Dynamics of alpha-helical subdomain rotation in the intact maltose ATP-binding cassette transporter

ATP-binding cassette (ABC) transporters are powered by a nucleotide-binding domain dimer that opens and closes during cycles of ATP hydrolysis. These domains consist of a RecA-like subdomain and an α-helical subdomain that is specific to the family. Many studies on isolated domains suggest that the helical subdomain rotates toward the RecA-like subdomain in response to ATP binding, moving the family signature motif into a favorable position to interact with the nucleotide across the dimer interface. Moreover, the transmembrane domains are docked into a cleft at the interface between these subdomains, suggesting a putative role of the rotation in interdomain communication. Electron paramagnetic resonance spectroscopy was used to study the dynamics of this rotation in the intact Escherichia coli maltose transporter MalFGK(2). This importer requires a periplasmic maltose-binding protein (MBP) that activates ATP hydrolysis by promoting the closure of the cassette dimer (MalK(2)). Whereas this rotation occurred during the transport cycle, it required not only trinucleotide, but also MBP, suggesting it is part of a global conformational change in the transporter. Interaction of AMP-PNP-Mg(2+) and a MBP that is locked in a closed conformation induced a transition from open MalK(2) to semiopen MalK(2) without significant subdomain rotation. Inward rotation of the helical subdomain and complete closure of MalK(2) therefore appear to be coupled to the reorientation of transmembrane helices and the opening of MBP, events that promote transfer of maltose into the transporter. After ATP hydrolysis, the helical subdomain rotates out as MalK(2) opens, resetting the transporter in an inward-facing conformation.     

Vanadate-catalyzed photocleavage of the signature motif of an ATP-binding cassette (ABC) transporter

The maltose transport complex of Escherichia coli, a member of the ATP-binding cassette (ABC) superfamily, is made up of two nucleotide-binding subunits, MalK(2), which hydrolyze ATP with positive cooperativity, and two transmembrane subunits, MalF and MalG. The ABC family is defined in part by the canonical signature motif LSGGQ whose exact function remains controversial. Taking advantage of the dual function of vanadate as a transition state analogue and as a photoactive chemical, we demonstrate that vanadate catalyzes the UV-dependent cleavage of the polypeptide backbone at both the LSGGQ motif and the nucleotide-binding, or Walker A, motif when it is trapped in the nucleotide-binding site of the bacterial maltose transporter. This highly specific cleavage pattern indicates that residues in both motifs are immediately adjacent to ATP during hydrolysis, and are therefore likely to participate directly in ATP-binding and/or hydrolysis. Because the LSGGQ motif is too distant from the nucleotide in the structure of an ABC monomer for cleavage to occur, these data support a model in which the LSGGQ motif contacts the nucleotide across the interface of a MalK dimer, as seen in the crystal structure of Rad50. This architecture provides a basis for the cooperativity observed in the nucleotide-binding domains of ABC transporters and a function for this highly conserved family signature motif.     

Snapshots of the maltose transporter during ATP hydrolysis

ATP-binding cassette transporters are powered by ATP, but the mechanism by which these transporters hydrolyze ATP is unclear. In this study, four crystal structures of the full-length wild-type maltose transporter, stabilized by adenosine 5′-(β,γ-imido)triphosphate or ADP in conjunction with phosphate analogs , , or , were determined to 2.2- to 2.4-Å resolution. These structures led to the assignment of two enzymatic states during ATP hydrolysis and demonstrate specific functional roles of highly conserved residues in the nucleotide-binding domain, suggesting that ATP-binding cassette transporters catalyze ATP hydrolysis via a general base mechanism.     

