Crystal structures of the MJ1267 ATP binding cassette reveal an induced-fit effect at the ATPase active site of an ABC transporter

BACKGROUND: ATP binding cassette (ABC) transporters are ubiquitously distributed transmembrane solute pumps that play a causative role in numerous diseases. Previous structures have defined the fold of the ABC and established the flexibility of its alpha-helical subdomain. But the nature of the mechanical changes that occur at each step of the chemical ATPase cycle have not been defined. RESULTS: Crystal structures were determined of the MJ1267 ABC from Methanococcus jannaschii in Mg-ADP-bound and nucleotide-free forms. Comparison of these structures reveals an induced-fit effect at the active site likely to be a consequence of nucleotide binding. In the Mg-ADP-bound structure, the loop following the Walker B moves toward the Walker A (P-loop) coupled to backbone conformational changes in the intervening "H-loop", which contains an invariant histidine. These changes affect the region believed to mediate intercassette interaction in the ABC transporter complex. Comparison of the Mg-ADP-bound structure of MJ1267 to the ATP-bound structure of HisP suggests that an outward rotation of the alpha-helical subdomain is coupled to the loss of a molecular contact between the gamma-phosphate of ATP and an invariant glutamine in a segment connecting this subdomain to the core of the cassette. CONCLUSIONS: The induced-fit effect and rotation of the alpha-helical subdomain may play a role in controlling the nucleotide-dependent change in cassette-cassette interaction affinity believed to represent the power-stroke of ABC transporters. Outward rotation of the alpha-helical subdomain also likely facilitates Mg-ADP release after hydrolysis. The MJ1267 structures therefore define features of the nucleotide-dependent conformational changes that drive transmembrane transport in ABC transporters.     

Nucleotide-dependent conformational changes in HisP: molecular dynamics simulations of an ABC transporter nucleotide-binding domain

ATP-binding cassette (ABC) transporters mediate the movement of molecules across cell membranes in both prokaryotes and eukaryotes. In ABC transporters, solute translocation occurs after ATP is either bound or hydrolyzed at the intracellular nucleotide-binding domains (NBDs). Molecular dynamics (MD) simulations have been employed to study the interactions of nucleotide with NBD. The results of extended (approximately 20 ns) MD simulations of HisP (total simulation time approximately 80 ns), the NBD of the histidine transporter HisQMP2J from Salmonella typhimurium, are presented. Analysis of the MD trajectories reveals conformational changes within HisP that are dependent on the presence of ATP in the binding pocket of the protein, and are sensitive to the presence/absence of Mg ions bound to the ATP. These changes are predominantly confined to the alpha-helical subdomain of HisP. Specifically there is a rotation of three alpha-helices within the subdomain, and a movement of the signature sequence toward the bound nucleotide. In addition, there is considerable conformational flexibility in a conserved glutamine-containing loop, which is situated at the interface between the alpha-helical subdomain and the F1-like subdomain. These results support the mechanism for ATP-induced conformational transitions derived from the crystal structures of other NBDs.     

Nucleotide-dependent allostery within the ABC transporter ATP-binding cassette: a computational study of the MJ0796 dimer

ATP-binding cassette transporters perform energy-dependent transmembrane solute trafficking in all organisms. These proteins often mediate cellular resistance to therapeutic drugs and are involved in a range of human genetic diseases. Enzymological studies have implicated a helical subdomain within the ATP-binding cassette nucleotide-binding domain in coupling ATP hydrolysis to solute transport in the transmembrane domains. Consistent with this, structural and computational analyses have indicated that the helical subdomain undergoes nucleotide-dependent movement relative to the core of the nucleotide-binding domain fold. Here we use theoretical methods to examine the allosteric nucleotide dependence of helical subdomain transitions to further elucidate its role in interactions between the transmembrane and nucleotide-binding domains. Unrestrained 30-ns molecular dynamics simulations of the ATP-bound, ADP-bound, and apo states of the MJ0796 monomer support the idea that interaction of a conserved glutamine residue with the catalytic metal mediates the rotation of the helical subdomain in response to nucleotide binding and hydrolysis. Simulations of the nucleotide-binding domain dimer revealed that ATP hydrolysis induces a large transition of one helical subdomain, resulting in an asymmetric conformation of the dimer not observed previously. A coarse-grained elastic network analysis supports this finding, revealing the existence of corresponding dynamic modes intrinsic to the contact topology of the protein. The implications of these findings for the coupling of ATP hydrolysis to conformational changes in the transmembrane domains required for solute transport are discussed in light of recent whole transporter structures.     

