Substrate transport activation is mediated through second periplasmic loop of transmembrane protein MalF in maltose transport complex of Escherichia coli

In a recent study we described the second periplasmic loop P2 of the transmembrane protein MalF (MalF-P2) of the maltose ATP-binding cassette transporter (MalFGK(2)-E) as an important element in the recognition of substrate by the maltose-binding protein MalE. In this study, we focus on MalE and find that MalE undergoes a structural rearrangement after addition of MalF-P2. Analysis of residual dipolar couplings (RDCs) shows that binding of MalF-P2 induces a semiopen state of MalE in the presence and absence of maltose, whereas maltose is retained in the binding pocket. These data are in agreement with paramagnetic relaxation enhancement experiments. After addition of MalF-P2, an increased solvent accessibility for residues in the vicinity of the maltose-binding site of MalE is observed. MalF-P2 is thus not only responsible for substrate recognition, but also directly involved in activation of substrate transport. The observation that substrate-bound and substrate-free MalE in the presence of MalF-P2 adopts a similar semiopen state hints at the origin of the futile ATP hydrolysis of MalFGK(2)-E.     

Sequential Action of MalE and Maltose Allows Coupling ATP Hydrolysis to Translocation in the MalFGK2 Transporter

ATP-binding cassette (ABC) transporters have evolved an ATP-dependent alternating-access mechanism to transport substrates across membranes. Despite important progress, especially in their structural analysis, it is still unknown how the substrate stimulates ATP hydrolysis, the hallmark of ABC transporters. In this study, we measure the ATP turnover cycle of MalFGK₂ in steady and pre-steady state conditions. We show that (i) the basal ATPase activity of MalFGK₂ is very low because the cleavage of ATP is rate-limiting, (ii) the binding of open-state MalE to the transporter induces ATP cleavage but leaves release of P_i limiting, and (iii) the additional presence of maltose stimulates release of P_i, and therefore increases the overall ATP turnover cycle. We conclude that open-state MalE stabilizes MalFGK₂ in the outward-facing conformation until maltose triggers return to the inward-facing state for substrate and P_i release. This concerted action explains why ATPase activity of MalFGK₂ depends on maltose, and why MalE is essential for transport.     

The mechanism of nucleotide‐binding domain dimerization in the intact maltose transporter as studied by all‐atom molecular dynamics simulations

The Escherichia coli maltose transporter MalFGK₂‐E belongs to the protein superfamily of ATP‐binding cassette (ABC) transporters. This protein is composed of heterodimeric transmembrane domains (TMDs) MalF and MalG, and the homodimeric nucleotide‐binding domains (NBDs) MalK₂. In addition to the TMDs and NBDs, the periplasmic maltose binding protein MalE captures maltose and shuttle it to the transporter. In this study, we performed all‐atom molecular dynamics (MD) simulations on the maltose transporter and found that both the binding of MalE to the periplasmic side of the TMDs and binding of ATP to the MalK₂ are necessary to facilitate the conformational change from the inward‐facing state to the occluded state, in which MalK₂ is completely dimerized. MalE binding suppressed the fluctuation of the TMDs and MalF periplasmic region (MalF‐P2), and thus prevented the incorrect arrangement of the MalF C‐terminal (TM8) helix. Without MalE binding, the MalF TM8 helix showed a tendency to intrude into the substrate translocation pathway, hindering the closure of the MalK₂. This observation is consistent with previous mutagenesis experimental results on MalF and provides a new point of view regarding the understanding of the substrate translocation mechanism of the maltose transporter.     