An ATP-binding cassette transporter-like complex governs cell-wall hydrolysis at the bacterial cytokinetic ring

ATP-binding cassette transporters are ubiquitous membrane protein complexes that move substrates across membranes. They do so using ATP-induced conformational changes in their nucleotide-binding domains to alter the conformation of the transport cavity formed by their transmembrane domains. In Escherichia coli, an ATP-binding cassette transporter-like complex composed of FtsE (nucleotide-binding domain) and FtsX (transmembrane domain) has long been known to be important for cytokinesis, but its role in the process has remained mysterious. Here we identify FtsEX as a regulator of cell-wall hydrolysis at the division site. Cell-wall material synthesized by the division machinery is shared initially by daughter cells and must be split by hydrolytic enzymes called "amidases" to drive daughter-cell separation. We recently showed that the amidases require activation at the cytokinetic ring by proteins with LytM domains, of which EnvC is the most critical. In this report, we demonstrate that FtsEX directly recruits EnvC to the septum via an interaction between EnvC and a periplasmic loop of FtsX. Importantly, we also show that FtsEX variants predicted to be ATPase defective still recruit EnvC to the septum but fail to promote cell separation. Our results thus suggest that amidase activation via EnvC in the periplasm is regulated by conformational changes in the FtsEX complex mediated by ATP hydrolysis in the cytoplasm. Since FtsE has been reported to interact with the tubulin-like FtsZ protein, our model provides a potential mechanism for coupling amidase activity with the contraction of the FtsZ cytoskeletal ring.     

Mechanism of ABC transporters: a molecular dynamics simulation of a well characterized nucleotide-binding subunit

ATP-binding cassette (ABC) transporters are membrane-bound molecular pumps that form one of the largest of all protein families. Several of them are central to phenomena of biomedical interest, including cystic fibrosis and resistance to chemotherapeutic drugs. ABC transporters share a common architecture comprising two hydrophilic nucleotide-binding domains (NBDs) and two hydrophobic transmembrane domains (TMDs) that form the substrate pathway across the membrane. The conformational changes in the NBDs induced by ATP hydrolysis and the means by which they are transmitted to the TMDs to effect substrate translocation remain largely unknown. We have performed a molecular dynamics simulation of HisP, the well studied NBD of the bacterial histidine permease, to identify hinges and switches of the NBD conformational transitions and subunit-subunit interfaces. This analysis reveals that the TMDs regulate ATP hydrolysis by controlling conformational transitions of the NBD helical domains, and identifies the conformational changes and the crucial TMD:NBD interface, by which the energy of ATP hydrolysis is transmitted to the TMDs. We also define the conformational transitions of the Q-loop, a key element of the NBD mechanism, and identify pathways by which Q-loop switching is coordinated with TMD and NBD conformational changes. We propose a model for the catalytic cycle of ABC transporters that shows how substrate-binding and transport by the TMDs may be coordinated and coupled with ATP binding and hydrolysis in the NBDs.     

Structures of P-glycoprotein reveal its conformational flexibility and an epitope on the nucleotide-binding domain

P-glycoprotein (P-gp) is one of the best-known mediators of drug efflux-based multidrug resistance in many cancers. This validated therapeutic target is a prototypic, plasma membrane resident ATP-Binding Cassette transporter that pumps xenobiotic compounds out of cells. The large, polyspecific drug-binding pocket of P-gp recognizes a variety of structurally unrelated compounds. The transport of these drugs across the membrane is coincident with changes in the size and shape of this pocket during the course of the transport cycle. Here, we present the crystal structures of three inward-facing conformations of mouse P-gp derived from two different crystal forms. One structure has a nanobody bound to the C-terminal side of the first nucleotide-binding domain. This nanobody strongly inhibits the ATP hydrolysis activity of mouse P-gp by hindering the formation of a dimeric complex between the ATP-binding domains, which is essential for nucleotide hydrolysis. Together, these inward-facing conformational snapshots of P-gp demonstrate a range of flexibility exhibited by this transporter, which is likely an essential feature for the binding and transport of large, diverse substrates. The nanobody-bound structure also reveals a unique epitope on P-gp.     

Trapping the transition state of an ATP-binding cassette transporter: evidence for a concerted mechanism of maltose transport

High-affinity uptake into bacterial cells is mediated by a large class of periplasmic binding protein-dependent transport systems, members of the ATP-binding cassette superfamily. In the maltose transport system of Escherichia coli, the periplasmic maltose-binding protein binds its substrate maltose with high affinity and, in addition, stimulates the ATPase activity of the membrane-associated transporter when maltose is present. Vanadate inhibits maltose transport by trapping ADP in one of the two nucleotide-binding sites of the membrane transporter immediately after ATP hydrolysis, consistent with its ability to mimic the transition state of the gamma-phosphate of ATP during hydrolysis. Here we report that the maltose-binding protein becomes tightly associated with the membrane transporter in the presence of vanadate and simultaneously loses its high affinity for maltose. These results suggest a general model explaining how ATP hydrolysis is coupled to substrate transport in which a binding protein stimulates the ATPase activity of its cognate transporter by stabilizing the transition state.     

