Carbene Footprinting and Mass Spectrometry Capture Global Conformational Changes in Elongation Factor-Tu Induced by Antibiotic Binding

Abstract

Carbene footprinting methodology, in combination with mass spectrometry, was employed to study the interactions of bacterial elongation factor-thermo-unstable (EF-Tu) with two antibiotics. EF-Tu, a GTPase, delivers aa-tRNA to the ribosome during protein synthesis where it undergoes a significant conformational switch. Due to structural differences from its eukaryotic homologue, EF-Tu represents a valuable antimicrobial target in pathogenic bacteria such as Acinetobacter baumannii. Binding of GE2270A, a thiazolyl peptide, to EF-Tu resulted in reduced carbene probe labeling (masking) at the protein’s aa-tRNA binding site on domain II, as predicted by the mode of action of this inhibitor. In contrast, binding of the polyketide enacyloxin IIa (ENX), which has a similar mechanism of action to kirromycin, caused global masking of EF-Tu, consistent with stabilization of the protein’s closed GTP-bound conformation. Moreover, these effects were found to correlate with the affinity of EF-Tu wild-type and resistant mutants for ENX. The findings presented here show that carbene footprinting can reveal global protein conformational changes associated with ligand binding as well as mapping direct binding sites. Thus, the methodology can be used to discriminate between inhibitory mechanisms of the EF-Tu target.