Discovery of an auto-regulation mechanism for the maltose ABC transporter MalFGK2

The maltose transporter MalFGK(2), together with the substrate-binding protein MalE, is one of the best-characterized ABC transporters. In the conventional model, MalE captures maltose in the periplasm and delivers the sugar to the transporter. Here, using nanodiscs and proteoliposomes, we instead find that MalE is bound with high-affinity to MalFGK2 to facilitate the acquisition of the sugar. When the maltose concentration exceeds the transport capacity, MalE captures maltose and dissociates from the transporter. This mechanism explains why the transport rate is high when MalE has low affinity for maltose, and low when MalE has high affinity for maltose. Transporter-bound MalE facilitates the acquisition of the sugar at low concentrations, but also captures and dissociates from the transporter past a threshold maltose concentration. In vivo, this maltose-forced dissociation limits the rate of transport. Given the conservation of the substrate-binding proteins, this mode of allosteric regulation may be universal to ABC importers.     

A New Class of Cobalamin Transport Mutants (btuF) Provides Genetic Evidence for a Periplasmic Binding Protein in Salmonella typhimurium

No periplasmic binding protein has been demonstrated for the ATP-binding cassette (ABC)-type cobalamin transporter BtuCD. New mutations (btuF) are described that affect inner-membrane transport. The BtuF protein has a signal sequence and resembles the periplasmic binding proteins of several other ABC transporters.     

ABC transporter architecture and regulatory roles of accessory domains

We present an overview of the architecture of ATP-binding cassette (ABC) transporters and dissect the systems in core and accessory domains. The ABC transporter core is formed by the transmembrane domains (TMDs) and the nucleotide binding domains (NBDs) that constitute the actual translocator. The accessory domains include the substrate-binding proteins, that function as high affinity receptors in ABC type uptake systems, and regulatory or catalytic domains that can be fused to either the TMDs or NBDs. The regulatory domains add unique functions to the transporters allowing the systems to act as channel conductance regulators, osmosensors/regulators, and assemble into macromolecular complexes with specific properties.     

ABC transporters: one,two or four extracytoplasmic substrate-binding sites?

Two families of ATP-binding cassette (ABC) transporters in which one or two extracytoplasmic substrate-binding domains are fused to either the N- or C-terminus of the translocator protein have been detected. This suggests that two, or even four, substrate-binding sites may function in the ABC transporter complex. This domain organization in ABC transporters, widely represented among microorganisms, raises new possibilities for how the substrate-binding protein(s) (SBPs) might interact with the translocator. One appealing hypothesis is that multiple substrate-binding sites in proximity to the entry site of the translocation pore enhance the transport capacity. We also discuss the implications of multiple substrate-binding sites in close proximity to the translocator in terms of broadened substrate specificity and possible cooperative interactions between SBPs and the translocator.     

Two ATP-Binding Cassette Transporters Involved in (S)-2-Aminoethyl-Cysteine Uptake in Thermus thermophilus

Thermus thermophilus exhibits hypersensitivity to a lysine analog, (S)-2-aminoethyl-cysteine (AEC). Cosmid libraries were constructed using genomes from two AEC-resistant mutants, AT10 and AT14, and the cosmids that conferred AEC resistance on the wild-type strain were isolated. When the cosmid library for mutant AT14 was screened, two independent cosmids, conferring partial AEC resistance to the wild type, were obtained. Two cosmids carried a common genomic region from TTC0795 to TTC0810. This region contains genes encoding an ATP-binding cassette (ABC) transporter consisting of TTC0806/TTC0795, using TTC0807 as the periplasmic substrate-binding protein. Sequencing revealed that AT14 carries mutations in TTC0795 and TTC0969, causing decreases in the thermostability of the products. TTC0969 encodes the nucleotide-binding protein of a different ABC transporter consisting of TTC0967/TTC0968/TTC0969/TTC0970 using TTC0966 as the periplasmic substrate-binding protein. By similar screening for cosmids constructed for the mutant AT10, mutations were found at TTC0807 and TTC0969. Mutation in either of the transporter components gave partial resistance to AEC in the wild-type strain, while mutations of both transporters conferred complete AEC resistance. This result indicates that both transporters are involved in AEC uptake in T. thermophilus. To elucidate the mechanism of AEC uptake, crystal structures of TTC0807 were determined in several substrate-binding forms. The structures revealed that TTC0807 recognizes various basic amino acids by changing the side-chain conformation of Glu19, which interacts with the side-chain amino groups of the substrates.     

