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Peripheral mutations underlie promiscuous transport of quaternary ammonium antiseptics by Small Multidrug Resistance transporters

Burata, O. E.; O'Donnell, E.; Hyun, J.; Lucero, R. M.; Thomas, J. E.; Gibbs, E. M.; Reacher, I.; Carney, N. A.; Stockbridge, R. B.

2024-02-07 biochemistry
10.1101/2024.02.06.579181 bioRxiv
Show abstract

The mechanistic basis of transport promiscuity in multidrug exporters is not well understood. We examine this question using the Small Multidrug Resistance (SMR) transporters. We engineer a selective SMR protein to promiscuously export quaternary ammonium antiseptics, similar to multidrug exporters in this family. Using combinatorial mutagenesis and deep sequencing, we identify the necessary and sufficient molecular determinants of this new activity. Using x-ray crystallography, electrophysiology, and a novel proteoliposome-based antiseptic transport assay, we tease apart the mechanistic roles that these residues play in transport polyspecificity. We find that substrate preference changes not through modification of the residues that directly interact with the substrate, but through mutations peripheral to the binding pocket. Our new molecular insights into substrate promiscuity among the SMRs can be applied to understand multidrug export and the evolution of novel transport functions more generally.

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