ATP-binding Cassette Exporters Research Articles

Sav1866 from Staphylococcus aureus and P-Glycoprotein: Similarities and Differences in ATPase Activity Assessed with Detergents as Allocrites

The ATP-binding cassette exporters Sav1866 from Staphylococcus aureus and P-glycoprotein are known to share a certain sequence similarity and disposition for cationic allocrites. Conversely, the two ATPases react very differently to neutral detergents that have previously been shown to be inhibitory allocrites for P-glycoprotein. To gain insight into the functional differences of the two proteins, we compared their basal and detergent-stimulated ATPase activity. P-Glycoprotein was investigated in NIH-MDR1-G185 plasma membrane vesicles and Sav1866 in lipid vesicles exhibiting a membrane packing density and a surface potential similar to those of the plasma membrane vesicles. Under basal conditions, Sav1866 revealed a lower catalytic efficiency and concomitantly a more pronounced sodium chloride and pH dependence than P-glycoprotein. As expected, the cationic allocrites (alkyltrimethylammonium chlorides) induced similar bell-shaped activity curves as a function of concentration for both exporters, suggesting stimulation upon binding of the first and inhibition upon binding of the second allocrite molecule. However, the neutral allocrites (n-alkyl-β-d-maltosides and n-ethylene glycol monododecyl ethers) reduced P-glycoprotein's ATPase activity at concentrations well below their critical micelle concentration (CMC) but strongly enhanced Sav1866's ATPase activity even at concentrations above their CMC. The lack of ATPase inhibition at high concentrations of neutral of detergents could be explained by their comparatively low binding affinity for the transmembrane domains of Sav1866, which seems to prevent binding of a second inhibitory molecule. The high ATPase activity in the presence of hydrophobic, long chain detergents moreover revealed that Sav1866, despite its lower basal catalytic efficiency, is a more efficient floppase for lipidlike amphiphiles than P-glycoprotein.

Boundary of the Nucleotide-Binding Domain of Streptococcus ComA Based on Functional and Structural Analysis

The ATP-binding cassette (ABC) transporter ComA is a key molecule essential for the first step of the quorum-sensing system of Streptococcus. The nucleotide binding domains (NBD) of Streptococcus mutans ComA with different N termini, NBD1 (amino acid residues 495-760), NBD2 (517-760), and NBD3 (528-760), were expressed, purified, and characterized. The shortest NBD3 corresponds to the region commonly defined as NBD in the database searches of ABC transporters. A kinetic analysis showed that the extra N-terminal region conferred a significantly higher ATP hydrolytic activity on the NBD at a neutral pH. Gel-filtration, X-ray crystallography, and mutational analyses suggest that at least four to five residues beyond the N-terminal boundary of NBD3 indeed participate in stabilizing the protein scaffold of the domain structure, thereby facilitating the ATP-dependent dimerization of NBD which is a prerequisite to the catalysis. These findings, together with the presence of a highly conserved glycine residue in this region, support the redefinition of the N-terminal boundary of the NBD of these types of ABC exporters.

Molecular Disruption of the Power Stroke in the ATP-binding Cassette Transport Protein MsbA

ATP-binding cassette transporters affect drug pharmacokinetics and are associated with inherited human diseases and impaired chemotherapeutic treatment of cancers and microbial infections. Current alternating access models for ATP-binding cassette exporter activity suggest that ATP binding at the two cytosolic nucleotide-binding domains provides a power stroke for the conformational switch of the two membrane domains from the inward-facing conformation to the outward-facing conformation. In outward-facing crystal structures of the bacterial homodimeric ATP-binding cassette transporters MsbA from gram-negative bacteria and Sav1866 from Staphylococcus aureus, two transmembrane helices (3 and 4) in the membrane domains have their cytoplasmic extensions in close proximity, forming a tetrahelix bundle interface. In biochemical experiments on MsbA from Escherichia coli, we show for the first time that a robust network of inter-monomer interactions in the tetrahelix bundle is crucial for the transmission of nucleotide-dependent conformational changes to the extracellular side of the membrane domains. Our observations are the first to suggest that modulation of tetrahelix bundle interactions in ATP-binding cassette exporters might offer a potent strategy to alter their transport activity.