The MalF P2 Loop of the ATP-Binding Cassette Transporter MalFGK2 from Escherichia coli and Salmonella enterica Serovar Typhimurium Interacts with Maltose Binding Protein (MalE) throughout the Catalytic Cycle

We have investigated the interaction of the uncommonly large periplasmic P2 loop of the MalF subunit of the maltose ATP-binding cassette transporter (MalFGK₂) from Escherichia coli and Salmonella enterica serovar Typhimurium with maltose binding protein (MalE) by site-specific chemical cross-linking in the assembled transport complex. We focused on possible distance changes between two pairs of residues of the P2 loop and MalE during the transport cycle. The distance between MalF(S205C) and MalE(T80C) (∼5 Å) remained unchanged under all conditions tested. Cross-linking did not affect the ATPase activity of the complex. The distance between MalF(T177C) and MalE(T31C) changed from ∼10 Å to ∼5 Å upon binding of ATP (or maltose, with a less pronounced result) and was reset to ∼10 Å after hydrolysis of one ATP. A cross-link (∼25 Å) between MalF(S205C) and MalE(T31C) was observed only when the transporter resided in a transition state-like conformation, as was the case after vanadate trapping or in a binding protein-independent mutant, both of which are characterized by tight binding of unliganded MalE to the transporter. Thus, we propose that the observed cross-link is indicative of catalytic intermediates of the transporter. Together, our results strengthen the notion that the MalF P2 loop plays an important role in intersubunit communication. In particular, this loop is involved in keeping MalE in close contact with the transporter. The data are discussed with respect to a crystal structure and current transport models.ATP-binding cassette (ABC) transporters utilize the free energy of ATP hydrolysis to translocate substrates across biological membranes and can function as import or export systems (17). ABC transporters are generally composed of two hydrophobic, pore-forming transmembrane subunits and transmembrane domains (TMDs) and two hydrophilic nucleotide-binding (or ABC) subunits and nucleotide-binding domains (NBDs) that hydrolyze ATP (9). The crystal structures of isolated NBDs (6, 23, 34, 43) revealed that NBDs can be divided into a RecA-like subdomain comprising both the Walker A and the Walker B motifs, which are involved in nucleotide binding, and a helical subdomain harboring the unique LSGGQ motif (35). Furthermore, in the physiologically relevant NBD dimer, the nucleotide is complexed between the Walker A and B sites of one monomer and the LSGGQ motif of the opposing monomer. Both subdomains are joined by the “Q loop” containing a conserved glutamine residue that binds to the Mg²⁺ ion and attacking water and is likely to be involved in communicating ATP binding to the TMDs (10, 20, 29). ATP-dependent closing of the NBD dimer is thought to provide one possibility of the power stroke of ABC transporters (38).ABC importers that are confined to prokaryotes mediate the uptake of a large variety of solutes, including inorganic ions, amino acids, sugars, vitamins, oligopeptides, and polyamines (5). They require an additional protein, the extracytoplasmic solute binding protein (SBP), in order to capture the substrate and to deliver it to the cognate ABC transporter (37). SBPs typically consist of two lobes that are connected by a linker region. The interface between the two lobes forms the substrate binding site. Upon binding of the ligand, the proteins undergo a conformational change from an open toward a closed state (33) which, by interaction with extracytoplasmic peptide regions of TMDs of the cognate ABC transporter, initiates the transport process (31). The molecular events by which binding of ATP to the NBDs and interaction of liganded binding proteins with the TMDs are communicated to eventually trigger substrate translocation are still poorly understood.The maltose ABC transporter of Escherichia coli and Salmonella enterica serovar Typhimurium is one of the best-characterized transporters and thus serves as a model system for studying the mechanism by which ABC transporters exert their functions in general (15). The transporter is composed of the extracytoplasmic (periplasmic) maltose binding protein (MalE), the membrane-spanning subunits MalF and MalG, and two copies of the ATP-hydrolyzing subunit (MalK) (Fig. ​(Fig.1A1A).Open in a separate windowFIG. 1.(A) Structure of the catalytic intermediate of the maltose transporter [MalFGK(E159Q)₂-E]. The complex is shown in a ribbon diagram. White horizontal bars mark the boundaries of the membrane. Color code: yellow, MalE; cyan, MalF; red, MalG; green and magenta, MalK dimer. (B) Close-up view of the contact site between MalF P2 and the N-terminal lobe of MalE. The color code is the same as that for panel A. Residues from regions I and II that were replaced by cysteines are indicated in pink (MalF) and green (MalE). Residue MalE-K179, which was used as a control, is shown in green. The figure was drawn with DS ViewerPro 6.0 (Accelrys, Cambridge, United Kingdom), using the coordinates from entry 2R6G in the Brookhaven Protein Data Bank.Recently, suppressor mutational analysis provided a first hint that substrate availability is communicated from MalE to the MalK dimer via periplasmic loop regions of MalFG (11). Moreover, by site-directed cross-linking based on previous genetic evidence (19, 40), we demonstrated a close proximity of MalE G13 to Pro-78 in the first periplasmic loop (P1) of MalG, independently of cofactors such as maltose or ATP. Interaction of both residues was also observed in intact cells (11). These findings led us to propose that a copy of MalE is permanently associated with the transporter throughout the catalytic cycle. Furthermore, we have found that a region of the large, periplasmic P2 loop of MalF around Ser-205 (Fig. ​(Fig.1)1) is in cross-linking distance from MalE in the presence of maltose and MgATP only or when the transporter resides in the vanadate-trapped transition state. These results were perfectly confirmed by the subsequently published crystal structure of the MalFGK(E159Q)₂-E complex, which represents a transport intermediate (32). Here, the MalK dimer is complexed with two ATP molecules, and MalE is tightly associated with MalFG, but maltose has already been released into a binding pocket formed by MalF only. In particular, the N-terminal lobe of MalE is in close contact with the P2 loop of MalF (Fig. ​(Fig.1A1A).In this communication, we have taken advantage of this structural information to gain further insight into the MalF P2-MalE interaction during the transport cycle. We demonstrate ATP- and maltose-dependent distance changes between selected pairs of residues of the loop and MalE in the assembled complex by site-specific cross-linking. Our data demonstrate for the first time that the MalF P2 loop is in close contact to MalE throughout the catalytic cycle.     