Mutations that change the position of the putative gamma-phosphate linker in the nucleotide binding domains of CFTR alter channel gating.

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is an ATP-binding cassette transporter that contains conserved nucleotide-binding domains (NBDs). In CFTR, the NBDs bind and hydrolyze ATP to open and close the channel. Crystal structures of related NBDs suggest a structural model with an important signaling role for a gamma-phosphate linker peptide that couples bound nucleotide to movement of an alpha-helical subdomain. We mutated two residues in CFTR that the structural model predicts will uncouple effects of nucleotide binding from movement of the alpha-helical subdomain. These residues are Gln-493 and Gln-1291, which may directly connect the ATP gamma-phosphate to the gamma-phosphate linker, and residues Asn-505 and Asn-1303, which may form hydrogen bonds that stabilize the linker. In NBD1, Q493A reduced the frequency of channel opening, suggesting a role for this residue in coupling ATP binding to channel opening. In contrast, N505C increased the frequency of channel opening, consistent with a role for Asn-505 in stabilizing the inactive state of the NBD. In NBD2, Q1291A decreased the effects of pyrophosphate without altering other functions. Mutations of Asn-1303 decreased the rate of channel opening and closing, suggesting an important role for NBD2 in controlling channel burst duration. These findings are consistent with both the bacterial NBD structural model and gating models for CFTR. Our results extend models of nucleotide-induced structural changes from bacterial NBDs to a functional mammalian ATP-binding cassette transporter.     

Molecular-dynamics simulations of the ATP/apo state of a multidrug ATP-binding cassette transporter provide a structural and mechanistic basis for the asymmetric occluded state

ATP-binding cassette transporters use the energy of ATP hydrolysis to transport substrates across cellular membranes. They have two transmembrane domains and two cytosolic nucleotide-binding domains. Biochemical studies have characterized an occluded state of the transporter in which nucleotide is tenaciously bound in one active site, whereas the opposite active site is empty or binds nucleotide loosely. Here, we report molecular-dynamics simulations of the bacterial multidrug ATP-binding cassette transporter Sav1866. In two simulations of the ATP/apo state, the empty site opened substantially by way of rotation of the nucleotide-binding domain (NBD) core subdomain, whereas the ATP-bound site remained occluded and intact. We correlate our findings with elastic network and molecular-dynamics simulation analyses of the Sav1866 NBD monomer, and with existing experimental data, to argue that the observed transition is physiological, and that the final structure observed in the ATP/apo simulations corresponds to the tight/loose state of the NBD dimer characterized experimentally.     

Opening of the ADP-bound active site in the ABC transporter ATPase dimer: evidence for a constant contact, alternating sites model for the catalytic cycle

ABC transporters are ubiquitous, ATP-dependent transmembrane pumps. The mechanism by which ATP hydrolysis in the nucleotide-binding domain (NBD) effects conformational changes in the transmembrane domain that lead to allocrite translocation remains largely unknown. A possible aspect of this mechanism was suggested by previous molecular dynamics simulations of the MJ0796 NBD dimer, which revealed a novel, nucleotide-dependent intrasubunit conformational change involving the relative rotation of the helical and catalytic subdomains. Here, we find that in four of five simulations of the ADP/ATP-bound dimer, the relative rotation of the helical and catalytic subdomains in the ADP-bound monomer results in opening of the ADP-bound active site, probably sufficient or close to sufficient to allow nucleotide exchange. We also observe that in all five simulations of the ADP/ATP-bound dimer, the intimate contact of the LSGGQ signature sequence with the ATP gamma-phosphate is weakened by the intrasubunit conformational change within the ADP-bound monomer. We discuss how these results support a constant contact model for the function of the NBD dimer in contrast to switch models, in which the NBDs are proposed to fully disassociate during the catalytic cycle.     