ATP Alone Triggers the Outward Facing Conformation of the Maltose ATP-binding Cassette Transporter

The maltose transporter MalFGK(2) is a study prototype for ABC importers. During catalysis, the MalFG membrane domain alternates between inward and outward facing conformations when the MalK dimer closes and hydrolyzes ATP. Because a rapid ATP hydrolysis depends on MalE and maltose, it has been proposed that closed liganded MalE facilitates the transition to the outward facing conformation. Here we find that, in contrast to the expected, ATP is sufficient for the closure of MalK and for the conversion of MalFG to the outward facing state. The outward facing transporter binds MalE with nanomolar affinity, yet neither MalE nor maltose is necessary or facilitates the transition. Thus, the rapid hydrolysis of ATP observed in the presence of MalE and maltose is not because closed liganded MalE accelerates the formation of the outward facing conformation. These findings have fundamental implications for the description of the transport reaction.

Outward- to Inward-Facing Transition of MsbA Transporter: A Mechanistic Picture at Atomic Resolution

MsbA is a structurally well-characterized, bacterial ATP-binding cassette (ABC) exporter. It undergoes a large-scale conformational change between the inward-facing (IF) and the outward-facing (OF) states during its transport cycle. Despite extensive experimental and computational studies on MsbA and its homologs, a unified mechanistic picture describing the OF-IF conformational transition of ABC exporters is still missing. In order to study this transition at an atomic level, we have used a novel approach based on a nonequilibrium driven scheme and performed an extensive set of molecular dynamics simulations that sample the OF-to-IF reaction-path ensemble of the apo MsbA in the presence of explicit solvent and membrane. using several distinct system-specific biasing protocols, we were able to steer the system along a large number of different paths in a low-dimensional holonomic coordinate space. Nonequilibrium work relations were employed to interpret the results and to determine the optimal transition path. Our results provide clear evidence that the opening of the cytoplasmic gate in the apo MsbA is prohibitively disfavored when the extracellular gate is open, an observation consistent with the “alternating-access mechanism”. More interestingly, we observe that the closure of the extracellular gate does not result in a stable IF conformation unless the nucleotide-binding domains (NBDs) undergo a “twisting” motion that involves a drastic change in their relative orientation. We thus propose a “door-knob mechanism” for the OF-to-IF transition of MsbA that asserts a crucial role for the “twisting” of NBDs in the opening of the cytoplasmic gate. More generally, our results call into question the simplistic models of NBD dissociation/dimerization that ignore the important role of the relative orientation between the NBDs in promoting the OF-IF conformational transitions of ABC exporters.

Association/Dissociation of the Nucleotide-Binding Domains of an ATP-Binding Cassette (ABC) Exporter during the ATP Hydrolysis Cycle

ATP-binding cassette (ABC) proteins constitute one of the largest protein families, and most are membrane transport proteins. Eukaryotic exporters in this group, including the multidrug resistance protein P-glycoprotein, have been linked to chemotherapy resistance due to their extrusion of anticancer agents from cells. Like its prokaryotic homolog MsbA, P-glycoprotein has a functional core composed of two transmembrane domains and two nucleotide-binding domains (NBDs). Substrate transport is driven by ATP hydrolysis, but the mechanism of hydrolysis is controversial. The proposed mechanisms can be broadly classified as: 1) monomer-dimer models, where alternating access depends on a large separation of the NBDs (up to 50 angstroms) during the ATP binding/hydrolysis cycle, or 2) constant-contact models, where the NBDs remain associated during the cycle. To address the ABC exporters' mechanism, we used lanthanide-based resonance energy transfer (LRET) to follow the intra-molecular movements of MsbA during the ATP binding/hydrolysis cycle. Although the MsbA NBDs sample three discrete positions during the cycle, the average NBD separation decreases dramatically after the addition of ATP to the Apo (nucleotide-free) protein. ATP binding reduced the predominant NBD separation distance from 50 to 35 angstroms. ATP hydrolysis elicited by MgATP relaxed a fraction of the NBD dimer population towards the Apo state. These steady-state distance measurements are consistent with a monomer-dimer model. The correlation between rates of NBDs dissociation (determined from kinetic LRET measurements) and ATP hydrolysis are consistent with monomer-dimer models, where the NBDs dissociate during each hydrolysis cycle. This work was supported by a grant from CPRIT grant RP101073 and an American Heart Association Pre-doctoral Fellowship 11PRE7360046 to RSC.

