Date of Award
Doctor of Philosophy
Physics and Astronomy
Edward W. Yu
Multidrug binding proteins are able to recognize structurally unrelated compounds. These proteins play a crucial role as drug resistant export proteins in both eukaryotic and prokaryotic cells. Furthermore, multidrug binding proteins exist that regulate transcription of multidrug transporters and play an important role in responding to incoming toxins. To study the function of multidrug binding proteins and exporters, AcrD of Escherichia coli (E. coli), CmeR of Campylobacter jejuni, and AcrR of E. coli were chosen as models. AcrD, a multidrug efflux pump, functions to rid the bacterial cell of incoming hydrophilic substrates by harboring a diverse ligand binding cavity capable of recognizing the deleterious compounds. By using fluorescence polarization and isothermal titration calorimetry, we show that AcrD specifically interacts with aminoglycosides and anthracyclines with dissociation constants in the low micromolar range. By modeling the AcrD structure, hydrophilic patches were identified that could allow substrates to be captured from the cytoplasm or periplams and exported completely out of the bacterial cell.
The transcriptional regulators CmeR and AcrR interact with inducing ligands by utilizing a similar mechanism, whereby a diverse binding pocket exists allowing recognition of various substrates. This work shows how CmeR recognizes large, negatively charged bile salts by harboring a large hydrophobic surface with appropriately spaced polar residues to stabilize bile acid binding. Furthermore, by comparing the structures of AcrR and CmeR, a model can be developed to describe transcriptional regulation. Upon ligand interaction in the C-terminal domain, adjacent N-terminal domains separate, breaking bonds with the DNA operator, thus releasing repression. Interestingly, kinetics studies reveal that AcrR, CmeR, and AcrD interact with ligands in the low micromolar range, which may be a critical feature of mutlidrug binding proteins.
Mathew David Routh
Routh, Mathew David, "Structure, function, and regulation of multidrug export proteins among the RND superfamily in Gram-negative bacteria" (2010). Graduate Theses and Dissertations. 11401.