ATP induces conformational changes of periplasmic loop regions of the maltose ATP-binding cassette transporter

We have studied cofactor-induced conformational changes of the maltose ATP-binding cassette transporter by employing limited proteolysis in detergent solution. The transport complex consists of one copy each of the transmembrane subunits, MalF and MalG, and of two copies of the nucleotide-binding subunit, MalK. Transport activity further requires the periplasmic maltose-binding protein, MalE. Binding of ATP to the MalK subunits increased the susceptibility of two tryptic cleavage sites in the periplasmic loops P2 of MalF and P1 of MalG, respectively. Lys(262) of MalF and Arg(73) of MalG were identified as probable cleavage sites, resulting in two N-terminal peptide fragments of 29 and 8 kDa, respectively. Trapping the complex in the transition state by vanadate further stabilized the fragments. In contrast, the tryptic cleavage profile of MalK remained largely unchanged. ATP-induced conformational changes of MalF-P2 and MalG-P1 were supported by fluorescence spectroscopy of complex variants labeled with 2-(4'-maleimidoanilino)naphthalene-6-sulfonic acid. Limited proteolysis was subsequently used as a tool to study the consequences of mutations on the transport cycle. The results suggest that complex variants exhibiting a binding protein-independent phenotype (MalF500) or containing a mutation that affects the "catalytic carboxylate" (MalKE159Q) reside in a transition state-like conformation. A similar conclusion was drawn for a complex containing a replacement of MalKQ140 in the signature sequence by leucine, whereas substitution of lysine for Gln(140) appears to lock the transport complex in the ground state. Together, our data provide the first evidence for conformational changes of the transmembrane subunits of an ATP-binding cassette import system upon binding of ATP.     

