18.03.2021 - Understanding drug efflux – Small Multidrug Resistance Family - Stoichiometry and Specificity
The transporters of the SMR family catalyze multidrug and toxin efflux in bacteria, energetically driven by proton influx. Not only are these proteins interesting in the context of novel antimicrobial strategies but due to their small size, they are valuable tools for a better understanding of transport mechanisms and substrate specificity.
In the first session of the transporter webinar series, we have the opportunity to learn from two experts in the field, Randy Stockbridge and Katherine Henzler-Wildman, about their approaches and insights working with this transporter family.
Title: Determining transport stoichiometry using SSME
Randy Stockbridge, Assistant Professor, Department of Molecular, Cellular, and Developmental Biology
(University of Michigan)
Transporters from the small multidrug resistance (SMR) family provide broad resistance to environmental biocides, driving the spread of multidrug resistance cassettes among bacterial populations. Understanding substrate specificity is essential to understand this process. Using solid-supported membrane electrophysiology, we measure the transport of different substrates by SMR family members, and show that promiscuous transport of hydrophobic substituted cations is a general feature of all SMR transporters, including those whose primary physiological role is in bacterial nitrogen metabolism.
Title: Basis of promiscuity in small multidrug resistance transporters
Katherine Henzler-Wildman, Professor Biochemistry, Department of Biochemistry
(University of Wisconsin-Madison)
Transport stoichiometry can provide great insight into the mechanism and function of ion-coupled transporters. Traditional reversal potential assays are a reliable, general method for determining the transport stoichiometry of ion-coupled transporters, but the time and material costs of this technique hinder investigations of transporter behavior under multiple experimental conditions. Our prior work on EmrE has demonstrated that it is not a tightly coupled transporter and that the net transport stoichiometry is likely to vary with pH and substrate identity. This has motivated us to develop an SSME-based assay for assessing transport stoichiometry that is rapid and easily adaptable to different substrates and pH conditions. Here we present results for Gdx and CLC-Ec1, two well-characterized transporters that demonstrate the success of our approach. Our SSME-based method reproduces the fixed 2H+:1 guanidinium+ antiport stoichiometry of Gdx, the 1H+:2Cl- antiport stoichiometry of CLC-ec1, and loose proton:nitrate coupling for CLC-ec1. This method requires only small amounts of transporter and provides a fast, easy method to characterize transport stoichiometry under varied conditions, which will facilitate future mechanistic and functional studies of ion-coupled transporters.
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