Structure of nucleotide-binding domain 1 of the cystic fibrosis transmembrane conductance regulator

Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that functions as a chloride channel. Nucleotide-binding domain 1 (NBD1), one of two ABC domains in CFTR, also contains sites for the predominant CF-causing mutation and, potentially, for regulatory phosphorylation. We have determined crystal structures for mouse NBD1 in unliganded, ADP- and ATP-bound states, with and without phosphorylation. This NBD1 differs from typical ABC domains in having added regulatory segments, a foreshortened subdomain interconnection, and an unusual nucleotide conformation. Moreover, isolated NBD1 has undetectable ATPase activity and its structure is essentially the same independent of ligand state. Phe508, which is commonly deleted in CF, is exposed at a putative NBD1-transmembrane interface. Our results are consistent with a CFTR mechanism, whereby channel gating occurs through ATP binding in an NBD1-NBD2 nucleotide sandwich that forms upon displacement of NBD1 regulatory segments.     

ATP binding to two sites is necessary for dimerization of nucleotide-binding domains of ABC proteins

ATP binding cassette (ABC) transporters have a functional unit formed by two transmembrane domains and two nucleotide binding domains (NBDs). ATP-bound NBDs dimerize in a head-to-tail arrangement, with two nucleotides sandwiched at the dimer interface. Both NBDs contribute residues to each of the two nucleotide-binding sites (NBSs) in the dimer. In previous studies, we showed that the prototypical NBD MJ0796 from Methanocaldococcus jannaschii forms ATP-bound dimers that dissociate completely following hydrolysis of one of the two bound ATP molecules. Since hydrolysis of ATP at one NBS is sufficient to drive dimer dissociation, it is unclear why all ABC proteins contain two NBSs. Here, we used luminescence resonance energy transfer (LRET) to study ATP-induced formation of NBD homodimers containing two NBSs competent for ATP binding, and NBD heterodimers with one active NBS and one binding-defective NBS. The results showed that binding of two ATP molecules is necessary for NBD dimerization. We conclude that ATP hydrolysis at one nucleotide-binding site drives NBD dissociation, but two binding sites are required to form the ATP-sandwich NBD dimer necessary for hydrolysis.     

ATP binding to the motor domain from an ABC transporter drives formation of a nucleotide sandwich dimer

It has been proposed that the reaction cycle of ATP binding cassette (ABC) transporters is driven by dimerization of their ABC motor domains upon binding ATP at their mutual interface. However, no such ATP sandwich complex has been observed for an ABC from an ABC transporter. In this paper, we report the crystal structure of a stable dimer formed by the E171Q mutant of the MJ0796 ABC, which is hydrolytically inactive due to mutation of the catalytic base. The structure shows a symmetrical dimer in which two ATP molecules are each sandwiched between the Walker A motif in one subunit and the LSGGQ signature motif in the other subunit. These results establish the stereochemical basis of the power stroke of ABC transporter pumps.     

Cooperative, ATP-dependent association of the nucleotide binding cassettes during the catalytic cycle of ATP-binding cassette transporters

ATP-binding cassette (ABC) transporters harvest the energy present in cellular ATP to drive the translocation of a structurally diverse set of solutes across the membrane barriers of eubacteria, archaebacteria, and eukaryotes. The positively cooperative ATPase activity (Hill coefficient, 1.7) of a model soluble cassette of known structure, MJ0796, from Methanococcus jannaschii indicates that at least two binding sites participate in the catalytic reaction. Mutation of the catalytic base in MJ0796, E171Q, produced a cassette that can bind but not efficiently hydrolyze ATP. The equivalent mutation (E179Q) in a homologous cassette, MJ1267, had an identical effect. Both mutant cassettes formed dimers in the presence of ATP but not ADP, indicating that the energy of ATP binding is first coupled to the transport cycle through a domain association reaction. The non-hydrolyzable nucleotides adenosine 5'-(beta,gamma-imino)triphosphate and adenosine 5'-3-O-(thio)triphosphate were poor analogues of ATP in terms of their ability to promote dimerization. Moreover, inclusion of MgCl2, substitution of KCl for NaCl, or alterations in the polarity of the side chain at the catalytic base all weakened the ATP-dependent dimer, suggesting that electrostatic interactions are critical for the association reaction. Thus, upon hydrolysis of bound ATP and the release of product, both electrostatic and conformational changes drive the cassettes apart, providing a second opportunity to couple free energy changes to the transport reaction.     