Both maltose-binding protein and ATP are required for nucleotide-binding domain closure in the intact maltose ABC transporter

The maltose transporter MalFGK₂ of Escherichia coli is a member of the ATP-binding cassette superfamily. A periplasmic maltose-binding protein (MBP) delivers maltose to MalFGK₂ and stimulates its ATPase activity. Site-directed spin labeling EPR spectroscopy was used to study the opening and closing of the nucleotide-binding interface of MalFGK₂ during the catalytic cycle. In the intact transporter, closure of the interface coincides not just with the binding of ATP, as seen with isolated nucleotide-binding domains, but requires both MBP and ATP, implying that MBP stimulates ATPase activity by promoting the closure of the nucleotide-binding interface. After ATP hydrolysis, with MgADP and MBP bound, the nucleotide-binding interface resides in a semi-open configuration distinct from the fully open configuration seen in the absence of any ligand. We propose that P_i release coincides with the reorientation of transmembrane helices to an inward-facing conformation and the final step of maltose translocation into the cell.     

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.     

Flexibility in the ABC transporter MsbA: Alternating access with a twist

ATP-binding cassette (ABC) transporters are integral membrane proteins that translocate a wide variety of substrates across cellular membranes and are conserved from bacteria to humans. Here we compare four x-ray structures of the bacterial ABC lipid flippase, MsbA, trapped in different conformations, two nucleotide-bound structures and two in the absence of nucleotide. Comparison of the nucleotide-free conformations of MsbA reveals a flexible hinge formed by extracellular loops 2 and 3. This hinge allows the nucleotide-binding domains to disassociate while the ATP-binding half sites remain facing each other. The binding of the nucleotide causes a packing rearrangement of the transmembrane helices and changes the accessibility of the transporter from cytoplasmic (inward) facing to extracellular (outward) facing. The inward and outward openings are mediated by two different sets of transmembrane helix interactions. Altogether, the conformational changes between these structures suggest that large ranges of motion may be required for substrate transport.     

Evolutionary and functional divergence between the cystic fibrosis transmembrane conductance regulator and related ATP-binding cassette transporters

The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily, an ancient family of proteins found in all phyla. In nearly all cases, ABC proteins are transporters that couple the hydrolysis of ATP to the transmembrane movement of substrate via an alternating access mechanism. In contrast, CFTR is best known for its activity as an ATP-dependent chloride channel. We asked why CFTR, which shares the domain architecture of ABC proteins that function as transporters, exhibits functional divergence. We compared CFTR protein sequences to those of other ABC transporters, which identified the ABCC4 proteins as the closest mammalian paralogs, and used statistical analysis of the CFTR-ABCC4 multiple sequence alignment to identify the specific domains and residues most likely to be involved in the evolutionary transition from transporter to channel activity. Among the residues identified as being involved in CFTR functional divergence, by virtue of being both CFTR-specific and conserved among all CFTR orthologs, was R352 in the sixth transmembrane helix (TM6). Patch-clamp experiments show that R352 interacts with D993 in TM9 to stabilize the open-channel state; D993 is absolutely conserved between CFTRs and ABCC4s. These data suggest that CFTR channel activity evolved, at least in part, by converting the conformational changes associated with binding and hydrolysis of ATP, as are found in true ABC Transporters, into an open permeation pathway by means of intraprotein interactions that stabilize the open state. This analysis sets the stage for understanding the evolutionary and functional relationships that make CFTR a unique ABC transporter protein.     

