Conformational motion of the ABC transporter MsbA induced by ATP hydrolysis

We measured the amplitude of conformational motion in the ATP-binding cassette (ABC) transporter MsbA upon lipopolysaccharide (LPS) binding and following ATP turnover by pulse double electron-electron resonance and fluorescence homotransfer. The distance constraints from both methods reveal large-scale movement of opposite signs in the periplasmic and cytoplasmic part of the transporter upon ATP hydrolysis. LPS induces distinct structural changes that are inhibited by trapping of the transporter in an ATP post-hydrolysis intermediate. The formation of this intermediate involves a 33-Å distance change between the two ABCs, which is consistent with a dimerization-dissociation cycle during transport that leads to their substantial separation in the absence of nucleotides. Our results suggest that ATP-powered transport entails LPS sequestering into the open cytoplasmic chamber prior to its translocation by alternating access of the chamber, made possible by 10–20-Å conformational changes.     

A novel catalytic mechanism for ATP hydrolysis employed by the N-terminal nucleotide-binding domain of Cdr1p, a multidrug ABC transporter of Candida albicans

Although essentially conserved, the N-terminal nucleotide-binding domain (NBD) of Cdr1p and other fungal transporters has some unique substitutions of amino acids which appear to have functional significance for the drug transporters. We have previously shown that the typical Cys193 in Walker A as well as Trp326 and Asp327 in the Walker B of N-terminal NBD (NBD-512) of Cdr1p has acquired unique roles in ATP binding and hydrolysis. In the present study, we show that due to spatial proximity, fluorescence resonance energy transfer (FRET) takes place between Trp326 of Walker B and MIANS [2-(4-maleimidoanilino) naphthalene-6-sulfonic acid] on Cys193 of Walker A motif. By exploiting FRET, we demonstrate how these critical amino acids are positioned within the nucleotide-binding pocket of NBD-512 to bind and hydrolyze ATP. Our results show that both Mg2+ coordination and nucleotide binding contribute to the formation of the active site. The entry of Mg2+ into the active site causes the first large conformational change that brings Trp326 and Cys193 in close proximity to each other. We also show that besides Trp326, typical Glu238 in the Q-loop also participates in coordination of Mg2+ by NBD-512. A second conformational change is induced when ATP, but not ADP, docks into the pocket. Asn328 does sensing of the gamma-phosphate of the substrate in the extended Walker B motif, which is essential for the second conformational change that must necessarily precede ATP hydrolysis. Taken together our results imply that the uniquely placed residues in NBD-512 have acquired critical roles in ATP catalysis, which drives drug extrusion.     

Diverse effects of phospholipids on lipoprotein sorting and ATP hydrolysis by the ABC transporter LolCDE complex

The LolCDE complex of Escherichia coli releases outer membrane-specific lipoproteins from the inner membrane. Lipoproteins with Asp at +2 remain in the inner membrane since this residue functions as a LolCDE avoidance signal depending on phosphatidylethanolamine. We examined the effects of other phospholipids on lipoprotein sorting in proteoliposomes reconstituted with LolCDE and various synthetic phospholipids. The lipoprotein release and ATP hydrolysis were both low at 2 mM Mg(2+) but very high at 10 mM Mg(2+) in proteoliposomes containing cardiolipin alone. However, the Lol avoidance function was abolished at 10 mM Mg(2+), and the release of lipoproteins with Asp at +2 was as efficient as that of outer membrane-specific lipoproteins. The addition of phosphatidylethanolamine to cardiolipin stimulated the ATP hydrolysis and increased the Lol avoidance function of Asp at +2 at 2 mM Mg(2+). The addition of phosphatidylglycerol to cardiolipin nearly completely inhibited the release of lipoproteins with Asp at +2 even at 10 mM Mg(2+), while that of outer membrane-specific lipoproteins was not. Taken together, these results indicate that three major phospholipids of E. coli differently affect lipoprotein sorting and the activity of LolCDE.     

