ATPase Activity of the MsbA Lipid Flippase of Escherichia coli

ATP-binding cassette exporters use the energy of ATP hydrolysis to transport substrates across membranes by switching between inward-and outward-facing conformations. Essentially all structural studies of these proteins have been performed with the proteins in detergent micelles, locked in specific conformations and/or at low temperature. Here, we used luminescence resonance energy transfer spectroscopy to study the prototypical ATP-binding cassette exporter MsbA reconstituted in nanodiscs at 37°C while it performs ATP hydrolysis. We found major differences when comparing MsbA in these native-like conditions with double electron-electron resonance data and the crystal structure of MsbA in the open inward-facing conformation. The most striking differences include a significantly smaller separation between the nucleotide-binding domains and a larger fraction of molecules with associated nucleotide-binding domains in the nucleotide-free apo state. These studies stress the importance of studying membrane proteins in an environment that approaches physiological conditions. ATP-binding cassette (ABC)2 transporters constitute one of the largest families of membrane proteins and are found in all domains of life (1-3). They can be either importers (most prokaryote ABC transporters) or exporters (most mammal ABC transporters) (1-3). The core structure of ABC proteins consists of two transmembrane domains that form the translocation pathway and two conserved nucleotide-binding domains (NBDs) that bind and hydrolyze ATP (1-3), a process essential for their function. Binding of ATP promotes the formation of an NBD dimer in a head to tail orientation (4, 5). In the resulting structure two ATP molecules are "sandwiched" at the dimer interface, and residues from both NBDs form each of the two nucleotide-binding sites (1, 4, 5). NBD dimerization is essential for ATP hydrolysis and requires ATP binding to both nucleotide-binding sites (6), whereas dissociation of the dimers occurs following hydrolysis at only one of the two sites (7). This ATP-dependent NBDs dimerization/dissociation process is coupled to rearrangements of the transmembrane helices, switching the transporters from an inward-facing conformation (dissociated NBDs) to an outward-facing conformation (dimeric NBDs), with the concomitant translocation of substrate (1, 3).Two of the most studied ABC exporters are the multidrug resistance protein P-glycoprotein (MDR1 and ABCB1) that plays a role in the resistance to chemotherapy of some forms of cancer (3),and its bacterial homolog, the lipid flippase MsbA (8). MsbA is located in the inner membrane of Gram-negative bacteria, where it transports lipid A from the inner to the outer leaflet (9, 10). MsbA has been crystallized in inward-and outward-facing conformations (11) and has also been extensively studied by different spectroscopic techniques (12-18). Several studies point to large motions between the nucleotide-free "open" inward-facing conformation (NBDs separated by tens of Angstroms) and the ATP-bound "closed" outw...

Section: Resultsmentioning

confidence: 99%

“…MsbA is located in the inner membrane of Gram-negative bacteria, where it transports lipid A from the inner to the outer leaflet (9,10). MsbA has been crystallized in inward-and outward-facing conformations (11) and has also been extensively studied by different spectroscopic techniques (12)(13)(14)(15)(16)(17)(18).…”

Section: Atp-binding Cassette (Abc)mentioning

confidence: 99%

The Lipid Bilayer Modulates the Structure and Function of an ATP-binding Cassette Exporter

Zoghbi

Cooper

Altenberg

2016

“…Patients with sitosterolemia lack the ABC transporters that pump absorbed sterols, including the plant-derived sitosterol, back into the gut or the bile (66). In bacteria, point mutants in the essential ABC transporter MsbA are defective in flipping nascent LPS across the inner membranes at elevated temperatures (35,36,67,68), as judged by accessibility to periplasmic lipid A modification enzymes. Such mutants are also defective in glycerophospholipid export to the outer membrane (35), but this phenomenon might be secondary to LPS accumulation within the inner membrane.…”

Section: Discussionmentioning

confidence: 99%

An Undecaprenyl Phosphate-Aminoarabinose Flippase Required for Polymyxin Resistance in Escherichia coli

Yan¹,

Guan²,

Raetz

2007

Self Cite

137

136

Modification of lipid A with the 4-amino-4-deoxy-L-arabinose (L-Ara4N) moiety is required for resistance to polymyxin and cationic antimicrobial peptides in Escherichia coli and Salmonella typhimurium. An operon of seven genes (designated pmrHFIJKLM in S. typhimurium), which is regulated by the PmrA transcription factor and is also present in E. coli, is necessary for the maintenance of polymyxin resistance. We previously elucidated the roles of pmrHFIJK in the biosynthesis and attachment of L-Ara4N to lipid A and renamed these genes arn-BCADT, respectively. We now propose functions for the last two genes of the operon, pmrL and pmrM. Chromosomal inactivation of each of these genes in an E. coli pmrA c parent switched its phenotype from polymyxin-resistant to polymyxin-sensitive. The lipid A moiety of lipopolysaccharide (LPS) 4 in the outer membranes of Gram-negative bacteria typically consists of a ␤,1Ј-6-linked disaccharide of glucosamine, modified with phosphate groups at the 1-and 4Ј-positions, and with R-3-hydroxyacyl chains at the 2-, 3-, 2Ј-, and 3Ј-positions (Fig.

“…In vitro studies of MsbA substrate specificity identify a broad range of substrates that stimulate ATPase activity (29). In addition to the putative physiological substrates lipid A and lipopolysaccharide (LPS), the ABCB1 substrates Ilmofosine, H33342, and verapamil differentially enhance ATP hydrolysis of MsbA (29,30). Intrinsic MsbA tryptophan (Trp) fluorescence quenching by these putative substrate molecules provides further support of interaction (29).…”

mentioning

confidence: 99%

“…In this study, we utilize a site-specific quenching approach to identify residues in the vicinity of the daunorubicin (DNR)-binding site (37). Although the data on DNR stimulation of ATP hydrolysis is inconclusive (20,29,30), the quenching of MsbA Trp fluorescence suggests a specific interaction. Spin labels were introduced along transmembrane helices 3, 4, and 6 of MsbA to assess their ATP-dependent quenching of DNR fluorescence.…”

mentioning

confidence: 99%

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

Smriti

Zou

Mchaourab

2009

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.