcrystalline preparation. Two torsional angle constraints are sufficient to eliminate the structural ambiguities associated with ¹³C–¹⁵N interatomic distance measurements, and thus provide a reliable representation of the conformation of glutamate in its receptor-bound state.