Diverse effects of phospholipids on lipoprotein sorting and ATP hydrolysis by the ABC transporter LolCDE complex

The LolCDE complex of Escherichia coli releases outer membrane-specific lipoproteins from the inner membrane. Lipoproteins with Asp at + 2 remain in the inner membrane since this residue functions as a LolCDE avoidance signal depending on phosphatidylethanolamine. We examined the effects of other phospholipids on lipoprotein sorting in proteoliposomes reconstituted with LolCDE and various synthetic phospholipids. The lipoprotein release and ATP hydrolysis were both low at 2 mM Mg²⁺ but very high at 10 mM Mg²⁺ in proteoliposomes containing cardiolipin alone. However, the Lol avoidance function was abolished at 10 mM Mg²⁺, and the release of lipoproteins with Asp at + 2 was as efficient as that of outer membrane-specific lipoproteins. The addition of phosphatidylethanolamine to cardiolipin stimulated the ATP hydrolysis and increased the Lol avoidance function of Asp at + 2 at 2 mM Mg²⁺. The addition of phosphatidylglycerol to cardiolipin nearly completely inhibited the release of lipoproteins with Asp at + 2 even at 10 mM Mg²⁺, while that of outer membrane-specific lipoproteins was not. Taken together, these results indicate that three major phospholipids of E. coli differently affect lipoprotein sorting and the activity of LolCDE.     

H662 is the linchpin of ATP hydrolysis in the nucleotide-binding domain of the ABC transporter HlyB

The ABC transporter HlyB is a central element of the HlyA secretion machinery, a paradigm of Type I secretion. Here, we describe the crystal structure of the HlyB-NBD (nucleotide-binding domain) with H662 replaced by Ala in complex with ATP/Mg2+. The dimer shows a composite architecture, in which two intact ATP molecules are bound at the interface of the Walker A motif and the C-loop, provided by the two monomers. ATPase measurements confirm that H662 is essential for activity. Based on these data, we propose a model in which E631 and H662, highly conserved among ABC transporters, form a catalytic dyad. Here, H662 acts as a 'linchpin', holding together all required parts of a complicated network of interactions between ATP, water molecules, Mg2+, and amino acids both in cis and trans, necessary for intermonomer communication. Based on biochemical experiments, we discuss the hypothesis that substrate-assisted catalysis, rather than general base catalysis might operate in ABC-ATPases.     

A novel catalytic mechanism for ATP hydrolysis employed by the N-terminal nucleotide-binding domain of Cdr1p, a multidrug ABC transporter of Candida albicans

Although essentially conserved, the N-terminal nucleotide-binding domain (NBD) of Cdr1p and other fungal transporters has some unique substitutions of amino acids which appear to have functional significance for the drug transporters. We have previously shown that the typical Cys193 in Walker A as well as Trp326 and Asp327 in the Walker B of N-terminal NBD (NBD-512) of Cdr1p has acquired unique roles in ATP binding and hydrolysis. In the present study, we show that due to spatial proximity, fluorescence resonance energy transfer (FRET) takes place between Trp326 of Walker B and MIANS [2-(4-maleimidoanilino) naphthalene-6-sulfonic acid] on Cys193 of Walker A motif. By exploiting FRET, we demonstrate how these critical amino acids are positioned within the nucleotide-binding pocket of NBD-512 to bind and hydrolyze ATP. Our results show that both Mg²⁺ coordination and nucleotide binding contribute to the formation of the active site. The entry of Mg²⁺ into the active site causes the first large conformational change that brings Trp326 and Cys193 in close proximity to each other. We also show that besides Trp326, typical Glu238 in the Q-loop also participates in coordination of Mg²⁺ by NBD-512. A second conformational change is induced when ATP, but not ADP, docks into the pocket. Asn328 does sensing of the γ-phosphate of the substrate in the extended Walker B motif, which is essential for the second conformational change that must necessarily precede ATP hydrolysis. Taken together our results imply that the uniquely placed residues in NBD-512 have acquired critical roles in ATP catalysis, which drives drug extrusion.     

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.     

Stereochemical reaction mechanism formulations for enzyme-catalyzed pyrophosphate hydrolysis,ATP hydrolysis,and ATP synthesis

Fundamental concepts pertaining to the stereochemistry paths of polar additionelimination (nucleophilic substitution) reactions at phosphate phosphorus centers are reviewed and employed to analyze ¹⁸O exchange reactions catalyzed by inorganic pyrophosphatase and mitochondrial ATP synthetase. The analysis suggests reasonable choices for the stereochemistry path of the ¹⁸O exchanges. This, in turn, permits reasonable choices for the stereochemistry paths of hydrolysis of pyrophosphate catalyzed by pyrophosphatase and of hydrolysis and synthesis of ATP catalyzed by ATP synthetase.     

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.     

