The structural basis of promiscuity in small multidrug resistance transporters

Ali A Kermani,Randy B Stockbridge,Christian B Macdonald,Akiko Koide,Eric Denbaum,B Ben Koff,Shohei Koide,Olive E Burata

doi:10.1038/s41467-020-19820-8

Abstract

By providing broad resistance to environmental biocides, transporters from the small multidrug resistance (SMR) family drive the spread of multidrug resistance cassettes among bacterial populations. A fundamental understanding of substrate selectivity by SMR transporters is needed to identify the types of selective pressures that contribute to this process. Using solid-supported membrane electrophysiology, we find that promiscuous transport of hydrophobic substituted cations is a general feature of SMR transporters. To understand the molecular basis for promiscuity, we solved X-ray crystal structures of a SMR transporter Gdx-Clo in complex with substrates to a maximum resolution of 2.3 Å. These structures confirm the family’s extremely rare dual topology architecture and reveal a cleft between two helices that provides accommodation in the membrane for the hydrophobic substituents of transported drug-like cations.

Highlights

By providing broad resistance to environmental biocides, transporters from the small multidrug resistance (SMR) family drive the spread of multidrug resistance cassettes among bacterial populations
Radioactive uptake assays confirm that EmrE transports methyl viologen, but not Gdm+; that Gdx-Clo transports Gdm+, but not methyl viologen; and that both proteins discriminate against a substituted guanidinyl metabolite, agmatine (Fig. 2a)
Environmental contamination by hydrophobic quaternary amines is associated with antiseptic use[9], and substituted guanidinium ions and biguanides have been identified as widespread, long-lived, environmentally disruptive contaminants that enter the biosphere as agricultural or industrial chemicals[40,41,42] or pharmaceuticals that impact the human microbiome and that are excreted in waste water[43,44,45,46]

Summary

Introduction

By providing broad resistance to environmental biocides, transporters from the small multidrug resistance (SMR) family drive the spread of multidrug resistance cassettes among bacterial populations. To understand the molecular basis for promiscuity, we solved X-ray crystal structures of a SMR transporter GdxClo in complex with substrates to a maximum resolution of 2.3 Å These structures confirm the family’s extremely rare dual topology architecture and reveal a cleft between two helices that provides accommodation in the membrane for the hydrophobic substituents of transported drug-like cations. The dispersal of drug resistance genes among bacteria, the transport capabilities of SMR proteins, and the distribution of SMR substrates in the biosphere are intimately linked Despite their importance, functional experiments to test the chemical scope of transported compounds have been limited to a narrow range of SMR homologs and drugs, and the overall fold has been determined[5], sidechainresolution structural data have not been reported for any family member. Is the functional dichotomy between the Qac and Gdx subtypes as strict as early experiments suggested? Or do proteins in the SMR family share transport capabilities that make them broadly adaptive in human-impacted environments?

Methods

Results

Conclusion