A conditional probability analysis of cystic fibrosis transmembrane conductance regulator gating indicates that ATP has multiple effects during the gating cycle

ATP-binding cassette (ABC) transporters bind and hydrolyze ATP. In the cystic fibrosis transmembrane conductance regulator Cl(-) channel, this interaction with ATP generates a gating cycle between a closed (C) and two open (O1 and O2) conformations. To understand better how ATP controls channel activity, we examined gating transitions from the C to the O1 and O2 states and from these open states to the C conformation. We made three main observations. First, we found that the channel can open into either the O1 or O2 state, that the frequency of transitions to both states was increased by ATP concentration, and that ATP increased the relative proportion of openings into O1 vs. O2. These results indicate that ATP can interact with the closed state to open the channel in at least two ways, which may involve binding to nucleotide-binding domains (NBDs) NBD1 and NBD2. Second, ATP prolonged the burst duration and altered the way in which the channel closed. These data suggest that ATP also interacts with the open channel. Third, the channel showed runs of specific types of open-closed transitions. This finding suggests a mechanism with more than one cycle of gating transitions. These data suggest models to explain how ATP influences conformational transitions in cystic fibrosis transmembrane conductance regulator and perhaps other ABC transporters.     

Crystal structure of the Holliday junction migration motor protein RuvB from Thermus thermophilus HB8

We report here the crystal structure of the RuvB motor protein from Thermus thermophilus HB8, which drives branch migration of the Holliday junction during homologous recombination. RuvB has a crescent-like architecture consisting of three consecutive domains, the first two of which are involved in ATP binding and hydrolysis. DNA is likely to interact with a large basic cleft, which encompasses the ATP-binding pocket and domain boundaries, whereas the junction-recognition protein RuvA may bind a flexible beta-hairpin protruding from the N-terminal domain. The structures of two subunits, related by a noncrystallographic pseudo-2-fold axis, imply that conformational changes of motor protein coupled with ATP hydrolysis may reflect motility essential for its translocation around double-stranded DNA.     

Analysis of the AAA sensor-2 motif in the C-terminal ATPase domain of Hsp104 with a site-specific fluorescent probe of nucleotide binding

Hsp104 from Saccharomyces cerevisiae is a hexameric protein with two AAA ATPase domains (N- and C-terminal nucleotide-binding domains NBD1 and NBD2, respectively) per monomer. Our previous analysis of the Hsp104 ATP hydrolysis cycle revealed that NBD1 and NBD2 have very different catalytic properties, but each shows positive cooperativity in hydrolysis. There is also communication between the two domains, in that ATP hydrolysis at NBD1 depends on the nucleotide that is bound to NBD2. Here, we extend our understanding of the Hsp104 ATP hydrolysis cycle through mutagenesis of the AAA sensor-2 motif in NBD2. To do so, we took advantage of the lack of tryptophan residues in Hsp104 to place a single tryptophan in the C-terminal domain (Y819W). The Y819W substitution has no significant effects on folding stability of the C-terminal domain or on ATP hydrolysis by NBD1 or NBD2. The fluorescence of this tryptophan changes in response to ATP and ADP binding, allowing the K(d) and Hill coefficient to be determined for each nucleotide. By using this site-specific probe of binding, we analyze the effect of mutating the conserved arginine residue in the sensor-2 motif in Hsp104 NBD2. An R826M mutation causes nearly equal decreases in affinity of NBD2 for both ATP and ADP, indicating that at this site, the sensor-2 provides binding energy, but does not act to sense the difference between these nucleotides. In addition, the rate of ATP hydrolysis at NBD1 is decreased by the R826M mutation, providing further evidence for interdomain communication in the Hsp104 ATP hydrolysis cycle.     

Crystal structures of actin-related protein 2/3 complex with bound ATP or ADP

Actin-related protein (Arp) 2/3 complex stimulates formation of actin filaments at the leading edge of motile cells. Nucleation of filaments depends on hydrolysis of ATP bound to Arp2. Here we report crystal structures of Arp2/3 complex with bound ATP or ADP. The nucleotides are immobilized on the face of subdomains 3 and 4 of Arp2, whereas subdomains 1 and 2 are flexible and absent from the electron density maps. This flexibility may explain why Arp2 does not hydrolyze ATP until the complex is activated. ATP stabilizes a relatively closed conformation of Arp3 with the gamma-phosphate bridging loops from opposite sides of the cleft. ADP binds Arp3 in a unique conformation that favors an open cleft, revealing a conformational change that may occur in actin and Arps when ATP is hydrolyzed and phosphate dissociates. These structures provide the an opportunity to compare all nucleotide-binding states in an actin-related protein and give insights into the function of both the Arp2/3 complex and actin.     