Modulation of ATPase activity by physical disengagement of the ATP-binding domains of an ABC transporter, the histidine permease.

The membrane-bound complex of the prokaryotic histidine permease, a periplasmic protein-dependent ABC transporter, is composed of two hydrophobic subunits, HisQ and HisM, and two identical ATP-binding subunits, HisP, and is energized by ATP hydrolysis. The soluble periplasmic binding protein, HisJ, creates a signal that induces ATP hydrolysis by HisP. The crystal structure of HisP has been resolved and shown to have an "L" shape, with one of its arms (arm I) being involved in ATP binding and the other one (arm II) being proposed to interact with the hydrophobic subunits (Hung, L.-W., Wang, I. X., Nikaido, K., Liu, P.-Q., Ames, G. F.-L., and Kim, S.-H. (1998) Nature 396, 703-707). Here we study the basis for the defect of several HisP mutants that have an altered signaling pathway and hydrolyze ATP constitutively. We use biochemical approaches to show that they produce a loosely assembled membrane complex, in which the mutant HisP subunits are disengaged from HisQ and HisM, suggesting that the residues involved are important in the interaction between HisP and the hydrophobic subunits. In addition, the mutant HisPs are shown to have lower affinity for ADP and to display no cooperativity for ATP. All of the residues affected in these HisP mutants are located in arm II of the crystal structure of HisP, thus supporting the proposed function of arm II of HisP as interacting with HisQ and HisM. A revised model involving a cycle of disengagement and reengagement of HisP is proposed as a general mechanism of action for ABC transporters.     

Nucleotide Binding Domain Interactions During the Mechanochemical Reaction Cycle of ATP-Binding Cassette Transporters

ATP-binding cassette (ABC) transporters serve as importers and exporters for a wide variety of solutes in both prokaryotes and eukaryotes, and are implicated in microbial drug resistance and a number of significant human genetic disorders. Initial crystal structures of the soluble nucleotide binding domains (NBDs) of ABC transporters, while a significant step towards understanding the coupling of ATP binding and hydrolysis to transport, presented researchers with important questions surrounding the role of the signature sequence residues, the composition of the nucleotide binding sites, and the mode of NBD dimerization during the transport reaction cycle. Recent studies have begun to address these concerns. This mini-review summarizes the biochemical and structural characterizations of two archaebacterial NBDs from Methanocaldococcus jannaschii, MJ0796 and MJ1267, and offers current perspectives on the functional mechanism of ABC transporters.     

Disulfide cross-linking reveals a site of stable interaction between C-terminal regulatory domains of the two MalK subunits in the maltose transport complex

Understanding the structure and function of the ATP-binding cassette (ABC) transporters is very important because defects in ABC transporters lie at the root of several serious diseases including cystic fibrosis. MalK, the ATP-binding cassette of the maltose transporter of Escherichia coli, is distinct from most other ATP-binding cassettes in that it contains an additional C-terminal regulatory domain. The published structure of a MalK dimer is elongated with C-terminal domains at opposite poles (Diederichs, K., Diez, J., Greller, G., Muller, C., Breed, J., Schnell, C., Vonrhein, C., Boos, W., and Welte, W. (2000) EMBO J. 19, 5951-5961). Some uncertainty exists as to whether the orientation of MalK in the dimer structure is correct. Superpositioning of the N-terminal domains of MalK onto the ATP-binding domains of an alternate ABC dimer, in which ATP is bound along the dimer interface between Walker A and LSGGQ motifs, places both N- and C-terminal domains of MalK along the dimer interface. Consistent with this model, a cysteine substitution at position 313 in the C-terminal domain of an otherwise cysteine-free MalK triggered disulfide bond formation between two MalK subunits in an intact maltose transporter. Disulfide bond formation did not inhibit the function of the transporter, suggesting that the C-terminal domains of MalK remain in close proximity throughout the transport cycle. Enzyme IIAglc still inhibited the ATPase activity of the disulfide-linked transporter indicating that the mechanism of inducer exclusion was unaffected. These data support a model for ATP hydrolysis in which the C-terminal domains of MalK remain in contact whereas the N-terminal domains of MalK open and close to allow nucleotide binding and dissociation.     

