Authors:	Rose-Sperling, Dania
Title:	Structure and dynamics of multidrug resistance ABC transporters : elucidating the driving force behind ABC transporters
Online publication date:	15-Feb-2023
Year of first publication:	2023
Language:	english
Abstract:	The ATP binding cassette (ABC) transporter family is a large membrane protein superfamily found in all phyla of life. ABC transporters are essential for the transport of nutrients, ions, peptides, lipids, and also antibiotics and other harmful compounds across the lipid bilayer. Some of these membrane transporters, such as LmrA from Lactococcus lactis, MsbA from Escherichia coli and BmrA from Bacillus subtilis are homologs of the human multidrug resistance (MDR)-related P glycoprotein. The general architecture of ABC transporters consists of two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs). ATP binding and hydrolysis within the NBDs lead to conformational changes in the TMDs that allow translocation of the substrate. ABC transporters have been studied for more than five decades, and a wealth of structural information is now available. However, it remains unclear how exactly these molecular machines mediate substrate transport across a membrane, how the substrates are recognized in molecular detail and how the TMD and NBD are allosterically coupled to enable progression through the catalytic cycle and the efficient export of substrates. This PhD thesis focuses on the role of the highly conserved ABC transporter NBD and its structure and dynamics during the substrate transport mechanism. The first part of the thesis deals with the question how nucleotide interaction affects NBD dynamics across timescales, and how this may affect transmission of signals (i.e. nucleotide occupancy within the NBD), from the nucleotide binding side (NBS) to the TMD or across the NBD-NBD interface. The isolated NBDs of the exporters LmrA, MsbA and BmrA could be expressed in E. coli in large quantities and showed to be properly folded and able to bind nucleotides via nuclear magnetic resonance (NMR) spectroscopy. Analysis of NMR spectra of LmrA-NBD, MsbA-NBD and BmrA-NBD WT showed that some residues in the apo-state have backbone fluctuations in the µs – ms timescale, which became rigidified in the ADP-bound state. Chemical shift perturbation (CSP), H/D exchange and hetNOE (heteronuclear Overhauser Effect) experiments were carried out with the LmrA-NBD to investigate the dynamics of individual amino acids upon nucleotide binding. For MsbA-NBD WT, photoelectron-transfer fluorescence correlation spectroscopy (PET-FCS), which gives information about global dynamics, was carried out. Nucleotide binding quenches the dynamics of the ATP binding site itself but also affects remote residues thus beginning to shed light on signal propagation pathways through the NBD. Conserved motifs known to be important for ATP-binding and/ or hydrolysis within the NBD, such as the A-loop, the Walker A motif, D-loop, Q-loop or the H-loop and other interesting regions as the coupling helix groove and the C-terminus of the NBD react to nucleotide binding. Overall, the hetNOE measurements showed that the secondary structural elements essentially remain identical on the ps - ns timescale for LmrA-NBD WT in the apo and ADP-bound state, however, while the dynamics of the catalytic subdomain are quenched in the presence of ADP, the α-helical subdomain became more mobile. H/D exchange and CSP experiments also show ADP binding induced rigidification of residues in the catalytic subdomain of LmrA-NBD in the ms - s timescale. These dynamic differences support the role of the α-helical and the catalytic subdomain in transmitting conformational changes between the NBD and the TMD. Considering that the data obtained for the NBD in the ADP-bound state reflect the post-hydrolytic state, the “unique” dynamic profile of this state may have far reaching consequences for substrate unloading and transporter switching from the outward open to the inward open state before a new transport cycle can begin again. Furthermore, backbone dynamic studies revealed differences in dynamics between residues of the D-loop, Q-loop and the C-terminal end of the X-loop, which are flexible in the apo and ADP states, and residues of the N-terminal end of the X-loop and a residue of the Walker B motif, which are rigid in both states. Investigation of the different catalytic