Mechanism of maltose transport in Escherichia coli: transmembrane signaling by periplasmic binding proteins.

Maltose transport across the cytoplasmic membrane of Escherichia coli is dependent on the presence of a periplasmic maltose-binding protein (MBP), the product of the malE gene. The products of the malF, malG, and malK genes form a membrane-associated complex that catalyzes the hydrolysis of ATP to provide energy for the transport event. Previously, mutants were isolated that had gained the ability to grow on maltose in the absence of MBP. After reconstitution of the transport complex into proteoliposomes, measurement of the ATPase activity of wild-type and mutant complexes in the presence and absence of MBP revealed that the wild-type complex hydrolyzed ATP rapidly only when MBP and maltose were both present. In contrast, the mutant complexes have gained the ability to hydrolyze ATP in the absence of maltose and MBP. The basal rate of hydrolysis by the different mutant complexes was directly proportional to the growth rate of that strain on maltose, a result indicating that the constitutive ATP hydrolysis and presumably the resultant cyclic conformational changes of the complex produce maltose transport in the absence of MBP. These results also suggest that ATP hydrolysis is not directly coupled to ligand transport even in wild-type cells and that one important function of MBP is to transmit a transmembrane signal, through the membrane-spanning MalF and MalG proteins, to the MalK protein on the other side of the membrane, so that ATP hydrolysis can occur.     

Structure of adeno-associated virus type 2 Rep40-ADP complex: insight into nucleotide recognition and catalysis by superfamily 3 helicases

We have determined the structure of adeno-associated virus type 2 (AAV2) Rep40 to 2.1-A resolution with ADP bound at the active site. The complex crystallizes as a monomer with one ADP molecule positioned in an unexpectedly open binding site. The nucleotide-binding pocket consists of the P-loop residues interacting with the phosphates and a loop (nucleoside-binding loop) that emanates from the last strand of the central beta-sheet and interacts with the sugar and base. As a result of the open nature of the binding site, one face of the adenine ring is completely exposed to the solvent, and consequently the number of protein-nucleotide contacts is scarce as compared with other P-loop nucleotide phosphohydrolases. The conformation of the ADP molecule in its binding site bears a resemblance to those found in only three other families of P-loop ATPases: the ATP-binding cassette transporter family, the bacterial RecA proteins, and the type II topoisomerase family. In all these cases, oligomerization is required to attain a competent nucleotide-binding pocket. We propose that this characteristic is native to superfamily 3 helicases and allows for an additional mechanism of regulation by these multifunctional proteins. Furthermore, it explains the strong tendency by members of this family such as simian virus 40 TAg to oligomerize after binding ATP.     

Distinct functions and cooperative interaction of the subunits of the transporter associated with antigen processing (TAP)

The ATP-binding cassette (ABC) transporter TAP translocates peptides from the cytosol to awaiting MHC class I molecules in the endoplasmic reticulum. TAP is made up of the TAP1 and TAP2 polypeptides, which each possess a nucleotide binding domain (NBD). However, the role of ATP in peptide binding and translocation is poorly understood. We present biochemical and functional evidence that the NBDs of TAP1 and TAP2 are non-equivalent. Photolabeling experiments with 8-azido-ATP demonstrate a cooperative interaction between the two NBDs that can be stimulated by peptide. The substitution of key lysine residues in the Walker A motifs of TAP1 and TAP2 suggests that TAP1-mediated ATP hydrolysis is not essential for peptide translocation but that TAP2-mediated ATP hydrolysis is critical, not only for translocation, but for peptide binding.     

