Deciphering Multidrug Efflux: Substrate Interactions and Transport Mechanisms in PatAB and NorM-VC

Multidrug efflux transporters are ubiquitously expressed in all kingdoms of life. In bacteria, they participate in several innate physiological functions to aid survival and adaptation. Given their crucial roles in conferring antimicrobial resistance (AMR), the understanding of the structural and functional aspects of these transporters is essential for developing strategies to combat AMR. Despite recent advances in structural biology, several key aspects of these transporters remain unclear. These include the molecular details of how they recognise, interact with, and transport substrates; the detailed mechanisms of energy coupling to substrate transport; and the functional implications of the structural asymmetry in heterodimeric ATP-binding cassette (ABC) multidrug transporters. To address these gaps, this work focuses on two distinct types of multidrug efflux transporters: the ABC transporter PatAB from Streptococcus pneumoniae and the multidrug and toxic compound extrusion (MATE) transporter NorM-VC from Vibrio cholerae. First, the study on PatAB revealed two classes of transported substrates based on their effects on nucleotide hydrolysis activity. While most substrates had little to no impact on nucleotide hydrolysis activity, a group of inhibitory substrates were identified, including ethidium, propidium, novobiocin, and coumermycin A1, which significantly decrease nucleotide hydrolysis activity at low concentrations through non-competitive inhibition. Analyses of nucleotide binding confirmed that these inhibitors do not compete with nucleotide binding but that they bind to allosteric sites on PatAB, possibly at the interface between the nucleotide-binding domains (NBDs) or near the coupling helices at the interface between NBDs and transmembrane domains (TMDs). Next, mutations in the consensus and degenerate D-loops of the NBDs were introduced to investigate the complex coupling between nucleotide hydrolysis and substrate transport of PatAB. The consensus D-loop mutation significantly decreased both nucleotide hydrolysis and substrate transport activities. Surprisingly, the degenerate D-loop mutation retained only 2-4% of nucleotide hydrolysis activity compared to the wildtype yet maintained transport capability in cells for most tested substrates. These observations led the proposal of a re-coupling model for PatAB, where substrate binding improves the coupling between NBDs and TMDs. This process likely reduces excessive uncoupled NTPase activity by disrupting the communications between NBDs, thus efficiently regulating energy consumptions and transport functions. A procedure for reconstituting PatAB into peptidiscs was also established and optimised. Both ATPase and GTPase activities of PatAB in peptidiscs were lower compared to those in detergent solution or when reconstituted into nanodiscs or proteoliposomes, similar to previous reports for Pdr5. While this presents challenges for functional studies, the successful reconstitution of PatAB into peptidiscs establishes a foundation for future structural studies, potentially providing new insights into the conformational states that PatAB adopts in a membrane-like environment. Finally, the structural basis and functional mechanisms of substrate recognition and transport in NorM-VC were explored with an international collaborator, with particular emphasis on doxorubicin as a substrate. Using a combination of cryo-electron microscopy (cryo-EM), site-directed mutagenesis and transport assays, important residues and relevant structure-activity relationships in NorM-VC were examined. In transport assays in cells and proteoliposomes, I confirmed the presence of a well-defined binding pocket with key residues in doxorubicin recognition. Although the ethidium-bound structure is yet to be determined, functional analysis suggest that the binding sites for doxorubicin and ethidium are non-overlapping. The investigation into transport energetics of NorM-VC revealed that both the membrane potential and the chemical proton gradient contribute to the driving force for doxorubicin and acriflavine efflux. Additionally, proteoliposome studies showed that efficient doxorubicin transport by NorM-VC requires both pH and sodium gradients. When taken together, these findings align well with previous observations on NorM-VC-mediated substrate transport, suggesting that the proton/sodium ion binding pockets near D36 and E255/D371 are involved in the transport of different substrates. In conclusion, this study provides novel insights into the molecular mechanisms of PatAB and NorM-VC and enhances our understanding of the membrane transport reactions that are catalysed. The findings not only have implications for the understanding of structurally similar human transporters but may potentially contribute to future research on inhibitor design for these transporters.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2024

Author dc:creator

• Hsieh, Pei-Yu

Advisor dc:contributor.advisor

• Van Veen, Hendrik

• ABC transporters
• Antimicrobial resistance
• MATE transporters
• Membrane transporters
• Multidrug efflux transporters

Licence

• https://www.repository.cam.ac.uk/bitstreams/399aee00-cd32-43b0-8588-facd2a716474/download
• http://purl.org/NET/rdflicense/allrightsreserved

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.115307

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/379117