Structural consequences of ATP hydrolysis on the ABC transporter NBD dimer: molecular dynamics studies of HlyB

ABC transporters are a large and important family of membrane proteins involved in substrate transport across the membrane. The transported substrates are quite diverse, ranging from monatomic ions to large biomolecules. Consequently, some ABC transporters are involved in biomedically relevant situations, from genetic diseases to multidrug resistance. The most conserved domains in ABC transporters are the nucleotide binding domains (NBDs), which form a dimer responsible for the binding and hydrolysis of ATP, concomitantly with substrate translocation. To elucidate how ATP hydrolysis structurally affects the NBD dimer, and consequently the transporter, we performed a molecular dynamics study on the NBD dimer of the HlyB ABC exporter. We have observed a change in the contact surface between the monomers after hydrolysis, even though we have not seen dimer opening in any of the five 100 ns simulations. We have also identified specific regions that respond to ATP hydrolysis, in particular the X-loop motif of ABC exporters, which has been shown to be in contact with the coupling helices of the transmembrane domains (TMDs). We propose that this motif is an important part of the NBD-TMD communication in ABC exporters. Through nonequilibrium analysis, we have also identified gradual conformational changes within a short time scale after ATP hydrolysis.     

A two-site kinetic mechanism for ATP binding and hydrolysis by E. coli Rep helicase dimer bound to a single-stranded oligodeoxynucleotide.

Escherichia coli Rep helicase catalyzes the unwinding of duplex DNA in reactions that are coupled to ATP binding and hydrolysis. We have investigated the kinetic mechanism of ATP binding and hydrolysis by a proposed intermediate in Rep-catalyzed DNA unwinding, the Rep "P2S" dimer (formed with the single-stranded (ss) oligodeoxynucleotide, (dT)16), in which only one subunit of a Rep homo-dimer is bound to ssDNA. Pre-steady-state quenched-flow studies under both single turnover and multiple turnover conditions as well as fluorescence stopped-flow studies were used (4 degrees C, pH 7.5, 6 mM NaCl, 5 mM MgCl2, 10 % (v/v) glycerol). Although steady-state studies indicate that a single ATPase site dominates the kinetics (kcat=17(+/-2) s-1; KM=3 microM), pre-steady-state studies provide evidence for a two-ATP site mechanism in which both sites of the dimer are catalytically active and communicate allosterically. Single turnover ATPase studies indicate that ATP hydrolysis does not require the simultaneous binding of two ATP molecules, and under these conditions release of product (ADP-Pi) is preceded by a slow rate-limiting isomerization ( approximately 0.2 s-1). However, product (ADP or Pi) release is not rate-limiting under multiple turnover conditions, indicating the involvement of a second ATP site under conditions of excess ATP. Stopped-flow fluorescence studies monitoring ATP-induced changes in Rep's tryptophan fluorescence displayed biphasic time courses. The binding of the first ATP occurs by a two-step mechanism in which binding (k+1=1.5(+/-0.2)x10(7) M-1 s-1, k-1=29(+/-2) s-1) is followed by a protein conformational change (k+2=23(+/-3) s-1), monitored by an enhancement of Trp fluorescence. The second Trp fluorescence quenching phase is associated with binding of a second ATP. The first ATP appears to bind to the DNA-free subunit and hydrolysis induces a global conformational change to form a high energy intermediate state with tightly bound (ADP-Pi). Binding of the second ATP then leads to the steady-state ATP cycle. As proposed previously, the role of steady-state ATP hydrolysis by the DNA-bound Rep subunit may be to maintain the DNA-free subunit in an activated state in preparation for binding a second fragment of DNA as needed for translocation and/or DNA unwinding. We propose that the roles of the two ATP sites may alternate upon binding DNA to the second subunit of the Rep dimer during unwinding and translocation using a subunit switching mechanism.     

The interplay of DNA binding, ATP hydrolysis and helicase activities of the archaeal MCM helicase

The MCM (minichromosome maintenance) proteins of archaea are widely believed to be the replicative DNA helicase of these organisms. Most archaea possess a single MCM orthologue that forms homo-multimeric assemblies with a single hexamer believed to be the active form. In the present study we characterize the roles of highly conserved residues in the ATPase domain of the MCM of the hyperthermophilic archaeon Sulfolobus solfataricus. Our results identify a potential conduit for communicating DNA-binding information to the ATPase active site.     

One intact ATP-binding subunit is sufficient to support ATP hydrolysis and translocation in an ABC transporter, the histidine permease.

