Poster Presentation BacPath 13: Molecular Analysis of Bacterial Pathogens Conference 2015

Conserved intramembranous acidic residues function in transport but not substrate binding in PACE family proteins (#115)

Karl Hassan 1 , Scott Jackson 2 , Qi Liu 1 , Liam Elbourne 1 , Alaska Pokhrel 1 , Emily Gibson 1 , Peter Henderson 2 , Ian Paulsen 1
  1. Macquarie University, North Ryde, NSW, Australia
  2. University of Leeds, Leeds, United Kingdom

The Proteobacterial Antimicrobial Compound Efflux (PACE) family of multidrug transport proteins was only recently described. Consequently the molecular details of the transport mechanism(s) operating in this group of proteins remain to determined. Membrane-embedded acidic residues participate in substrate binding and/or coupling reactions in several classes of drug efflux pumps. PACE family proteins typically have two glutamic acid residues that are predicted to be in transmembrane regions. We examined the functional importance of these highly-conserved glutamic acid residues in the prototypical PACE members AceI from Acinetobacter baumannii and VP1155 from Vibrio parahaemolyticus using a series of mutant proteins. Mutants in which these residues were neutralised were either unable to confer resistance to antimicrobials or had compromised resistance function. Corresponding to this loss of resistance function, these mutants displayed reduced or abolished efflux activity, assayed using either fluorimetric or radioactive transport experiments. In contrast, the mutant proteins maintained a capacity to interact with substrates. This phenotype suggests that the transport defect in these mutants may be related to a coupling reaction rather than direct substrate recognition.