Hydrolysis at One of the Two Nucleotide-binding Sites Drives the Dissociation of ATP-binding Cassette Nucleotide-binding Domain Dimers

The functional unit of ATP-binding cassette (ABC) transporters consists of two transmembrane domains and two nucleotide-binding domains (NBDs). ATP binding elicits association of the two NBDs, forming a dimer in a head-to-tail arrangement, with two nucleotides “sandwiched” at the dimer interface. Each of the two nucleotide-binding sites is formed by residues from the two NBDs. We recently found that the prototypical NBD MJ0796 from Methanocaldococcus jannaschii dimerizes in response to ATP binding and dissociates completely following ATP hydrolysis. However, it is still unknown whether dissociation of NBD dimers follows ATP hydrolysis at one or both nucleotide-binding sites. Here, we used luminescence resonance energy transfer to study heterodimers formed by one active (donor-labeled) and one catalytically defective (acceptor-labeled) NBD. Rapid mixing experiments in a stop-flow chamber showed that NBD heterodimers with one functional and one inactive site dissociated at a rate indistinguishable from that of dimers with two hydrolysis-competent sites. Comparison of the rates of NBD dimer dissociation and ATP hydrolysis indicated that dissociation followed hydrolysis of one ATP. We conclude that ATP hydrolysis at one nucleotide-binding site drives NBD dimer dissociation.     

Dissociation of ATP-binding cassette nucleotide-binding domain dimers into monomers during the hydrolysis cycle

ATP-binding cassette (ABC) proteins have two nucleotide-binding domains (NBDs) that work as dimers to bind and hydrolyze ATP, but the molecular mechanism of nucleotide hydrolysis is controversial. In particular, it is still unresolved whether hydrolysis leads to dissociation of the ATP-induced dimers or opening of the dimers, with the NBDs remaining in contact during the hydrolysis cycle. We studied a prototypical ABC NBD, the Methanococcus jannaschii MJ0796, using spectroscopic techniques. We show that fluorescence from a tryptophan positioned at the dimer interface and luminescence resonance energy transfer between probes reacted with single-cysteine mutants can be used to follow NBD association/dissociation in real time. The intermonomer distances calculated from luminescence resonance energy transfer data indicate that the NBDs separate completely following ATP hydrolysis, instead of opening. The results support ABC protein NBD association/dissociation, as opposed to constant-contact models.     

The A-loop, a novel conserved aromatic acid subdomain upstream of the Walker A motif in ABC transporters, is critical for ATP binding

ATP-binding cassette (ABC) transporters represent one of the largest families of proteins, and transport a variety of substrates ranging from ions to amphipathic anticancer drugs. The functional unit of an ABC transporter is comprised of two transmembrane domains and two cytoplasmic ABC ATPase domains. The energy of the binding and hydrolysis of ATP is used to transport the substrates across membranes. An ABC domain consists of conserved regions, the Walker A and B motifs, the signature (or C) region and the D, H and Q loops. We recently described the A-loop (Aromatic residue interacting with the Adenine ring of ATP), a highly conserved aromatic residue approximately 25 amino acids upstream of the Walker A motif that is essential for ATP-binding. Here, we review the mutational analysis of this subdomain in human P-glycoprotein as well as homology modeling, structural and data mining studies that provide evidence for a functional role of the A-loop in ATP-binding in most members of the superfamily of ABC transporters.     