The membrane-bound complex of the Salmonella typhimurium histidine permease, a member of the ABC transporters (or traffic ATPases) superfamily, is composed of two integral membrane proteins, HisQ and HisM, and two copies of an ATP-binding subunit, HisP, which hydrolyze ATP, thus supplying the energy for translocation. The three-dimensional structure of HisP has been resolved. Extensive evidence indicates that the HisP subunits form a dimer. We investigated the mechanism of action of such a dimer, both within the complex and in soluble form, by creating heterodimers between the wild type and mutant HisP proteins. The data strongly suggest that within the complex both subunits hydrolyze ATP and that one subunit is activated by the other. In a heterodimer containing one wild type and one hydrolysis defective subunit both hydrolysis and ligand translocation occur at half the rate of the wild type. Soluble HisP also hydrolyzes ATP if one subunit is inactive; its specific activity is identical to that of the wild type, indicating that only one of the subunits in a soluble dimer is involved in hydrolysis. We show that the activating ability varies depending on the nature of the substitution of a well conserved residue, His-211.     

Coupling between ATP hydrolysis and protein conformational change in maltose transporter

As the intracellular part of maltose transporter, MalK dimer utilizes the energy of ATP hydrolysis to drive protein conformational change, which then facilitates substrate transport. Free energy evaluation of the complete conformational change before and after ATP hydrolysis is helpful to elucidate the mechanism of chemical‐to‐mechanical energy conversion in MalK dimer, but is lacking in previous studies. In this work, we used molecular dynamics simulations to investigate the structural transition of MalK dimer among closed, semi‐open and open states. We observed spontaneous structural transition from closed to open state in the ADP‐bound system and partial closure of MalK dimer from the semi‐open state in the ATP‐bound system. Subsequently, we calculated the reaction pathways connecting the closed and open states for the ATP‐ and ADP‐bound systems and evaluated the free energy profiles along the paths. Our results suggested that the closed state is stable in the presence of ATP but is markedly destabilized when ATP is hydrolyzed to ADP, which thus explains the coupling between ATP hydrolysis and protein conformational change of MalK dimer in thermodynamics. Proteins 2017; 85:207–220. © 2016 Wiley Periodicals, Inc.     

RNA aptamers to initiation factor 4A helicase hinder cap-dependent translation by blocking ATP hydrolysis

The mammalian translation initiation factor 4A (eIF4A) is a prototype member of the DEAD-box RNA helicase family that couples ATPase activity to RNA binding and unwinding. In the crystal form, eIF4A has a distended "dumbbell" structure consisting of two domains, which probably undergo a conformational change, on binding ATP, to form a compact, functional structure via the juxtaposition of the two domains. Moreover, additional conformational changes between two domains may be involved in the ATPase and helicase activity of eIF4A. The molecular basis of these conformational changes, however, is not understood. Here, we generated RNA aptamers with high affinity for eIF4A by in vitro RNA selection-amplification. On binding, the RNAs inhibit ATP hydrolysis. One class of RNAs contains members that exhibit dissociation constant of 27 nM for eIF4A and severely inhibit cap-dependent in vitro translation. The binding affinity was increased on Arg substitution in the conserved motif Ia of eIF4A, which probably improves a predicted arginine network to bind RNA substrates. Selected RNAs, however, failed to bind either domain of eIF4A that had been split at the linker site. These findings suggest that the selected RNAs interact cooperatively with both domains of eIF4A, either in the dumbbell or the compact form, and entrap it into a dead-end conformation, probably by blocking the conformational change of eIF4A. The selected RNAs, therefore, represent a new class of specific inhibitors that are suitable for the analysis of eukaryotic initiation, and which pose a potential therapeutic against malignancies that are caused by aberrant translational control.     

