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.     

The crystal structure of the MJ0796 ATP-binding cassette. Implications for the structural consequences of ATP hydrolysis in the active site of an ABC transporter

The crystal structure of the MJ0796 ATP-binding cassette, a member of the o228/LolD transporter family, has been determined at 2.7-A resolution with MgADP bound at its active site. Comparing this structure with that of the ATP-bound form of the HisP ATP-binding cassette (Hung, L. W., Wang, I. X., Nikaido, K., Liu, P. Q., Ames, G. F., and Kim, S. H. (1998) Nature 396, 703-707) shows a 5-A withdrawal of a phylogenetically invariant glutamine residue from contact with the gamma-phosphate of ATP in the active site. This glutamine is located in a protein segment that links the rigid F(1)-type ATP-binding core of the enzyme to an ABC transporter-specific alpha-helical subdomain that moves substantially away from the active site in the MgADP-bound structure of MJ0796 compared with the ATP-bound structure of HisP. A similar conformational effect is observed in the MgADP-bound structure of MJ1267 (Karpowich, N., et al. (2001) Structure, in press), establishing the withdrawal of the glutamine and the coupled outward rotation of the alpha-helical subdomain as consistent consequences of gamma-phosphate release from the active site of the transporter. Considering this subdomain movement in the context of a leading model for the physiological dimer of cassettes present in ABC transporters indicates that it produces a modest mechanical change that is likely to play a role in facilitating nucleotide exchange out of the ATPase active site. Finally, it is noteworthy that one of the intersubunit packing interactions in the MJ0796 crystal involves antiparallel beta-type hydrogen bonding interactions between the outermost beta-strands in the two core beta-sheets, leading to their fusion into a single extended beta-sheet, a type of structural interaction that has been proposed to play a role in mediating the aggregation of beta-sheet-containing proteins.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Allosteric transitions of the maltose transporter studied by an elastic network model

The maltose transporter from Escherichia coli is one of the ATP‐binding cassette (ABC) transporters that utilize the energy from ATP hydrolysis to translocate substrates across cellular membranes. Until 2011, three crystal structures have been determined for maltose transporter at different states in the process of transportation. Here, based on these crystal structures, the allosteric pathway from the resting state (inward‐facing) to the catalytic intermediate state (outward‐facing) is studied by applying an adaptive anisotropic network model. The results suggest that the allosteric transitions proceed in a coupled way. The closing of the nucleotide‐binding domains occurs first, and subsequently this conformational change is propagated to the transmembrane domains (TMD) via the EAA and EAS loops, and then to the maltose‐binding protein, which facilitates the translocation of the maltose. It is also found that there exist nonrigid‐body and asymmetric movements in the TMD. The cytoplasmic gate may only play the role of allosteric propagation during the transition from the pretranslocation to outward‐facing states. In addition, the results show that the movment of the helical subdomain towards the RecA‐like subdomain mainly occurs in the earlier stages of the transition. These results can provide some insights into the understanding of the mechanism of ABC transporters. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 758–768, 2014.     

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.     

Structural insights into ABC transporter mechanism

ATP-binding cassette (ABC) transporters utilize the energy from ATP hydrolysis to transport substances across the membrane. In recent years, crystal structures of several ABC transporters have become available. These structures show that both importers and exporters oscillate between two conformations: an inward-facing conformation with the substrate translocation pathway open to the cytoplasm and an outward-facing conformation with the translocation pathway facing the opposite side of the membrane. In this review, conformational differences found in the structures of homologous ABC transporters are analyzed to understand how alternating-access is achieved. It appears that rigid-body rotations of the transmembrane subunits, coinciding with the opening and closing of the nucleotide-binding subunits, couples ATP hydrolysis to substrate translocation.     

Structure and mechanism of ABC transporter proteins

ATP-binding cassette (ABC) transporters are ubiquitous membrane proteins that couple the transport of diverse substrates across cellular membranes to the hydrolysis of ATP. The crystal structures of four ABC transporters have recently been determined. They reveal similar arrangements of the conserved ATP-hydrolyzing nucleotide-binding domains, but unrelated architectures of the transmembrane domains, with the notable exception of a common 'coupling helix' that is essential for transmitting conformational changes. The structures suggest a mechanism that rationalizes ATP-driven transport: While binding of ATP appears to trigger an outward-facing conformation, dissociation of the hydrolysis products may promote an inward-facing conformation. This basic scheme can, in principle, explain nutrient import by ABC importers and drug extrusion by ABC exporters.     

