Structural and functional study of multidrug transporters

Long, Feng
Major Professor
Edward W. Yu
Committee Member
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Physics and Astronomy
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Physics and Astronomy

Active efflux of antimicrobial agents is one of the most important strategies that bacteria use to ensure their survival in a toxic extracellular environment. Our interest is to elucidate the structural and functional relationships of bacterial multidrug efflux transporters that give rise to antibiotic resistance. In this dissertation, I focus on studying two specific groups, the multidrug and toxic compound extrusion (MATE) family and the resistance-nodulation-division (RND) family, of multidrug efflux transporters. The functions of the two MATE efflux pumps, NorM of Neisseria gonorrhoeae and YdhE of Escherichia coli, are characterized using a variety of biochemical assays, including drug susceptibility and transport assays. Direct measurements of efflux allow us to confirm that NorM behaves as a Na+-dependent transporter. The capacity of NorM and YdhE to recognize structurally divergent compounds is also examined using steady-state fluorescence polarization assays. The results suggest that NorM and YdhE bind these antimicrobials with dissociation constants (KDs) in the micromolar range.

In addition, I present the crystal structures of the CusA transporter, the only heavy-metal efflux (HME) pump of the RND family in E. coli, in the absence and presence of Cu(I) and Ag(I) ions. These are the first structures of any efflux pumps belonging to the HME-RND family. Surprisingly, the binding of Cu(I) and Ag(I) triggers significant conformational changes in both the periplasmic and transmembrane domains of the pump. Based on the crystal structures and biochemical studies, I put forward my hypothesis that CusA is capable of actively picking up metal ions from the cytoplasm as well as the periplasmic space, utilizing the methionine pairs/clusters in the pump to bind and export metal ions. A stepwise shuttle mechanism is probably employed by the pump to extrude these heavy metals.