Overlay-independent comparisons of X-ray structures reveal small, systematic conformational changes in liganded acetylcholinesterase

Background and Purpose: Comparisons of macromolecular 3D structures by minimizing corresponding atom RMSDs can by virtue of averaging, hide small unexpected deviations caused by ligand binding or site-directed mutagenesis. We developed an overlay-independent comparison approach and used it to compare selected, PDB structures of acetylcholinesterase (AChE; EC 3.1.1.7) in the apo state, in complexes with reversible ligands, in covalent conjugates or upon AChE mutagenesis. Methods: The active serine alpha carbon of the AChE monomer was used as a frame of reference for each of the structures and distances calculated to remaining alpha carbons of the monomer backbone. Differences in distances at corresponding backbone positions from pairwise comparisons with apo-AChE structures were taken as a measure of changes in tertiary structures of liganded or mutant AChE monomers. Results: This analysis revealed small, yet significant backbone shifts in monomeric tertiary structures of AChEs. While reversible complexes with neurotoxin Fas2 showed global expansion of AChE monomer backbones, covalent conjugates at the active serine and high affinity reversible inhibitor hupA complexes revealed backbone compression towards the active serine. In some of AChE homodimers 0.6 - 1 angstrom shifts of monomer backbone affected crystallographic quaternary structures by causing significant, up to seven degree tilts in monomers, relative to angles measured in the apo-AChE. Conclusions: Small but systematic differences detected in tertiary structures of liganded or mutagenized AChEs appear consistent with larger magnitude alterations of several AChE homodimer quaternary structures. Those observations vaildate our overlay-independent approach for detecting small, yet significant, tertiary backbone shifts in macromolecular 3D structures.