states (apo, (Mg)ADP-bound and (Mg)ATP-bound) with the different methods yielded a “dynamic fingerprint” of model ABC transporter NBDs in all physiologically relevant states. The second part of this PhD thesis examines the structural and dynamic consequences of mutations in the conserved D-loop, Q-loop and H-loop motifs of the LmrA-NBD using NMR spectroscopy. The experiments show that there is crosstalk between the three conserved motifs, with the H-loop as a key team player, and remote NBD regions. Furthermore, D-, Q- and H-loop communicate with other conserved motifs such as the Walker A and Walker B motifs. However, no or only minor effects on the structure and dynamics in A-loop, X-loop and C-loop were observed by the inserted mutations. To identify the role of the D-loop residues in substrate transport and ATP hydrolysis functional assays (i.e. ATPase activity and substrate transport) were carried out using isolated NBDs and the full-length B. subtilis transporter BmrA. Substrate transport and ATP hydrolysis were reduced or fully abrogated when mutating the conserved D-loop. The classic ABC transporter D-loop consensus sequence “SALD” is only maintained in MsbA, but slightly modified in LmrA (ASLD) and BmrA (SSLD). Interestingly, when the D-loop mutations ASLD or SALD were introduced into full-length BmrA, substrate transport was still possible. However, for the ASLD mutant, an increase in basal ATP hydrolysis was observed but at the same time, this mutant did not react with an increase of ATP hydrolysis upon substrate addition, i.e. it lost its ability to carry out “stimulated ATPase activity”, a common feature of ABC multidrug transporters. These observations indicate that the N terminal serine and conserved aspartic acid residues of the D-loop (507S SLD510) in BmrA are particularly important for ATP hydrolysis. The functional consequences of the D-loop mutants in combination with CSP experimetns lead to the hypothesis that conformational changes and dynamics are incorrectly transmitted to important conserved motifs such as H- and Q-loop and residues in regions important for processing substrate transport, i.e. coupling helix groove and the C-terminus within the NBD. The third part of this PhD thesis describes the discovery of a novel “communication hinge” linking the Walker A motif to the coupling helix groove and thus connecting NBD and TMD via highly conserved residues in the NBD. NMR spectroscopic studies (i.e. CSP and 19F NMR) of LmrA-NBD WT, MsbA-NBD WT and BmrA-NBD WT revealed that the C-terminal end of the Walker A helix, which is sequentially highly conserved in type I ABC exporters (type IV), interacts with an opposing bulky hydrophobic/aromatic residue. Mutating the conserved arginine residue at the C-terminal end of the Walker A helix in full-length BmrA was found to disrupt substrate transport for all tested mutants. ATPase activity, on the other hand, showed different results depending on the amino acid inserted at the position of the arginine, thereby showing that substrate transport in the TMD and ATP hydrolysis in the NBD can be selectively decoupled at this site. Furthermore, it was evaluated in this thesis that mutations in this region impact protein stability of type I ABC exporters (type IV). However, none of the NMR experiments conducted in this thesis for the D-, Q- or H-loop motif revealed a link to any residue of our identified allosterically affected region (R-W/L region), suggesting that this is an NBD intradomain signaling pathway independent of these conserved motifs. The results presented in this thesis for three archetypical bacterial ABC exporters show that the substrate transport and ATP hydrolysis mechanisms are enabled through complex interaction networks.
DDC:	500 Naturwissenschaften 500 Natural sciences and mathematics 540 Chemie 540 Chemistry and allied sciences 570 Biowissenschaften 570 Life sciences
Institution:	Johannes Gutenberg-Universität Mainz
Department:	FB 09 Chemie, Pharmazie u. Geowissensch.
Place:	Mainz
ROR:	https://ror.org/023b0x485
DOI:	http://doi.org/10.25358/openscience-8529
URN:	urn:nbn:de:hebis:77-openscience-0a42e724-ca43-433e-b4d0-39b59bb542a49
Version:	Original work
Publication type:	Dissertation
License:	In Copyright
Information on rights of use:	http://rightsstatements.org/vocab/InC/1.0/
Extent:	XVIII, 240 Seiten ; Illustrationen, Diagramme
Appears in collections:	JGU-Publikationen