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

Abstract

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.

Emergence of mass spectrometry detergents for membrane proteomics

“…The outcome was condensed into design guidelines with which the structure of OGDs can now predictably be tuned for individual experimental parameters, including protein yields, delipidation, and charge reduction inside the vacuum of a mass spectrometer [ 58 ]. Conveniently, OGDs enable the straightforward nMS analysis of membrane proteins and their lipid complexes, which represents a new enabling step for the investigation of drug targets [ 27 , 51 , 58 , 68 – 70 , 72 , 73 ].…”

Role of PatAB Transporter in Efflux of Levofloxacin in Streptococcus pneumoniae

“…Substitutions F16L, F30L and S33A in the SPN1852 protein can be considered conservative, and indeed do not affect the TMH prediction. However, residues from all TMHs are believed to contribute to the surface of drug translocation pathway across the membrane [ 39 , 40 , 41 , 42 ], and mutations in these residues could alter this process. In fact, the aromatic F has about a 12% larger hydrophobic surface area than L [ 43 ], and substitution of L by F in class A amphipathic helical peptide affected its interaction with lipids [ 44 ].…”