In Vitro Reassembly of the Ribose ATP-binding Cassette Transporter Reveals a Distinct Set of Transport Complexes

Bacterial ATP-binding cassette (ABC) importers are primary active transporters that are critical for nutrient uptake. Based on structural and functional studies, ABC importers can be divided into two distinct classes, type I and type II. Type I importers follow a strict alternating access mechanism that is driven by the presence of the substrate. Type II importers accept substrates in a nucleotide-free state, with hydrolysis driving an inward facing conformation. The ribose transporter in Escherichia coli is a tripartite complex consisting of a cytoplasmic ATP-binding cassette protein, RbsA, with fused nucleotide binding domains; a transmembrane domain homodimer, RbsC₂; and a periplasmic substrate binding protein, RbsB. To investigate the transport mechanism of the complex RbsABC₂, we probed intersubunit interactions by varying the presence of the substrate ribose and the hydrolysis cofactors, ATP/ADP and Mg²⁺. We were able to purify a full complex, RbsABC₂, in the presence of stable, transition state mimics (ATP, Mg²⁺, and VO₄); a RbsAC complex in the presence of ADP and Mg²⁺; and a heretofore unobserved RbsBC complex in the absence of cofactors. The presence of excess ribose also destabilized complex formation between RbsB and RbsC. These observations suggest that RbsABC₂ shares functional traits with both type I and type II importers, as well as possessing unique features, and employs a distinct mechanism relative to other ABC transporters.     

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.     

Receptor-transporter interactions of canonical ATP-binding cassette import systems in prokaryotes

ATP-binding cassette (ABC) transport systems mediate the translocation of solutes across biological membranes at the expense of ATP. They share a common modular architecture comprising two pore-forming transmembrane domains and two nucleotide binding domains. In prokaryotes, ABC transporters are involved in the uptake of a large variety of chemicals, including nutrients, osmoprotectants and signal molecules. In pathogenic bacteria, some ABC importers are virulence factors. Canonical ABC import systems require an additional component, a substrate-specific receptor or binding protein for function. Interaction of the liganded receptor with extracytoplasmic loop regions of the transmembrane domains initiate the transport cycle. In this review we summarize the current knowledge on receptor-transporter interplay provided by crystal structures as well as by biochemical and biophysical means. In particular, we focus on the maltose/maltodextrin transporter of enterobacteria and the transporters for positively charged amino acids from the thermophile Geobacillus stearothermophilus and Salmonella enterica serovar Typhimurium.     

EPR Spectroscopy of MolB2C2-A Reveals Mechanism of Transport for a Bacterial Type II Molybdate Importer

In bacteria, ATP-binding cassette (ABC) transporters are vital for the uptake of nutrients and cofactors. Based on differences in structure and activity, ABC importers are divided into two types. Type I transporters have been well studied and employ a tightly regulated alternating access mechanism. Less is known about Type II importers, but much of what we do know has been observed in studies of the vitamin B₁₂ importer BtuC₂D₂. MolB₂C₂ (formally known as HI1470/71) is also a Type II importer, but its substrate, molybdate, is ∼10-fold smaller than vitamin B₁₂. To understand mechanistic differences among Type II importers, we focused our studies on MolBC, for which alternative conformations may be required to transport its relatively small substrate. To investigate the mechanism of MolBC, we employed disulfide cross-linking and EPR spectroscopy. From these studies, we found that nucleotide binding is coupled to a conformational shift at the periplasmic gate. Unlike the larger conformational changes in BtuCD-F, this shift in MolBC-A is akin to unlocking a swinging door: allowing just enough space for molybdate to slip into the cell. The lower cytoplasmic gate, identified in BtuCD-F as “gate I,” remains open throughout the MolBC-A mechanism, and cytoplasmic gate II closes in the presence of nucleotide. Combining our results, we propose a peristaltic mechanism for MolBC-A, which gives new insight in the transport of small substrates by a Type II importer.     