The "LSGGQ" motif in each nucleotide-binding domain of human P-glycoprotein is adjacent to the opposing walker A sequence

The human multidrug resistance P-glycoprotein (P-gp, ABCB1), a member of the ATP-binding cassette (ABC) family of transport proteins, actively transports many cytotoxic compounds out of the cell. ABC transporters have two nucleotide-binding domains (NBD) and two transmembrane domains. The presence of the conserved "signature" sequence (LSGGQ) in each NBD is a unique feature in these transporters. The function of the signature sequences is unknown. In this study, we tested whether the signature sequences ((531)LSGGQ(535) in NBD1; (1176)LSGGQ(1180) in NBD2) in P-gp are in close proximity to the opposing Walker A consensus nucleotide-binding sequences ((1070)GSSGCGKS(1077) in NBD2; (427)GNSGCGKS(434) in NBD1). Pairs of cysteines were introduced into a Cys-less P-gp at the signature and "Walker A" sites and the mutant P-gps were subjected to oxidative cross-linking. At 4 degrees C, when thermal motion is low, P-gp mutants (L531C(Signature)/C1074(Walker A) and C431(Walker A)/L1176C(Signature) were cross-linked. Cross-linking inhibited the drug-stimulated ATPase activities of these two mutants. Their activities were restored, however, after addition of the reducing agent, dithiothreitol. Vanadate trapping of nucleotide at the ATP-binding sites prevented cross-linking of the mutants. These results indicate that the signature sequences are adjacent to the opposing Walker A site. They likely participate in forming the ATP-binding sites and are displaced upon ATP hydrolysis. The resulting conformational change may be the signal responsible for coupling ATP hydrolysis to drug transport by inducing conformational changes in the transmembrane segments.     

Purification and characterization of the N-terminal nucleotide binding domain of an ABC drug transporter of Candida albicans: uncommon cysteine 193 of Walker A is critical for ATP hydrolysis

The Candida drug resistance protein Cdr1p (approximately 170 kDa) is a member of ATP binding cassette (ABC) superfamily of drug transporters, characterized by the presence of 2 nucleotide binding domains (NBD) and 12 transmembrane segments (TMS). NBDs of these transporters are the hub of ATP hydrolysis activity, and their sequence contains a conserved Walker A motif (GxxGxGKS/T). Mutations of the lysine residue within this motif abrogate the ability of NBDs to hydrolyze ATP. Interestingly, the sequence alignments of Cdr1p NBDs with other bacterial and eukaryotic transporters reveal that its N-terminal NBD contains an unusual Walker A sequence (GRPGAGCST), as the invariant lysine is replaced by a cysteine. In an attempt to understand the significance of this uncommon positioning of cysteine within the Walker A motif, we for the first time have purified and characterized the N-terminal NBD (encompassing first N-terminal 512 amino acids) of Cdr1p as well as its C193A mutant protein. The purified NBD-512 protein could exist as an independent functional general ribonucleoside triphosphatase with strong divalent cation dependence. It exhibited ATPase activity with an apparent K(m) in the 0.8-1.0 mM range and V(max) in the range of 147-160 nmol min(-)(1) (mg of protein)(-)(1). NBD-512-associated ATPase activity was also sensitive to inhibitors such as vanadate, azide, and NEM. The Mut-NBD-512 protein (C193A) showed a severe impairment in its ability to hydrolyze ATP (95%); however, no significant effect on ATP (TNP-ATP) binding was observed. Our results show that C193 is critical for N-terminal NBD-mediated ATP hydrolysis and represents a unique feature distinguishing the ATP-dependent functionality of the ABC transporters of fungi from those found in bacteria and other eukaryotes.     

A tweezers-like motion of the ATP-binding cassette dimer in an ABC transport cycle

The ATPase components of ATP binding cassette (ABC) transporters power the transporters by binding and hydrolyzing ATP. Major conformational changes of an ATPase are revealed by crystal structures of MalK, the ATPase subunit of the maltose transporter from Escherichia coli, in three different dimeric configurations. While other nucleotide binding domains or subunits display low affinity for each other in the absence of the transmembrane segments, the MalK dimer is stabilized through interactions of the additional C-terminal domains. In the two nucleotide-free structures, the N-terminal nucleotide binding domains are separated to differing degrees, and the dimer is maintained through contacts of the C-terminal regulatory domains. In the ATP-bound form, the nucleotide binding domains make contact and two ATPs lie buried along the dimer interface. The two nucleotide binding domains of the dimer open and close like a pair of tweezers, suggesting a regulatory mechanism for ATPase activity that may be tightly coupled to translocation.     

