Transport proteins from the small multidrug resistance (SMR) family play a crucial role in protecting bacteria from a wide range of quaternary ammonium antiseptics (QACs), which are commonly used in antibacterial hand soaps and cleaning solutions. However, the exact molecular mechanisms behind their ability to transport such a diverse array of substances have been unclear.

A recent study by Randy Stockbridge’s group successfully delineated the molecular basis for substrate promiscuity in SMR transporters.

The study found that substrate specificity in SMR transporters is influenced by mutations at peripheral positions rather than direct changes in the substrate-binding pocket.

The authors set out to identify a minimal set of mutations that converts a selective guanidinium exporter, Gdx-Clo, into a promiscuous transporter of quaternary ammoniums (engineered variant, Gdx-Clo-7x). Through combinatorial mutagenesis (as opposed to single-site mutational scans), they were able to identify mutations that work together synergistically to confer such changes in transport activity. Interestingly, these mutations were peripheral to the substrate-binding site.

Structural analysis revealed how these peripheral mutations influence the overall protein conformation, whereas solid-supported membrane electrophysiology (SURFE2R) and binding assays confirmed that the engineered transporter Gdx-Clo-7x exhibited a complete switch in substrate preference, binding QACs with high affinity while losing affinity for its original substrate, guanidinium (Gdm+).

These findings reveal that the key factors determining substrate specificity are not confined to the binding site itself but include distant regions of the protein. Thus, by targeting peripheral regions, scientists can potentially design transport proteins with tailored specificities for various applications in biotechnology and medicine.

Overall, this study underscores the importance of peripheral mutations in evolving new transport functions, offering a broader understanding of multidrug resistance mechanisms and enabling the development of new strategies to combat bacterial drug resistance.

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Find the full article here: https://www.pnas.org/doi/10.1073/pnas.2403273121

Learn more about SSM-Based Electrophysiology and SURFE²R devices here: https://www.nanion.de/products/surfe2r-n1/