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Despite their biomedical importance, the mechanisms underlying substrate transport by multidrug efflux transporters remain poorly understood. Here, we describe the first characterization of a multidrug transporter belonging to the major facilitator superfamily from the genus\n                    <jats:italic>Salmonella<\/jats:italic>\n                    . We show that several clinical\n                    <jats:italic>Salmonella<\/jats:italic>\n                    Typhi (\n                    <jats:italic>S<\/jats:italic>\n                    . Typhi) isolates constitutively express the\n                    <jats:italic>styMdtM<\/jats:italic>\n                    (\n                    <jats:italic>STY4874<\/jats:italic>\n                    ) gene, which encodes a known multidrug\u2010resistance (MDR) transporter. Guided by the structure of the\n                    <jats:italic>Escherichia coli<\/jats:italic>\n                    (\n                    <jats:italic>E. coli<\/jats:italic>\n                    ) homolog, we studied two residues critical for substrate transport, Asp25 and Arg111. Mutation of Asp25 to glutamate did not affect the transport function of styMdtM, whereas mutation to alanine reduced its transport activity, suggesting that a negative charge at this position is critical for substrate translocation across the membrane. Substrate\u2010affinity measurements by intrinsic fluorescence spectroscopy showed that the Asp25Ala mutant retained its capacity to bind substrate, albeit at a lower level. Mutation of Arg111 to alanine resulted in a decrease in secondary structure content of the transporter, and mutation to lysine completely destabilized the structure of the transporter. A homology model of styMdtM suggests that Arg111 is important for stabilizing the transmembrane domain by mediating necessary interactions between neighboring helices. Together, our studies provide new structural and mechanistic insights into the\n                    <jats:italic>Salmonella<\/jats:italic>\n                    MDR transporter styMdtM.\n                  <\/jats:p>","DOI":"10.1002\/prot.26141","type":"journal-article","created":{"date-parts":[[2021,5,13]],"date-time":"2021-05-13T10:06:54Z","timestamp":1620900414000},"page":"1193-1204","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Characterization of the multidrug efflux transporter\n                    <scp>styMdtM<\/scp>\n                    from\n                    <scp>\n                      <i>Salmonella enterica<\/i>\n                    <\/scp>\n                    serovar Typhi"],"prefix":"10.1002","volume":"89","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7887-677X","authenticated-orcid":false,"given":"Aqsa","family":"Shaheen","sequence":"first","affiliation":[{"name":"Drug Discovery and 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