On the role of the two extracytoplasmic substrate-binding domains in the ABC transporter OpuA

Members of two transporter families of the ATP-binding cassette (ABC) superfamily use two or even four extracytoplasmic substrate-binding domains (SBDs) for transport. We report on the role of the two SBDs in the translocation cycle of the ABC transporter OpuA from Lactococcus lactis. Heterooligomeric OpuA complexes with only one SBD or one functional and one non-functional SBD (inactivated by covalent linkage of a substrate mimic) have been constructed, and the substrate binding and transport kinetics of the purified transporters, reconstituted in liposomes, have been determined. The data indicate that the two SBDs of OpuA interact in a cooperative manner in the translocation process by stimulating either the docking of the SBDs onto the translocator or the delivery of glycine betaine to the translocator. It appears that one of these initial steps, but not the later steps in translocation or resetting of the system to the initial state, is rate determining for transport. These new insights on the functional role of the extracytoplasmic SBDs are discussed in the light of the current knowledge of substrate-binding-protein-dependent ABC transporters.     

Maltose binding protein (MalE) interacts with periplasmic loops P2 and P1 respectively of the MalFG subunits of the maltose ATP binding cassette transporter (MalFGK(2)) from Escherichia coli/Salmonella during the transport cycle

The ATP binding cassette (ABC-) transporter mediating the uptake of maltose/maltodextrins in Escherichia coli/Salmonella enterica serovar Typhimurium is one of the best characterized systems and serves as a model for studying the molecular mechanism by which ABC importers exert their functions. The transporter is composed of a periplasmic maltose binding protein (MalE), and a membrane-bound complex (MalFGK(2)), comprising the pore-forming hydrophobic subunits, MalF and MalG, and two copies of the ABC subunit, MalK. We report on the isolation of suppressor mutations within malFG that partially restore transport of a maltose-negative mutant carrying the malK809 allele (MalKQ140K). The mutation affects the conserved LSGGQ motif that is involved in ATP binding. Three out of four suppressor mutations map in periplasmic loops P2 and P1 respectively of MalFG. Cross-linking data revealed proximity of these regions to MalE. In particular, as demonstrated in vitro and in vivo, Gly-13 of substrate-free and substrate-loaded MalE is in close contact to Pro-78 of MalG. These data suggest that MalE is permanently in close contact to MalG-P1 via its N-terminal domain. Together, our results are interpreted in favour of the notion that substrate availability is communicated from MalE to the MalK dimer via extracytoplasmic loops of MalFG, and are discussed with respect to a current transport model.     

TGD1, -2, and -3 proteins involved in lipid trafficking form ATP-binding cassette (ABC) transporter with multiple substrate-binding proteins

Members of the ATP-binding cassette (ABC) transporter family are essential proteins in species as diverse as archaea and humans. Their domain architecture has remained relatively fixed across these species, with rare exceptions. Here, we show one exception to be the trigalactosyldiacylglycerol 1, 2, and 3 (TGD1, -2, and -3) putative lipid transporter located at the chloroplast inner envelope membrane. TGD2 was previously shown to be in a complex of >500 kDa. We demonstrate that this complex also contains TGD1 and -3 and is very stable because it cannot be broken down by gentle denaturants to form a "core" complex similar in size to standard ABC transporters. The complex was purified from Pisum sativum (pea) chloroplast envelopes by native gel electrophoresis and examined by mass spectrometry. Identified proteins besides TGD1, -2, or -3 included a potassium efflux antiporter and a TIM17/22/23 family protein, but these were shown to be in separate high molecular mass complexes. Quantification of the complex components explained the size of the complex because 8-12 copies of the substrate-binding protein (TGD2) were found per functional transporter.     

