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Research data supporting "Drug-dependent inhibition of nucleotide hydrolysis in the heterodimeric ABC multidrug transporter PatAB from Streptococcus pneumoniae"

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Guffick, Charlotte 
Hsieh, Pei-Yu 
Ali, Anam 
Shi, Wilma 
Howard, Julie 


The bacterial heterodimeric ATP-binding cassette (ABC) multidrug exporter PatAB has a critical role in conferring antibiotic resistance in multidrug-resistant infections by Streptococcus pneumoniae. As with other heterodimeric ABC exporters, PatAB contains two transmembrane domains that form a drug translocation pathway for efflux and two nucleotide-binding domains that bind ATP, one of which is hydrolysed during transport. The structural and functional elements in heterodimeric ABC multidrug exporters that determine interactions with drugs and couple drug binding to nucleotide hydrolysis are not fully understood. Here, we used mass spectrometry techniques to determine the subunit stoichiometry in PatAB in our lactococcal expression system and investigate locations of drug binding using the fluorescent drug-mimetic azido-ethidium. Surprisingly, our analyses of azido-ethidium-labelled PatAB peptides point to ethidium binding in PatA nucleotide-binding domain, with the azido moiety crosslinked to residue Q521 in the H-like loop of the degenerate nucleotide-binding site. Investigation into this compound and residue’s role in nucleotide hydrolysis pointed to a reduction in the activity for a Q521A mutant and ethidium-dependent inhibition in both mutant and wild type. Most transported drugs did not stimulate or inhibit nucleotide hydrolysis of PatAB in detergent solution or lipidic nanodiscs. However, further examples for ethidium-like inhibition were found with propidium, novobiocin and coumermycin A1, which all inhibit nucleotide hydrolysis by a non-competitive mechanism. These data cast light on potential mechanisms by which drugs can regulate nucleotide hydrolysis by PatAB, which might involve a novel drug binding site near the nucleotide-binding domains. The research data in this dataset record support the publication by Guffick et al. in FEBS J. and refer to the figures that are incorporated in the paper, and the DNA and protein sequences under study. Descriptions of the experimental details and statistical analyses are included in the Materials and Methods and figure legends of the paper.


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Microsoft Excel v16.53


ABC transporter, antibiotic resistance, drug transport, nucleotide hydrolysis, Streptococcus


British Society for Antimicrobial Chemotherapy (GA2011-19R)
Biotechnology and Biological Sciences Research Council (BB/K017713/1)
Biotechnology and Biological Sciences Research Council (BB/R00224X/1)
Biotechnology and Biological Sciences Research Council (2114197)