Author ORCID Identifier

https://orcid.org/0000-0003-0190-5891

Date of Award

12-10-2018

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Dr. Parjit Kaur

Second Advisor

Dr. Zehava Eichenbaum

Third Advisor

Dr. Eric Gilbert

Abstract

Multidrug resistance (MDR) is a serious problem for treatment of cancers and infectious diseases. A leading cause of MDR is energy-dependent drug efflux by membrane transport proteins. This work focuses on an ABC family drug transporter DrrAB found in soil bacterium Streptomyces peucetius. This organism produces two widely used chemotherapeutic drugs, doxorubicin and daunorubicin. Self-resistance in S. peucetius is conferred by DrrA and DrrB, which together form a dedicated efflux pump for export of these drugs. DrrA hydrolyzes ATP, and the energy is transduced via conformational changes to DrrB for drug export. Interestingly, our laboratory recently showed that, in spite of its dedicated nature, DrrAB system can also export other known MDR substrates. To understand the molecular basis of MDR in DrrAB and determine its mechanism of function, it is critical to understand the range of substrates recognized by DrrB and the energy transduction pathway in DrrAB. In this work, we used fluorescence-based approaches to investigate substrate binding properties of DrrAB and to analyze inter-subunit conformational changes between DrrA and DrrB. We show that DrrB binds drugs with variable affinities and contains multiple drug binding sites. Nucleotide binding analysis provided evidence of two asymmetric nucleotide binding sites in DrrA with strikingly different affinities. This study also provided clear evidence of long-range conformational changes occurring between DrrA and DrrB and led to identification of conserved regions involved in the transduction pathway, including Q-loop in the nucleotide binding domain (NBD) of DrrA, CREEM motif in the C-terminal domain (CTD) of DrrA, and EAA-like motif in DrrB. This work also describes identification of a novel conserved domain ‘GATE’ in the CTD of DrrA. Critical mutations in GATE diminished ATP catalysis and overall transport function of DrrAB. Therefore, although located outside of the NBD, GATE may also be involved in ATP catalysis and bidirectional communication between DrrA and DrrB. These studies lay solid groundwork for examining roles of various conserved regions of DrrAB in transduction of conformational changes. Finally, the last part of this dissertation focuses on metagenomic analysis of a soil DNA library with the goal to identify novel ABC transporters.

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