Kapoor, Parth
(2020)
Elucidating the molecular mechanism of drug binding and translocation by the multidrug transporter ABCG2.
PhD thesis, University of Nottingham.
Abstract
Many drug resistant tumours express one or more of the three main drug efflux transporters, P-glycoprotein (ABCB1), multidrug resistance protein 1 (MRP1; ABCC1) and breast cancer resistance protein (ABCG2). These transporters facilitate the export of diverse range of structurally unrelated compounds from the cell. ABCG2, is widely distributed in tissues, contributes towards normal physiology and pharmacokinetic profiles of many exogenous drugs. It is this promiscuous drug binding and transport that makes it an appealing target for research. The aim of this project is to better understand the molecular mechanism of multidrug binding and transport by ABCG2. Here we focus on two specific regions of the protein: transmembrane helix 3 (TM3) and the NBD: NBD (nucleotide binding domain) interface to elucidate their role in the function of ABCG2 transporter.
Earlier research associated residues R482 and P485 located within TM3 helix, with drug binding and/or transport. HEK293T stable cell lines for wild type protein and point mutants along the length of TM3 (residues D477- L497) were generated and characterized for cell surface expression using confocal microscopy and flow cytometry. The transport function of all mutants was investigated using flow cytometry to determine the efflux of three fluorescent substrates (mitoxantrone, pheophorbide A and daunorubicin). This revealed at least 16 isoforms of ABCG2 protein associated with altered transport for one of the three fluorescent substrates. The majority of the mutants showed reduced efflux for the fluorescent substrates mitoxantrone and pheophorbide A. D477A showed impaired efflux for pheophorbide A and M496A showed increased efflux for mitoxantrone. However, R482A showed significant gain in function for daunorubicin efflux, in accordance with the published literature, suggesting substrate specificity. Only three mutants altered both Mx and pheophorbide A transport, suggesting overlapping sites for substrate interaction. T490A showed altered phenotypic effects for mitoxantrone and pheophorbide A transport, indicating the importance of drug specific interactions. The recognition and transport mechanism is distinct for daunorubicin compared to mitoxantrone and pheophorbide A. On the basis of these results we hypothesise TM3 helix as a putative gatekeeper for hydrophobic substrates. We propose a tentative drug recognition/transport mechanism at a groove site, involving potential interactions with
TM3 residues by means of molecular docking, thereby reinstating the importance of TM3 in substrate recognition and translocation.
The structural revolution for ABCG2 and its related family members (ABCG5/G8) enabled us to focus on the NBDs close contact interface. The structures revealed two interfaces between the NBDs, one being the canonical ATP sandwich dimer interface and the other, a unique constant contact interface at the NPXDF motif and in its close proximity. We explore the importance of this unique NBD interface motif and some surrounding residues of ABCG2 in forming a closed conformation at the cytoplasmic interface between the NBD: NBD interface. Point mutations at this interface showed altered drug specificity (E285K), altered drug transport (E285K, D292A and D292K) and loss of ATPase activity (E285K, D292A and D292K). The study demonstrates the importance of this interface with impact on local NBD events (ATP hydrolysis) and allosteric communication to the transmembrane domains and drug transport.
This research forms the basis to further investigate substrate and inhibitor binding of ABGC2 using complementary techniques such as fluorescence correlation spectroscopy, microscale thermophoresis and carbene footprinting - mass spectrometry in order better understand its mechanistic details and potentially lead to therapeutic approaches towards drug resistant cancers.
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