Crystal structure of the multidrug transporter P-glycoprotein from Caenorhabditis elegans.

SummaryP-glycoprotein (P-gp) is an ATP-binding cassette (ABC) transporter that confers multidrug resistance in cancer cells1,2. It also affects the absorption, distribution, and clearance of cancer-unrelated drugs and xenobiotics. For these reasons, the structure and function of P-gp have been studied extensively for decades3. Here we present biochemical characterization of P-gp from C. elegans and its crystal structure at 3.4 Å resolution. This work provides the following new information towards a mechanistic understanding of P-gp: 1. The apparent affinities of P-gp for anticancer drugs actinomycin D and paclitaxel are approximately 4,000 and 100 times higher, respectively, in the membrane bilayer than in detergent. This affinity enhancement highlights the importance of membrane partitioning when drug accesses the transporter in the membrane4. 2. The transporter in the crystal structure opens its drug pathway at the level of the membrane’s inner leaflet. In the helices flanking the opening to the membrane we observe extended loops that may possibly mediate drug binding and/or function as hinges to gate the pathway. 3. The interface between the transmembrane and nucleotide-binding domains, which couples ATP hydrolysis to transport, contains a ball-and-socket joint and salt bridges similar to the ABC importers5, suggesting that ABC exporters and importers may share a similar mechanism to achieve alternating access for transport. 4. A carefully derived model of human P-gp, based on the C. elegans P-gp structure, not only is compatible with decades of biochemical analysis6–12, but also provides insights to explain perplexing functional data regarding the F335A mutant13,14.

Role of the Iron Axial Ligands of Heme Carrier HasA in Heme Uptake and Release

The hemophore protein HasA from Serratia marcescens cycles between two states as follows: the heme-bound holoprotein, which functions as a carrier of the metal cofactor toward the membrane receptor HasR, and the heme-free apoprotein fishing for new porphyrin to be taken up after the heme has been delivered to HasR. Holo- and apo-forms differ for the conformation of the two loops L1 and L2, which provide the axial ligands of the iron through His(32) and Tyr(75), respectively. In the apo-form, loop L1 protrudes toward the solvent far away from loop L2; in the holoprotein, closing of the loops on the heme occurs upon establishment of the two axial coordination bonds. We have established that the two variants obtained via single point mutations of either axial ligand (namely H32A and Y75A) are both in the closed conformation. The presence of the heme and one out of two axial ligands is sufficient to establish a link between L1 and L2, thanks to the presence of coordinating solvent molecules. The latter are stabilized in the iron coordination environment by H-bond interactions with surrounding protein residues. The presence of such a water molecule in both variants is revealed here through a set of different spectroscopic techniques. Previous studies had shown that heme release and uptake processes occur via intermediate states characterized by a Tyr(75)-iron-bound form with open conformation of loop L1. Here, we demonstrate that these states do not naturally occur in the free protein but can only be driven by the interaction with the partner proteins.

