Date of Award

Spring 5-10-2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Dr. Parjit Kaur

Second Advisor

Dr. Phang C. Tai

Third Advisor

Dr. John Houghton

Abstract

Multi-drug resistance (MDR) has become a serious clinical problem for both cancer and infectious disease treatment. One of the leading causes of MDR is the drug efflux mediated by the ATP-binding cassette (ABC) super-family of proteins which are found among diverse groups of organisms. In our laboratory, we are interested in studying an ABC transporter DrrAB from Streptomyces peucetius. It confers resistance to two anticancer antibiotics doxorubicin and daunorubicin by its drug efflux function. Insights into the function of the DrrAB complex are expected to facilitate a better understanding of mechanisms of MDR.

The normal function of the DrrAB complex depends on the cis expression of both subunits, DrrA and DrrB. DrrA forms the nucleotide binding domain (NBD) and provides energy for drug translocation, while the DrrB protein functions as the transmembrane domain (TMD) and forms the substrate translocation pathway. Studies on the detailed mechanisms of communication between DrrA and DrrB are critical for understanding the coupling of energy usage and substrate translocation. The present studies revealed the existence of two novel and functionally important modules in the C-terminal domain of DrrA that might be essential for conformational interplay between DrrA and DrrB during the catalytic cycle. One module present at the extreme C terminus of DrrA consists of two separate motifs, DEF and CREEM. CREEM motif together with its upstream region up to residue S319, interacts with the N-terminal cytoplasmic tail region of DrrB and forms an DrrA-DrrB interface, while the DEF motif regulates this interaction. The second novel module GATE is present 104 amino acids upstream of DEF. Based on our biochemical and structural analyses, we propose that GATE functions as a transducer of conformational changes (resulting from ATP binding) from DrrA to DrrB. Our studies provide important insights into the interplay between the NBD and TMD of the DrrAB drug transporter and their roles in communicating long range conformational changes during the catalytic cycle. These findings further deepen our understanding of the mechanisms of drug resistance.

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