Cooperative binding of ATP and RNA induces a closed conformation in a DEAD box RNA helicase

RNA helicases couple the energy from ATP hydrolysis with structural changes of their RNA substrates. DEAD box helicases form the largest class of RNA helicases and share a helicase core comprising two RecA-like domains. An opening and closing of the interdomain cleft during RNA unwinding has been postulated but not shown experimentally. Single-molecule FRET experiments with the Bacillus subtilis DEAD box helicase YxiN carrying donor and acceptor fluorophores on different sides of the interdomain cleft reveal an open helicase conformation in the absence of nucleotides, or in the presence of ATP, or ADP, or RNA. In the presence of ADP and RNA, the open conformation is retained. By contrast, cooperative binding of ATP and RNA leads to a compact helicase structure, proving that the ATP- and ADP-bound states of RNA helicases display substantially different structures only when the RNA substrate is bound. These results establish a closure of the interdomain cleft in the helicase core at the beginning of the unwinding reaction, and suggest a conserved mechanism of energy conversion among DEAD box helicases across kingdoms.     

Structural basis for substrate specificity in the Escherichia coli maltose transport system

ATP-binding cassette (ABC) transporters are molecular pumps that harness the chemical energy of ATP hydrolysis to translocate solutes across the membrane. The substrates transported by different ABC transporters are diverse, ranging from small ions to large proteins. Although crystal structures of several ABC transporters are available, a structural basis for substrate recognition is still lacking. For the Escherichia coli maltose transport system, the selectivity of sugar binding to maltose-binding protein (MBP), the periplasmic binding protein, does not fully account for the selectivity of sugar transport. To obtain a molecular understanding of this observation, we determined the crystal structures of the transporter complex MBP-MalFGK₂ bound with large malto-oligosaccharide in two different conformational states. In the pretranslocation structure, we found that the transmembrane subunit MalG forms two hydrogen bonds with malto-oligosaccharide at the reducing end. In the outward-facing conformation, the transmembrane subunit MalF binds three glucosyl units from the nonreducing end of the sugar. These structural features explain why modified malto-oligosaccharides are not transported by MalFGK₂ despite their high binding affinity to MBP. They also show that in the transport cycle, substrate is channeled from MBP into the transmembrane pathway with a polarity such that both MBP and MalFGK₂ contribute to the overall substrate selectivity of the system.The ATP-binding cassette (ABC) transporter family contains more than 2,000 members sharing a common architecture of two transmembrane domains (TMDs) that form the translocation pathway and two cytoplasmic nucleotide-binding domains (NBDs) that hydrolyze ATP (1). Importers found in prokaryotes require additional soluble proteins that bind substrates with high affinity and deliver them to the TMDs. Some ABC transporters recognize only a single substrate, whereas others are more promiscuous. For example, ABC transporters that secrete toxins, hydrolytic enzymes, and antibiotic peptides are dedicated to one specific substrate (2), but in contrast, the multidrug transporter P-glycoprotein interacts with more than 200 chemically diverse compounds (3). MRP1, ABCG2, and TAP also have broad substrate spectra (2).Regardless of substrate specificity, the ATPase activity of ABC transporters is regulated by the presence of substrates. Thus, substrate binding must generate a signal that enables ATP hydrolysis. Understanding how ABC transporters interact with their substrates has been a major challenge in the field.A controversial issue in the ABC transporter field is whether the transmembrane components contain a well-defined substrate-binding site. It has been suggested that for binding protein-dependent ABC transporters, substrate specificity is defined exclusively by the binding protein, which interacts with the substrate with high affinity. The transmembrane components act as a nonspecific pore for substrate to diffuse through the membrane (4). However, for the Escherichia coli maltose transporter, it has been well established that the selectivity of sugar binding to the maltose-binding protein (MBP) does not fully account for the selectivity of sugar transport. For example, cyclic maltodextrins, maltodextrins containing more than seven glucosyl units, and maltose analogs with a modified reducing end are not transported despite their high-affinity binding to MBP (5, 6). Further evidence for selectivity through the ABC transporter MalFGK₂ itself comes from mutant transporters that function independently of MBP. In the absence of MBP, these mutants constitutively hydrolyze ATP and specifically transport maltodextrins (7, 8).In this study, we determined the crystal structures of the maltose transport complex MBP-MalFGK₂ bound with large maltodextrin in two conformational states. The determination of these structures, along with previous studies of maltoporin and MBP, allow us to define how overall substrate specificity is achieved for the maltose transport system.