Dynamics of a bacterial multidrug ABC transporter in the inward- and outward-facing conformations

The study of membrane proteins remains a challenging task, and approaches to unravel their dynamics are scarce. Here, we applied hydrogen/deuterium exchange (HDX) coupled to mass spectrometry to probe the motions of a bacterial multidrug ATP-binding cassette (ABC) transporter, BmrA, in the inward-facing (resting state) and outward-facing (ATP-bound) conformations. Trypsin digestion and global or local HDX support the transition between inward- and outward-facing conformations during the catalytic cycle of BmrA. However, in the resting state, peptides from the two intracellular domains, especially ICD2, show a much faster HDX than in the closed state. This shows that these two subdomains are very flexible in this conformation. Additionally, molecular dynamics simulations suggest a large fluctuation of the Cα positions from ICD2 residues in the inward-facing conformation of a related transporter, MsbA. These results highlight the unexpected flexibility of ABC exporters in the resting state and underline the power of HDX coupled to mass spectrometry to explore conformational changes and dynamics of large membrane proteins.

YknWXYZ Is an Unusual Four-Component Transporter with a Role in Protection against Sporulation-Delaying-Protein-Induced Killing of Bacillus subtilis

YknXYZ is the ATP-binding cassette export complex from Bacillus subtilis, where YknX is a membrane fusion protein, YknY is an ATPase, and YknZ is a permease. The yknXYZ genes are arranged into an operon that also includes yknW, encoding a membrane protein with four putative transmembrane segments. Previous studies suggested that the yknWXYZ operon belongs to the σ(w) regulon and protects cells from the endogenous toxin SDP (sporulation-delaying protein) encoded by sdpC. In this study, we investigated the composition and function of YknW and YknXYZ. We report that the yknWXYZ operon is constitutively expressed in growing B. subtilis cells independently from sdpC. Chemical cross-linking in vivo and copurification approaches established that YknX interacts with YknYZ, whereas YknW binds YknXYZ, indicating that all four proteins form a complex in vivo. The complex assembly is modulated by YknW but proceeds in the absence of SdpC. When overproduced alone, YknW provides partial protection against SDP toxin, but all four Ykn proteins are required for full protection against both endogenous and exogenous SDP. We conclude that YknWXYZ is an unusual four-component transporter with a role in the starvation-induced killing of B. subtilis cells.

Yeast ATP-Binding Cassette Transporters Conferring Multidrug Resistance

Overexpression of the ATP-binding cassette (ABC) drug transporter P-glycoprotein (P-gp) is often responsible for the failure of chemotherapy as a treatment for human tumors. The presence of proteins homologous to P-gp in organisms ranging from prokaryotes to eukaryotes indicates that drug export is a general mechanism of multidrug resistance. Yeasts are no exception. They have developed a large subfamily of ABC exporters involved in pleiotropic drug resistance (PDR) and in the cellular efflux of a wide variety of drugs. The PDR transporters Pdr5p of Saccharomyces cerevisiae and Cdr1p of Candida albicans are important members of this PDR subfamily, which comprises up to 10 phylogenetic clusters in fungi. Here, we review current achievements concerning the structure, molecular mechanism, and physiological functions of yeast Pdr transporters.

Crystal structure of a heterodimeric ABC transporter in its inward-facing conformation

ATP-binding cassette (ABC) transporters shuttle a wide variety of molecules across cell membranes by alternating between inward- and outward-facing conformations, harnessing the energy of ATP binding and hydrolysis at their nucleotide binding domains (NBDs). Here we present the 2.9-Å crystal structure of the heterodimeric ABC transporter TM287-TM288 (TM287/288) from Thermotoga maritima in its inward-facing state. In contrast to previous studies, we found that the NBDs only partially separate, remaining in contact through an interface involving conserved motifs that connect the two ATP hydrolysis sites. We observed AMP-PNP binding to the degenerate catalytic site, which deviates from the consensus sequence in the same positions as the eukaryotic homologs CFTR and TAP1-TAP2 (TAP1/2). The TM287/288 structure provides unprecedented insights into the mechanism of heterodimeric ABC exporters and will enable future studies on this large transporter superfamily.