Insight in eukaryotic ABC transporter function by mutation analysis

With regard to structure-function relations of ATP-binding cassette (ABC) transporters several intriguing questions are in the spotlight of active research: Why do functional ABC transporters possess two ATP binding and hydrolysis domains together with two ABC signatures and to what extent are the individual nucleotide-binding domains independent or interacting? Where is the substrate-binding site and how is ATP hydrolysis functionally coupled to the transport process itself? Although much progress has been made in the elucidation of the three-dimensional structures of ABC transporters in the last years by several crystallographic studies including novel models for the nucleotide hydrolysis and translocation catalysis, site-directed mutagenesis as well as the identification of natural mutations is still a major tool to evaluate effects of individual amino acids on the overall function of ABC transporters. Apart from alterations in characteristic sequence such as Walker A, Walker B and the ABC signature other parts of ABC proteins were subject to detailed mutagenesis studies including the substrate-binding site or the regulatory domain of CFTR. In this review, we will give a detailed overview of the mutation analysis reported for selected ABC transporters of the ABCB and ABCC subfamilies, namely HsCFTR/ABCC7, HsSUR/ABCC8,9, HsMRP1/ABCC1, HsMRP2/ABCC2, ScYCF1 and P-glycoprotein (Pgp)/MDR1/ABCB1 and their effects on the function of each protein.     

The conformational coupling and translocation mechanism of vitamin B12 ATP-binding cassette transporter BtuCD

ATP-binding cassette transporter BtuCD mediating vitamin B₁₂ uptake in Escherichia coli couples the energy of ATP hydrolysis to the translocation of vitamin B₁₂ across the membrane into the cell. Elastic normal mode analysis of BtuCD demonstrates that the simultaneous substrate trapping at periplasmic cavity and ATP binding at the ATP-binding cassette (BtuD) dimer proceeds readily along the lowest energy pathway. The transport power stroke is attributed to ATP-hydrolysis-induced opening of the nucleotide-binding domain dimer, which is coupled to conformational rearrangement of transmembrane domain (BtuC) helices leading to the closing at the periplasmic side and opening at the cytoplasmic gate. Simultaneous hydrolysis of two ATP is supported by the fact that antisymmetric movement of BtuD dimer implying alternating hydrolysis cannot induce effective conformational change of the translocation pathway. A plausible mechanism of translocation cycle is proposed in which the possible effect of the association of periplasmic binding protein BtuF to the transporter is also considered.     

Powering the peptide pump: TAP crosstalk with energetic nucleotides

ATP-binding cassette (ABC) transporters represent a large family of membrane-spanning proteins that have a shared structural organization and conserved nucleotide-binding domains (NBDs). They transport a large variety of solutes, and defects in these transporters are an important cause of human disease. TAP (tmacr;ransporter associated with āntigen pmacr;rocessing) is a heterodimeric ABC transporter that uses nucleotides to drive peptide transport from the cytoplasm into the endoplasmic reticulum lumen, where the peptides then bind major histocompatibility complex (MHC) class I molecules. TAP plays an essential role in the MHC class I antigen presentation pathway. Recent studies show that the two NBDs of TAP fulfil distinct functions in the catalytic cycle of this transporter. In this opinion article, a model of alternating ATP binding and hydrolysis is proposed, in which nucleotide interaction with TAP2 primarily controls substrate binding and release, whereas interaction with TAP1 controls structural rearrangements of the transmembrane pathway. Viral proteins that inhibit TAP function cause arrests at distinct points of this catalytic cycle.