Mapping Daunorubicin-binding Sites in the ATP-binding Cassette Transporter MsbA Using Site-specific Quenching by Spin Labels

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.

Direct Spectroscopic Detection of ATP Turnover Reveals Mechanistic Divergence of ABC Exporters

“…The cysteine-less BmrCD (BmrCD-WT*)(Mishra et al, 2014), cysteine-less MsbA (MsbA-WT*)(Dong et al, 2005; Smriti et al, 2009; Zou and McHaourab, 2009), hereafter referred to as BmrCD and MsbA, and their double cysteine mutants were purified by sequential nickel affinity and size-exclusion chromatography (SEC) as previously described. Double cysteine mutants of BmrCD and MsbA were incubated with a 20-fold excess of (1-Oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate (MTSSL, Enzo Life Sciences) for 4 h at 23 °C and placed on ice for ~12 h. The labeled proteins were then separated from free label by SEC on a Superdex 200 column equilibrated with respective SEC buffer.…”

Conformational dynamics of the nucleotide binding domains and the power stroke of a heterodimeric ABC transporter

“…MsbA-WT* and cysteine substituted mutants were expressed, purified and labeled as previously described (Smriti et al, 2009; Zou and Mchaourab, 2009). …”

Time-resolved Fourier Transform Infrared Spectroscopy of the Nucleotide-binding Domain from the ATP-binding Cassette Transporter MsbA

“…Fluorescence quenching was used to identify allocrites (reviewed in Ref. 6) and their putative binding sites (22,23). Infrared spectroscopy with detection of secondary structure, hydrogen/deuterium exchange rates, and linear dichroism have been used to determine orientation and accessibility profiles of helices in lipid bilayers, e.g.…”