Characterization of the LSGGQ and H motifs from the Escherichia coli lipid A transporter MsbA

ATP-binding cassette (ABC) transporters make up one of the largest superfamilies of proteins known and have been shown to transport substrates ranging from lipids and antibiotics to sugars and amino acids. The dysfunction of ABC transporters has been linked to human pathologies such as cystic fibrosis, hyperinsulinemia, and macular dystrophy. Several bacterial ABC transporters are also necessary for bacterial survival and transport of virulence factors in an infected host. MsbA is a 65 kDa protein that forms a functional homodimer consisting of two six-helix transmembrane domains and two approximately 250 amino acid nucleotide-binding domains (NBD). The NBDs contain several conserved regions such as the Walker A, LSGGQ, and H motif that bind directly to ATP and align it for hydrolysis. MsbA transports lipid A, its native substrate, across the inner membrane of Gram-negative bacteria. The loss or dysfunction of MsbA results in a toxic accumulation of lipid A inside the cell, leading to cell-membrane instability and cell death. Using site-directed spin labeling electron paramagnetic resonance spectroscopy, conserved motifs within the MsbA NBD have been evaluated for structure and dynamics upon substrate binding. It has been determined that the LSGGQ NBD consensus sequence is consistent with an alpha-helical conformation and that these residues maintain extensive tertiary contacts throughout hydrolysis. The dynamics of the LSGGQ and the H-motif region have been studied in the presence of ATP, ADP, and ATP plus vanadate to identify the residues that are directly affected by interactions with the substrate before, after, and during hydrolysis, respectively.     

Multidrug resistance ABC transporters

Clinical multidrug resistance is caused by a group of integral membrane proteins that transport hydrophobic drugs and lipids across the cell membrane. One class of these permeases, known as multidrug resistance ATP binding cassette (ABC) transporters, translocate these molecules by coupling drug/lipid efflux with energy derived from the hydrolysis of ATP. In this review, we examine both the structures and conformational changes of multidrug resistance ABC transporters. Together with the available biochemical and structural evidence, we propose a general mechanism for hydrophobic substrate transport coupled to ATP hydrolysis.     

Characterization of the Walker A motif of MsbA using site-directed spin labeling electron paramagnetic resonance spectroscopy

MsbA is an ABC transporter that transports lipid A across the inner membrane of Gram-negative bacteria such as Escherichia coli. Without functional MsbA present, bacterial cells accumulate a toxic amount of lipid A within their inner membranes. A crystal structure of MsbA was recently obtained that provides an excellent starting point for functional dynamics studies in membranes [Chang and Roth (2001) Science 293, 1793-1800]. Although a structure of MsbA is now available, several functionally important motifs common to ABC transporters are unresolved in the crystal structure. The Walker A domain, one of the ABC transporter consensus motifs that is directly involved in ATP binding, is located within a large unresolved region of the MsbA ATPase domain. Site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy is a powerful technique for characterizing local areas within a large protein structure in addition to detecting and following changes in local structure due to dynamic interactions. MsbA reconstituted into lipid membranes has been evaluated by EPR spectroscopy, and it has been determined that the Walker A domain forms an alpha-helical structure, which is consistent with the structure of this motif observed in other crystallized ABC transporters. In addition, the interaction of the Walker A residues with ATP before, during, and after hydrolysis was followed using SDSL EPR spectroscopy in order to identify the residues directly involved in substrate binding and hydrolysis.     

Targeted degradation of ABC transporters in health and disease

ATP binding cassette (ABC) transporters comprise an extended protein family involved in the transport of a broad spectrum of solutes across membranes. They consist of a common architecture including two ATP-binding domains converting chemical energy into conformational changes and two transmembrane domains facilitating transport via alternating access. This review focuses on the biogenesis, and more precisely, on the degradation of mammalian ABC transporters in the endoplasmic reticulum (ER). We enlighten the ER-associated degradation pathway in the context of misfolded, misassembled or tightly regulated ABC transporters with a closer view on the cystic fibrosis transmembrane conductance regulator (CFTR) and the transporter associated with antigen processing (TAP), which plays an essential role in the adaptive immunity. Three rather different scenarios affecting the stability and degradation of ABC transporters are discussed: (1) misfolded domains caused by a lack of proper intra- and intermolecular contacts within the ABC transporters, (2) deficient assembly with auxiliary factors, and (3) arrest and accumulation of an intermediate or ‘dead-end’ state in the transport cycle, which is prone to be recognized by the ER-associated degradation machinery.