An Asymmetric Post-Hydrolysis State of the ABC Transporter ATPase Dimer

ABC transporters are a superfamily of enzyme pumps that hydrolyse ATP in exchange for translocation of substrates across cellular membranes. Architecturally, ABC transporters are a dimer of transmembrane domains coupled to a dimer of nucleotide binding domains (NBDs): the NBD dimer contains two ATP-binding sites at the intersubunit interface. A current controversy is whether the protomers of the NBD dimer separate during ATP hydrolysis cycling, or remain in constant contact. In order to investigate the ABC ATPase catalytic mechanism, MD simulations using the recent structure of the ADP+Pi-bound MJ0796 isolated NBD dimer were performed. In three independent simulations of the ADP+Pi/apo state, comprising a total of >0.5 µs, significant opening of the apo (empty) active site was observed; occurring by way of intrasubunit rotations between the core and helical subdomains within both NBD monomers. In contrast, in three equivalent simulations of the ATP/apo state, the NBD dimer remained close to the crystal structure, and no opening of either active site occurred. The results thus showed allosteric coupling between the active sites, mediated by intrasubunit conformational changes. Opening of the apo site is exquisitely tuned to the nature of the ligand, and thus to the stage of the reaction cycle, in the opposite site. In addition to this, in also showing how one active site can open, sufficient to bind nucleotide, while the opposite site remains occluded and bound to the hydrolysis products ADP+Pi, the results are consistent with a Constant Contact Model. Conversely, they show how there may be no requirement for the NBD protomers to separate to complete the catalytic cycle.     

Structure of the multidrug ABC transporter Sav1866 from Staphylococcus aureus in complex with AMP-PNP

Staphylococcus aureus Sav1866 is a bacterial homolog of the human ABC transporter Mdr1 that causes multidrug resistance in cancer cells. We report the crystal structure of Sav1866 in complex with adenosine-5'-(beta,gamma-imido)triphosphate (AMP-PNP) at 3.4A resolution and compare it with the previously determined structure of Sav1866 with bound ADP. Besides differences in the ATP-binding sites, no significant conformational changes were observed. The results confirm that the ATP-bound state of multidrug ABC transporters is coupled to an outward-facing conformation of the transmembrane domains.     

Subunit interactions in ABC transporters: towards a functional architecture

The ABC superfamily is a diverse group of integral membrane proteins involved in the ATP-dependent transport of solutes across biological membranes in both prokaryotes and eukaryotes. Although ABC transporters have been studied for over 30 years, very little is known about the mechanism by which the energy of ATP hydrolysis is used to transport substrate across the membrane. The recent report of the high resolution crystal structure of HisP, the nucleotide-binding subunit of the histidine permease complex of Salmonella typhimurium, represents a significant breakthrough toward the elucidation of the mechanism of solute translocation by ABC transporters. In this review, we use data from the crystallographic structures of HisP and other nucleotide-binding proteins, combined with sequence analysis of a subset of atypical ABC transporters, to argue a new model for the dimerisation of the nucleotide-binding domains that embraces the notion that the C motif from one subunit forms part of the ATP-binding site in the opposite subunit. We incorporate this dimerisation of the ATP-binding domains into our recently reported beta-barrel model for P-glycoprotein and present a general model for the cooperative interaction of the two nucleotide-binding domains and the translocation of mechanical energy to the transmembrane domains in ABC transporters.     

Dynamics of ATP-binding cassette contribute to allosteric control, nucleotide binding and energy transduction in ABC transporters

ATP-binding cassette (ABC) transporters move solutes across membranes and are associated with important diseases, including cystic fibrosis and multi-drug resistance. These molecular machines are energized by their charateristic ABC modules, molecular engines fuelled by ATP hydrolysis. A solution NMR study of a model ABC, Methanococcus jannaschii protein MJ1267, reveals that ADP-Mg binding alters the flexibilities of key ABC motifs and induces allosteric changes in conformational dynamics in the LivG insert, over 30A away from the ATPase active site. (15)N spin relaxation data support a "selected-fit" model for nucleotide binding. Transitions between rigidity and flexibility in key motifs during the ATP hydrolysis cycle may be crucial to mechanochemical energy transduction in ABC transporters. The restriction of correlated protein motions is likely a central mechanism for allosteric communications. Comparison between dynamics data from NMR and X-ray crystallography reveals their overall consistency and complementarity.