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      Drug‐dependent inhibition of nucleotide hydrolysis in the heterodimeric ABC multidrug transporter PatAB from Streptococcus pneumoniae


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          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 the 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.


          Heterodimeric ABC multidrug transporters are important contributors to antibiotic resistance in pathogenic bacteria. However, the structural and functional elements determining interactions with drugs and coupling drug binding to nucleotide hydrolysis are not fully understood. Using mass spectrometry and biochemical approaches, we determine the subunit composition of PatAB from Streptococcus pneumoniae and probe drug binding locations to explain the inhibition of nucleotide hydrolysis by a subclass of antibiotics and cytotoxic agents.

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          Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold.

          The alpha- and beta-subunits of membrane-bound ATP synthase complex bind ATP and ADP: beta contributes to catalytic sites, and alpha may be involved in regulation of ATP synthase activity. The sequences of beta-subunits are highly conserved in Escherichia coli and bovine mitochondria. Also alpha and beta are weakly homologous to each other throughout most of their amino acid sequences, suggesting that they have common functions in catalysis. Related sequences in both alpha and beta and in other enzymes that bind ATP or ADP in catalysis, notably myosin, phosphofructokinase, and adenylate kinase, help to identify regions contributing to an adenine nucleotide binding fold in both ATP synthase subunits.
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            ABC transporters catalyze transport reactions, such as the high-affinity uptake of micronutrients into bacteria and the export of cytotoxic compounds from mammalian cells. Crystal structures of ABC domains and full transporters have provided a framework for formulating reaction mechanisms of ATP-driven substrate transport, but recent studies have suggested remarkable mechanistic diversity within this protein family. This review evaluates the differing mechanistic proposals and outlines future directions for the exploration of ABC-transporter-catalyzed reactions.
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              Bayesian deconvolution of mass and ion mobility spectra: from binary interactions to polydisperse ensembles.

              Interpretation of mass spectra is challenging because they report a ratio of two physical quantities, mass and charge, which may each have multiple components that overlap in m/z. Previous approaches to disentangling the two have focused on peak assignment or fitting. However, the former struggle with complex spectra, and the latter are generally computationally intensive and may require substantial manual intervention. We propose a new data analysis approach that employs a Bayesian framework to separate the mass and charge dimensions. On the basis of this approach, we developed UniDec (Universal Deconvolution), software that provides a rapid, robust, and flexible deconvolution of mass spectra and ion mobility-mass spectra with minimal user intervention. Incorporation of the charge-state distribution in the Bayesian prior probabilities provides separation of the m/z spectrum into its physical mass and charge components. We have evaluated our approach using systems of increasing complexity, enabling us to deduce lipid binding to membrane proteins, to probe the dynamics of subunit exchange reactions, and to characterize polydispersity in both protein assemblies and lipoprotein Nanodiscs. The general utility of our approach will greatly facilitate analysis of ion mobility and mass spectra.

                Author and article information

                FEBS J
                FEBS J
                The Febs Journal
                John Wiley and Sons Inc. (Hoboken )
                11 February 2022
                July 2022
                : 289
                : 13 ( doiID: 10.1111/febs.v289.13 )
                : 3770-3788
                [ 1 ] Department of Pharmacology University of Cambridge UK
                [ 2 ] Cambridge Centre for Proteomics Department of Biochemistry University of Cambridge UK
                [ 3 ] Department of Chemistry University of Oxford UK
                [ 4 ] Department of Chemistry University of Cambridge UK
                [ 5 ] Department of Physiology Faculty of Biological Sciences Pontificia Universidad Católica de Chile Santiago Chile
                [ 6 ]Present address: School of Biosciences University of Birmingham UK
                Author notes
                [*] [* ] Correspondence

                H. W. van Veen, Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, UK

                Tel: +441223765295

                E‐mail: hwv20@ 123456cam.ac.uk

                © 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies

                This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                Page count
                Figures: 9, Tables: 1, Pages: 3788, Words: 13405
                Funded by: Biotechnology and Biological Sciences Research Council (BBSRC) , doi 10.13039/501100000268;
                Award ID: BB/K017713/1
                Award ID: BB/R00224X/1
                Award ID: 2114197
                Funded by: British Society for Antimicrobial Chemotherapy , doi 10.13039/501100000313;
                Award ID: GA2011‐19R
                Funded by: Croucher Foundation , doi 10.13039/501100001692;
                Original Article
                Original Articles
                Custom metadata
                July 2022
                Converter:WILEY_ML3GV2_TO_JATSPMC version:6.2.0 mode:remove_FC converted:07.10.2022

                Molecular biology
                abc transporter,antibiotic resistance,drug transport,nucleotide hydrolysis,streptococcus


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