Multipronged approach to characterize the promiscuity and mechanism of PACE transporters

About This Grant

PROJECT SUMMARY/ABSTRACT The PACE (proteobacterial antiseptic efflux) family of transporters was only discovered about 10 years ago through functional genomics studies in Acinetobacter baumannii identifying genes upregulated in response to chlorhexidine exposure. Short chain diamines such as putrescine and cadaverine have been identified as likely native substrates for these transporters, which are conserved with the core genome. Since then, several homologs have been profiled, revealing that most (but not all) PACE transporters confer resistance to chlorhexidine, and about half confer resistance to short chain diamines. However, the full substrate profile and native substrate of PACE transporters that do not transport short chain diamines are not known. Mutagenesis and in vitro transport assays have identified a glutamate residue in the first transmembrane helix as important for proton coupling, but the substrate binding site(s) has not been identified. Transport is electrogenic, but the H+/substrate stoichiometry and transport mechanism is not yet determined. The sequence indicates that the PACE transporters likely have 4 transmembrane helices, but native mass spectrometry in detergent and native PAGE in solubilized membranes suggest different degrees of oligomerization and there is no experimentally determined three dimensional structure. The PACE transporters are only slightly larger than the more well- studied SMR (small multidrug resistance) transporters, which also have 4 transmembrane helices and a functionally critical glutamate in transmembrane helix 1. Based on this similarity, we tested the ability of two PACE transporters from different clades, PA2880 and AceI, to confer resistance to known SMR substrates. We identified additional substrates and observed that PA2880 confers resistance to some substrates but enhances susceptibility to others. Our lab has previously characterized the transport mechanism of the SMR transporters, which have similar functional promiscuity, and the proposed work adapts the methods we have developed to study the PACE transporters. Here we combine resistance/susceptibility assays in bacteria to more broadly characterize the substrate profile of AceI and PA2880 (Aim 1), cross-linking to determine topology in vitro and in the bacterial membrane (Aim 2), and in vitro liposomal transport assays (Aim 2) and NMR studies (Aim 3) to, identify key residues in proton- and substrate- binding and determine the mechanism by which these transporters confer susceptibility rather than resistance to some substrates.