Identification of additional mechanistically important residues in the multidrug transporter styMdtM of Salmonella Typhi

Multidrug efflux is a well-established mechanism of drug resistance in bacterial pathogens like Salmonella Typhi. styMdtM (locus name; STY4874) is a multidrug efflux transporter of the major facilitator superfamily expressed in S. Typhi. Functional assays identified several residues important for its transport activity. Here, we used an AlphaFold model to identify additional residues for analysis by mutagenesis. Mutation of peripheral residue Cys185 had no effect on the structure or function of the transporter. However, substitution of channel-lining residues Tyr29 and Tyr231 completely abolished transport function. Finally, mutation of Gln294, which faces peripheral helices of the transporter, resulted in the loss of transport of some substrates. Crystallization studies yielded diffraction data for the wild-type protein at 4.5 angstrom resolution and allowed the unit cell parameters to be established as a = b = 64.3 angstrom, c = 245.4 angstrom, alpha = beta = gamma = 90(degrees), in space group P4. Our studies represent a further stepping stone towards a mechanistic understanding of the clinically important multidrug transporter styMdtM.

Automated summary

This study used the AlphaFold model to identify residues of the multidrug efflux transporter styMdtM, and confirmed their importance through mutagenesis experiments. The findings revealed that mutation of specific residues can result in loss of transport function, while mutation of peripheral residues has no effect on structure or function. Additionally, crystallization studies provided valuable data on the protein's structure.