Probing the Conformational Ensemble of a Bacterial Antitoxin through Molecular Dynamics Simulations and Mass Spectrometry

Abstract

Intrinsic disorder plays a key role in the regulation of cell death by bacterial toxin-antitoxin (TA) modules. In TA modules, an unstable antitoxin normally inhibits a protein toxin. Cellular stressors trigger increased degradation of the labile antitoxin, thereby releasing the toxin. When not inhibited, the toxin disrupts essential cellular processes, causing cell death or quiescence. The CcdA-CcdB TA module in E. coli consists of the antitoxin CcdA and the toxin CcdB. CcdA is comprised of a folded DNA-binding domain and two intrinsically disordered regions (IDRs), which regulate binding to CcdB. NMR studies suggest that CcdA, in the absence of CcdB, predominantly samples conformations belonging to either a closed or an open state, distinguished by the distance from the IDR termini to the folded domain. The potential for CcdA to sample multiple partially stable states provokes the question of the role these states play in facilitating the IDRs’ functions. We use all-atom explicit-water molecular dynamics (MD) simulations and native ion mobility-mass spectrometry (MS) to determine the conformational ensemble of unbound CcdA, with the goal of inferring functional roles from structural details. Both MD and MS indicate that CcdA samples metastable states of varying compactness. In one state CcdA can adopt compact, relatively closed conformations, as predicted by NMR. In a separate state, that has similar energy, one IDR extends away from the central folded domain. Further analysis of IDR helicity and solvent exposure within each substate provides insight into the functional role of these states. As intrinsically disordered antitoxin proteins like CcdA are plentiful in bacteria, understanding how disorder facilitates their functions could lead to novel antibiotics that harness TA modules to kill bacterial cells.