Substrate Recognition of Guanidinium Exporters (Gdx) from the Small Multidrug Resistance (SMR) Family

Abstract

The small multidrug resistance (SMR) proteins are a bacterial family known to confer both antimicrobial and antiseptic resistance against a wide array of unnatural drugs. A small protein cluster containing drug exporters exist in this family and are highly promiscuous in exporting polyaromatic, cationic substrates. Interestingly, the majority of the protein population within the SMRs are guanidinium exporters (Gdx), which are structurally and sequentially similar to the drug exporters. Despite the similarities, both drug exporters and Gdx differ in their functionality. Although Gdx can export its native substrate, guanidinium (Gdm+), it is also selective for mono-substituted Gdm+ analogues. Drug exporters, on the other hand, cannot transport Gdm+, but are highly promiscuous in exporting polyaromatic, cationic, unnatural substrates, including disubstituted Gdm+ analogues. At the lens of substrate specificity, it was very natural to observe an evolutionary pathway from Gdx to drug exporters, but how this event occurred remains a mystery. Here we explore how Gdx proteins evolved a more specialized function to becoming drug exporters. To elucidate this evolutionary process, we will provide a step-wise bridge by mutating key residues on a representative Eco Gdx protein from Escherichia coli,thereby potentially yielding mutants that behave more like EmrE, the most well-studied drug exporter. We will rely on both rational design and directed evolution to generate these mutants, which will then be tested for their transport functionality of cationic, polyaromatic substrates using solid surface membrane (SSM) electrophysiology.