Complete Dissociation of an ATP-Binding Cassette Nucleotide-Binding Domain during the Hydrolysis Cycle

ATP-binding cassette (ABC) proteins catalyze the transport of substrates across membranes. Many ABC proteins, including P-glycoprotein, pump substrates out of the cells. There are two competing models to explain the mechanism of ABC exporters: 1) AThe alternating-access model, where large conformational changes take place during the transport cycle as a consequence of ATP-induced nucleotide-binding domain (NBD) dimerization. 2) An alternative model, which suggests that the power stroke triggered by ATP binding consists of only moderate rearrangements, with the NBDs in contact at all times during the transport cycle. Here, we performed experiments on isolated bacterial NBDs using luminescence resonance energy transfer (LRET) and tryptophan fluorescence spectroscopy, to assess monomer association/dissociation in real time. We used MJ0976, a NBD from the thermophile M. jannaschii, as a model. We found that: 1) The dependence of dimerization for MgATP is stronger (Kd of ∼ 5 μM) than that for NaATP (∼80 μM). 2) The rate of dimerization increased >10-fold in MgATP vs. NaATP. 3) A steady-state dynamic equilibrium is reached in MgATP, with ∼50% of the protein in dimeric form. 4) The dimers completely dissociate into monomers during the hydrolysis cycle. Our observations support ABC protein models involving NBDs association/dissociation. This work was supported grants from CPRIT (RP101073) and NIH (R01GM79629 and R01GM079629-03S1R01).

Transmembrane Gate Movements in the Type II ATP-binding Cassette (ABC) Importer BtuCD-F during Nucleotide Cycle

ATP-binding cassette (ABC) transporters are ubiquitous integral membrane proteins that translocate substrates across cell membranes. The alternating access of their transmembrane domains to opposite sides of the membrane powered by the closure and reopening of the nucleotide binding domains is proposed to drive the translocation events. Despite clear structural similarities, evidence for considerable mechanistic diversity starts to accumulate within the importers subfamily. We present here a detailed study of the gating mechanism of a type II ABC importer, the BtuCD-F vitamin B(12) importer from Escherichia coli, elucidated by EPR spectroscopy. Distance changes at key positions in the translocation gates in the nucleotide-free, ATP- and ADP-bound conformations of the transporter were measured in detergent micelles and liposomes. The translocation gates of the BtuCD-F complex undergo conformational changes in line with a "two-state" alternating access model. We provide the first direct evidence that binding of ATP drives the gates to an inward-facing conformation, in contrast to type I importers specific for maltose, molybdate, or methionine. Following ATP hydrolysis, the translocation gates restore to an apo-like conformation. In the presence of ATP, an excess of vitamin B(12) promotes the reopening of the gates toward the periplasm and the dislodgment of BtuF from the transporter. The EPR data allow a productive translocation cycle of the vitamin B(12) transporter to be modeled.

Catalytic and transport cycles of ABC exporters

ABC (ATP-binding cassette) transporters are arguably the most important family of ATP-driven transporters in biology. Despite considerable effort and advances in determining the structures and physiology of these transporters, their fundamental molecular mechanisms remain elusive and highly controversial. How does ATP hydrolysis by ABC transporters drive their transport function? Part of the problem in answering this question appears to be a perceived need to formulate a universal mechanism. Although it has been generally hoped and assumed that the whole superfamily of ABC transporters would exhibit similar conserved mechanisms, this is proving not to be the case. Structural considerations alone suggest that there are three overall types of coupling mechanisms related to ABC exporters, small ABC importers and large ABC importers. Biochemical and biophysical characterization leads us to the conclusion that, even within these three classes, the catalytic and transport mechanisms are not fully conserved, but continue to evolve. ABC transporters also exhibit unusual characteristics not observed in other primary transporters, such as uncoupled basal ATPase activity, that severely complicate mechanistic studies by established methods. In this chapter, I review these issues as related to ABC exporters in particular. A consensus view has emerged that ABC exporters follow alternating-access switch transport mechanisms. However, some biochemical data suggest that alternating catalytic site transport mechanisms are more appropriate for fully symmetrical ABC exporters. Heterodimeric and asymmetrical ABC exporters appear to conform to simple alternating-access-type mechanisms.

