Abstract
ATP-binding cassette (ABC) transporters transduce the free energy of ATP hydrolysis to power the mechanical work of substrate translocation across cell membranes. MsbA is an ABC transporter implicated in trafficking lipid A across the inner membrane of Escherichia coli. It has sequence similarity and overlapping substrate specificity with multidrug ABC transporters that export cytotoxic molecules in humans and prokaryotes. Despite rapid advances in structure determination of ABC efflux transporters, little is known regarding the location of substrate-binding sites in the transmembrane segment and the translocation pathway across the membrane. In this study, we have mapped residues proximal to the daunorubicin (DNR)-binding site in MsbA using site-specific, ATP-dependent quenching of DNR intrinsic fluorescence by spin labels. In the nucleotide-free MsbA intermediate, DNR-binding residues cluster at the cytoplasmic end of helices 3 and 6 at a site accessible from the membrane/water interface and extending into an aqueous chamber formed at the interface between the two transmembrane domains. Binding of a nonhydrolyzable ATP analog inverts the transporter to an outward-facing conformation and relieves DNR quenching by spin labels suggesting DNR exclusion from proximity to the spin labels. The simplest model consistent with our data has DNR entering near an elbow helix parallel to the water/membrane interface, partitioning into the open chamber, and then translocating toward the periplasm upon ATP binding.
Highlights
Grant R01-GM077659 from the NIGMS. □S The on-line version of this article contains supplemental Figs
Two highly conserved ATP-binding cassette (ABC) or nucleotide-binding domains (NBDs) bind and hydrolyze ATP to supply the active energy for transport [7]
Bacterial ABC exporters are expressed as monomers, each consisting of one NBD and one transmembrane domains (TMDs), that dimerize to form the active transporter [3]
Summary
Materials—n-Dodecyl-␣-D-maltoside (␣-DDM) was from Anatrace Inc.; 1-oxyl-2,2,5,5-tetramethylpyrrolinyl-3-methylmethanethiosulfonate spin label was from Toronto Research Chemicals Inc.; nickel-nitrilotriacetic resin was from Qiagen Inc.; Superdex 200 column was from GE Healthcare; DNA oligonucleotides were from Integrated DNA Technologies (IDT); tris(2-carboxyethyl)phosphine (TCEP) was from Molecular Probes; and DNR, ATP, and AMP-PNP were from Sigma. Trp Fluorescence Quenching Studies—Trp fluorescence quenching by DNR was measured following incubation of MsbA (2 M) or free Trp (1 M) in buffer (50 mM HEPES, 50 mM NaCl, and 0.05% ␣-DDM, pH 7.2) with 0 –200 M of DNR (with and without 5 mM AMP-PNP for 30 min at 37 °C) for 15 min at 37 °C. Reversal in DNR quenching on releasing the spin label from spin-labeled proteins (10 M) was measured by preincubating the samples with TCEP (100 M) for 15 min at room temperature followed by incubation with DNR (5 M) for 15 min at 37 °C. To release the spin label from labeled proteins, samples (100 M) were incubated with TCEP (1 mM) for 15 min at room temperature. The amount of phosphate released was determined by comparing with inorganic phosphate standards
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