Nucleotide-binding domains of cystic fibrosis transmembrane conductance regulator, an ABC transporter, catalyze adenylate kinase activity but not ATP hydrolysis

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter family. CFTR consists of two transmembrane domains, two nucleotide-binding domains (NBD1 and NBD2), and a regulatory domain. Previous biochemical reports suggest NBD1 is a site of stable nucleotide interaction with low ATPase activity, whereas NBD2 is the site of active ATP hydrolysis. It has also been reported that NBD2 additionally possessed adenylate kinase (AK) activity. Knowledge about the intrinsic biochemical activities of the NBDs is essential to understanding the Cl(-) ion gating mechanism. We find that purified mouse NBD1, human NBD1, and human NBD2 function as adenylate kinases but not as ATPases. AK activity is strictly dependent on the addition of the adenosine monophosphate (AMP) substrate. No liberation of [(33)P]phosphate is observed from the gamma-(33)P-labeled ATP substrate in the presence or absence of AMP. AK activity is intrinsic to both human NBDs, as the Walker A box lysine mutations abolish this activity. At low protein concentration, the NBDs display an initial slower nonlinear phase in AK activity, suggesting that the activity results from homodimerization. Interestingly, the G551D gating mutation has an exaggerated nonlinear phase compared with the wild type and may indicate this mutation affects the ability of NBD1 to dimerize. hNBD1 and hNBD2 mixing experiments resulted in an 8-57-fold synergistic enhancement in AK activity suggesting heterodimer formation, which supports a common theme in ABC transporter models. A CFTR gating mechanism model based on adenylate kinase activity is proposed.     

The yeast a-factor transporter Ste6p, a member of the ABC superfamily, couples ATP hydrolysis to pheromone export

ATP-binding cassette (ABC) proteins transport a diverse collection of substrates. It is presumed that these proteins couple ATP hydrolysis to substrate transport, yet ATPase activity has been demonstrated for only a few. To provide direct evidence for such activity in Ste6p, the yeast ABC protein required for the export of a-factor mating pheromone, we established conditions for purification of Ste6p in biochemical quantities from both yeast and Sf9 insect cells. The basal ATPase activity of purified and reconstituted Ste6p (V(max) = 18 nmol/mg/min; K(m) for MgATP = 0.2 mm) compares favorably with several other ABC proteins and was inhibited by orthovanadate in a profile diagnostic of ABC transporters (apparent K(I) = 12 microm). Modest stimulation (approximately 40%) was observed upon the addition of a-factor either synthetic or in native form. We also used an 8-azido-[alpha-(32)P]ATP binding and vanadate-trapping assay to examine the behavior of wild-type Ste6p and two different double mutants (G392V/G1087V and G509D/G1193D) shown previously to be mating-deficient in vivo. Both mutants displayed a diminished ability to hydrolyze ATP, with the latter uncoupled from pheromone transport. We conclude that Ste6p catalyzes ATP hydrolysis coupled to a-factor transport, which in turn promotes mating.     

鼠脑驱动蛋白ATP水解机制的晶体学分析

鼠脑驱动蛋白是一类利用ATP水解释放的能量在微管系统上高连续性运动的常规驱动蛋白。了解ATP水解的化学能如何转化为机械动能是驱动蛋白研究中的重大课题。为此,鼠脑驱动蛋白单体(rK354)的晶体通过浸泡的方式引入ATP的结构类似物AMPPNP。rK354-AMPPNP复合物和rK354-ADP复合物结构的比较,揭示了开关区域Ⅱ的Glu237起连接ATP的γ-磷酸和驱动蛋白微管结合区的枢纽作用。     

Uncoupling substrate transport from ATP hydrolysis in the Escherichia coli maltose transporter

Members of the ATP-binding cassette superfamily couple the energy from ATP hydrolysis to the active transport of substrates across the membrane. The maltose transporter, a well characterized model system, consists of a periplasmic maltose-binding protein (MBP) and a multisubunit membrane transporter, MalFGK(2). On the basis of the structure of the MBP-MalFGK(2) complex in an outward-facing conformation (Oldham, M. L., Khare, D., Quiocho, F. A., Davidson, A. L., and Chen, J. (2007) Nature 450, 515-521), we identified two mutants in transmembrane domains MalF and MalG that generated futile cycling; although interaction with MBP stimulated the ATPase activity of the transporter, maltose was not transported. Both mutants appeared to disrupt the normal transfer of maltose from MBP to MalFGK(2). In the first case, substitution of aspartate for glycine in the maltose-binding site of MalF likely generated a futile cycle by preventing maltose from binding to MalFGK(2) during the catalytic cycle. In the second case, a four-residue deletion of a periplasmic loop of MalG limited its reach into the maltose-binding pocket of MBP, allowing maltose to remain associated with MBP during the catalytic cycle. Retention of maltose in the MBP binding site in the deletion mutant, as well as insertion of this loop into the binding site in the wild type, was detected by EPR as a change in mobility of a nitroxide spin label positioned near the maltose-binding pocket of MBP.