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As with other heterodimeric ABC exporters, PatAB contains two transmembrane domains that form a drug translocation pathway for efflux and two nucleotide\u2010binding 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\u2010mimetic azido\u2010ethidium. Surprisingly, our analyses of azido\u2010ethidium\u2010labelled PatAB peptides point to ethidium binding in the PatA nucleotide\u2010binding domain, with the azido moiety crosslinked to residue Q521 in the H\u2010like loop of the degenerate nucleotide\u2010binding site. Investigation into this compound and residue\u2019s role in nucleotide hydrolysis pointed to a reduction in the activity for a Q521A mutant and ethidium\u2010dependent inhibition in both mutant and wild type. Most transported drugs did not stimulate or inhibit nucleotide\u2009hydrolysis of PatAB in detergent solution or lipidic nanodiscs. However, further examples for ethidium\u2010like inhibition were found with propidium, novobiocin and coumermycin A1, which all inhibit nucleotide hydrolysis by a non\u2010competitive 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\u2010binding domains.<\/jats:p>","DOI":"10.1111\/febs.16366","type":"journal-article","created":{"date-parts":[[2022,1,23]],"date-time":"2022-01-23T16:18:17Z","timestamp":1642954697000},"page":"3770-3788","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Drug\u2010dependent inhibition of nucleotide hydrolysis in the heterodimeric ABC multidrug transporter PatAB from <i>Streptococcus pneumoniae<\/i>"],"prefix":"10.1111","volume":"289","author":[{"given":"Charlotte","family":"Guffick","sequence":"first","affiliation":[{"name":"Department of Pharmacology University of Cambridge  UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pei\u2010Yu","family":"Hsieh","sequence":"additional","affiliation":[{"name":"Department of Pharmacology 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