Structure of ABC transporters

ABC (ATP-binding cassette) transporters are primary active membrane proteins that translocate solutes (allocrites) across lipid bilayers. The prototypical ABC transporter consists of four domains: two cytoplasmic NBDs (nucleotide-binding domains) and two TMDs (transmembrane domains). The NBDs, whose primary sequence is highly conserved throughout the superfamily, bind and hydrolyse ATP to power the transport cycle. The TMDs, whose primary sequence and protein fold can be quite disparate, form the translocation pathway across the membrane and generally (but not always) determine allocrite specificity. Structure determination of ABC proteins initially took advantage of the relative ease of expression and crystallization of the hydrophilic bacterial NBDs in isolation from the transporter complex, and revealed detailed information on the structural fold of these domains, the amino acids involved in the binding and hydrolysis of nucleotide, and the head-to-tail arrangement of the NBD-NBD dimer interface. More recently, several intact transporters have been crystallized and three types have, so far, been characterized: type I and II ABC importers, and ABC exporters. All three are present in prokaryotes, but only the ABC exporters appear to be present in eukaryotes. Their structural determination has provided insight into the mechanisms of energy and signal transduction between the NBDs and TMDs (i.e. between the ATP- and allocrite-binding sites) and, for some, the nature of the allocrite-binding site(s) within the TMDs. In this chapter, we focus primarily on the ABC exporters and describe the structural, biochemical and biophysical evidence for and against the controversial bellows-like mechanism proposed for allocrite efflux.

Conformational changes induced by ATP‐hydrolysis in an ABC transporter: A molecular dynamics study of the Sav1866 exporter

ATP-Binding Cassette (ABC) transporters are ubiquitous membrane proteins that use energy from ATP binding or/and hydrolysis to actively transport allocrites across membranes. In this study, we identify ATP-hydrolysis induced conformational changes in a complete ABC exporter (Sav1866) from Staphylococcus aureaus, using molecular dynamics (MD) simulations. By performing MD simulations on the ATP and ADP+IP bound states, we identify the conformational consequences of hydrolysis, showing that the major rearrangements are not restricted to the NBDs, but extend to the transmembrane domains (TMDs) external regions. For the first time, to our knowledge, we see, within the context of a complete transporter, NBD dimer opening in the ADP+IP state in contrast with all ATP-bound states. This opening results from the dissociation of the ABC signature motif from the nucleotide. In addition, in both states, we observe the opening of a gate entrance in the intracellular loop region leading to the exposure of the TMDs internal cavity to the cytoplasm. To see if this opening was large enough to allow allocrite transport, the adiabatic energy profile for doxorubicin passage was determined. For both states, this profile, although an approximation, is overall downhill from the cytoplasmatic to the extracellular side, and the local energy barriers along the TMDs are relatively small, evidencing the exporter nature of Sav1866. The major difference between states is an energy barrier located in the cytoplasmic gate region, which becomes reduced upon hydrolysis, suggesting that allocrite passage is facilitated, and evidencing a possible molecular mechanism for the active transport in these proteins.

