Evidence that the C-terminal region is involved in the stability and functionality of OprM in E. coli

Abstract

In order to understand the specificity of interactions between the components of multidrug-resistant (MDR) efflux pumps and how they are recruited/assembled, we analyzed the effect of C-terminal truncation, deletion, and peptide swapping on the stability and functionality of OprM in Escherichia coli. The efflux activity of OprM was not affected by removing up to 19 amino acid residues from the C-terminus, while depletion of more than 20 residues or disruption the ₄₆₃LGGG₄₆₆ motif diminished both the stability and activity of OprM. The replacement of the OprM C-terminus 23 residues with the corresponding part of TolC or VceC did not affect the stability and the functionality of OprM. Therefore, it is confirmed that the C-terminal ₄₆₃LGGG₄₆₆ motif is one of the crucial components for the stability of OprM and for the functionality of the OprM-VceAB chimeric pump in E.coli. The results also indicate that one residue substitution on the hairpin domain of the membrane fusion protein (MFP) VceA could suppress the null like mutations on the C-terminal modified OprM. This finding will be the direct genetic evidence that the C-terminal domain of outer efflux protein (OEP) is involved in the functional assembly of OEP-MFP.

Keywords: C-terminal domain; Gain-of-function mutations; Multidrug-resistant efflux pumps; OprM.