Spectroscopic Studies of the Dimerization of ATP-Binding Cassette Nucleotide-Binding Domains

Cancer cells are frequently resistant against chemically unrelated anticancer agents or develop resistance to those agents during treatment (multidrug resistance). ATP-binding cassette (ABC) proteins such as P-glycoprotein are key mediators of multidrug resistance. They catalyze the efflux of chemotherapeutic agents out of cancer cells, preventing their intracellular accumulation and cytotoxic effect. There are two competing models to explain the mechanism of ABC exporters: 1) The alternating-access model, where large conformational changes take place during the transport cycle as a consequence of ATP-induced nucleotide-binding domain (NBD) dimerization. 2) An alternative model, which suggests that the power stroke triggered by ATP binding consists of only moderate rearrangements, with the NBDs in contact at all times during the transport cycle. Here, we performed experiments on isolated bacterial NBDs using luminescence resonance energy transfer (LRET) and tryptophan fluorescence quenching, to determine the ATP dependence on NBD dimerization. As a model, we used MJ0976, a NBD from the thermophile M. jannaschii. Under non-hydrolysis conditions (in the absence of Mg2+), tryptophan fluorescence quenching and LRET experiments showed a similar apparent affinity for ATP, in the 20-40 micromolar range. A mutation that abolishes ATPase activity increases the apparent affinity for ATP approximately 10 folds. Under conditions of ATP hydrolysis, in the presence of Mg2+-ATP, the decrease in LRET indicates that there was an increase in the proportion of NBDs in the monomeric form. The results show: 1) The feasibility of LRET studies to determine dimerization of ABC protein NBDs, and 2) That ATP-induced dimerization is a transient phenomenon. This work was supported by CPRIT grant RP101073.

Dissection of the conformational cycle of the multidrug/lipidA ABC exporter MsbA

Recent crystal structures of the multidrug ATP-binding cassette (ABC) exporters Sav1866 from Staphylococcus aureus, MsbA from Escherichia coli, Vibrio cholera, and Salmonella typhimurium, and mouse ABCB1a suggest a common alternating access mechanism for export. However, the molecular framework underlying this mechanism is critically dependent on assumed conformational relationships between nonidentical crystal structures and therefore requires biochemical verification. The structures of homodimeric MsbA reveal a pair of glutamate residues (E208 and E208') in the intracellular domains of its two half-transporters, close to the nucleotide-binding domains (NBDs), which are in close proximity of each other in the outward-facing state but not in the inward-facing state. Using intermolecular cysteine crosslinking between E208C and E208C' in E. coli MsbA, we demonstrate that the NBDs dissociate in nucleotide-free conditions and come close on ATP binding and ADP·vanadate trapping. Interestingly, ADP alone separates the half-transporters like a nucleotide-free state, presumably for the following catalytic cycle. Our data fill persistent gaps in current studies on the conformational dynamics of a variety of ABC exporters. Based on a single biochemical method, the findings describe a conformational cycle for a single ABC exporter at major checkpoints of the ATPase reaction under experimental conditions, where the exporter is transport active.

ABC Transporters Involved in Export of Cell Surface Glycoconjugates

Complex glycoconjugates play critical roles in the biology of microorganisms. Despite the remarkable diversity in glycan structures and the bacteria that produce them, conserved themes are evident in the biosynthesis-export pathways. One of the primary pathways involves representatives of the ATP-binding cassette (ABC) transporter superfamily. These proteins are responsible for the export of a wide variety of cell surface oligo- and polysaccharides in both Gram-positive and Gram-negative bacteria. Recent investigations of the structure and function of ABC transporters involved in the export of lipopolysaccharide O antigens have revealed two fundamentally different strategies for coupling glycan polymerization to export. These mechanisms are distinguished by the presence (or absence) of characteristic nonreducing terminal modifications on the export substrates, which serve as chain termination and/or export signals, and by the presence (or absence) of a discrete substrate-binding domain in the nucleotide-binding domain polypeptide of the ABC transporter. A bioinformatic survey examining ABC exporters from known oligo- and polysaccharide biosynthesis loci identifies conserved nucleotide-binding domain protein families that correlate well with themes in the structures and assembly of glycans. The familial relationships among the ABC exporters generate hypotheses concerning the biosynthesis of structurally diverse oligo- and polysaccharides, which play important roles in the biology of bacteria with different lifestyles.