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.     

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.     

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.     

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.     

Maltose transport in membrane vesicles of Escherichia coli is linked to ATP hydrolysis.

We examined the energy requirement for maltose transport in right-side-out membrane vesicles derived from Escherichia coli. When membrane vesicles were made from strains producing tethered maltose-binding proteins by dilution of spheroplasts into phosphate buffer, those from an F0F1 ATPase-containing (unc+) strain transported maltose in the presence of an exogenous electron donor, such as ascorbate/phenazine methosulfate, at a rate of 1-5 nmol/min per mg of protein, whereas those from an isogenic unc- strain failed to transport maltose. Transport in vesicles obtained from the latter strain could be restored in the presence of electron donors if the vesicles were made to contain NAD+ and either ATP or an ATP-regenerating system. ATP hydrolysis was apparently required for transport, since nonhydrolyzable ATP analogues did not sustain transport. Maltose transport significantly increased ATP hydrolysis in ATP-containing vesicles from unc- cells. Finally, ATP-containing vesicles from unc- strains producing normal maltose-binding proteins could accumulate maltose in the absence of electron donors. These results provide convincing evidence that it is the hydrolysis of ATP that drives maltose transport, and probably also other periplasmic-binding-protein-dependent transport systems.     

A single intact ATPase site of the ABC transporter BtuCD drives 5% transport activity yet supports full in vivo vitamin B12 utilization

In all kingdoms of life, ATP binding cassette (ABC) transporters are essential to many cellular functions. In this large superfamily of proteins, two catalytic sites hydrolyze ATP to power uphill substrate translocation. A central question in the field concerns the relationship between the two ATPase catalytic sites: Are the sites independent of one another? Are both needed for function? Do they function cooperatively? These issues have been resolved for type I ABC transporters but never for a type II ABC transporter. The many mechanistic differences between type I and type II ABC transporters raise the question whether in respect to ATP hydrolysis the two subtypes are similar or different. We have addressed this question by studying the Escherichia coli vitamin B₁₂ type II ABC transporter BtuCD. We have constructed and purified a series of BtuCD variants where both, one, or none of the ATPase sites were rendered inactive by mutation. We find that, in a membrane environment, the ATPase sites of BtuCD are highly cooperative with a Hill coefficient of 2. We also find that, when one of the ATPase sites is inactive, ATP hydrolysis and vitamin B₁₂ transport by BtuCD is reduced by 95%. These exact features are also shared by the archetypical type I maltose ABC transporter. Remarkably, mutants that have lost 95% of their ATPase and transport capabilities still retain the ability to fully use vitamin B₁₂ in vivo. The results demonstrate that, despite the many differences between type I and type II ABC transporters, the fundamental mechanism of ATP hydrolysis remains conserved.     

ATP hydrolysis is required to reset the ATP-binding cassette dimer into the resting-state conformation

ATP-binding cassette (ABC) transporters couple ATP binding and hydrolysis to the movement of substances across the membrane; conformational changes clearly play an important role in the transporter mechanism. Previously, we have shown that a dimer of MalK, the ATPase subunit of the maltose transporter from Escherichia coli, undergoes a tweezers-like motion in a transport cycle. The MalK monomer consists of an N-terminal nucleotide binding domain and a C-terminal regulatory domain. The two nucleotide-binding domains in a dimer are either open or closed, depending on whether ATP is present, while the regulatory domains maintain contacts to hold the dimer together. In this work, the structure of MalK in a posthydrolysis state is presented, obtained by cocrystallizing MalK with ATP-Mg(2+). ATP was hydrolyzed in the crystallization drop, and ADP-Mg(2+) was found in the resulting crystal structure. In contrast to the ATP-bound form where two ATP molecules are buried in a closed interface between the nucleotide-binding domains, the two nucleotide-binding domains of the ADP-bound form are open, indicating that ADP, unlike ATP, cannot stabilize the closed form. This conclusion is further supported by oligomerization studies of MalK in solution. At low protein concentrations, ATP promotes dimerization of MalK, whereas ADP does not. The structures of dimeric MalK in the nucleotide-free, ATP-bound, and ADP-bound forms provide a framework for understanding the nature of the conformational changes that occur in an ATP-binding cassette transporter hydrolysis cycle, as well as how conformational changes in MalK are coupled to solute transport.     

Reconstitution of a bacterial periplasmic permease in proteoliposomes and demonstration of ATP hydrolysis concomitant with transport.

The histidine periplasmic permease of Salmonella typhimurium has been partially purified and reconstituted into proteoliposomes. In this in vitro preparation, transport activity is completely dependent on the presence of all four permease proteins (HisJ, HisQ, HisM, and HisP) and on internal ATP. The reconstituted system shows initial rates of transport that are comparable to those obtained with right-side-out membrane vesicles and it establishes a 100-fold concentration gradient for histidine. Proteoliposomes also transport histidine when GTP replaces ATP. Proteoliposomes do not catalyze significant ATP hydrolysis until histidine transport is initiated by addition of substrate along with HisJ, the water-soluble histidine-binding protein. Both initially and throughout the course of substrate transport there is a concomitant hydrolysis of ATP, with an apparent stoichiometry (ATP/histidine) of 5:1. These experiments demonstrate directly that ATP is the source of energy for periplasmic permeases, thus resolving previous controversies on this topic.     

Conformational plasticity of the type I maltose ABC importer

ATP-binding cassette (ABC) transporters couple the translocation of solutes across membranes to ATP hydrolysis. Crystal structures of the Escherichia coli maltose importer (MalFGK₂) in complex with its substrate binding protein (MalE) provided unprecedented insights in the mechanism of substrate translocation, leaving the MalE–transporter interactions still poorly understood. Using pulsed EPR and cross-linking methods we investigated the effects of maltose and MalE on complex formation and correlated motions of the MalK₂ nucleotide-binding domains (NBDs). We found that both substrate-free (open) and liganded (closed) MalE interact with the transporter with similar affinity in all nucleotide states. In the apo-state, binding of open MalE occurs via the N-lobe, leaving the C-lobe disordered, but upon maltose binding, closed MalE associates tighter to the transporter. In both cases the NBDs remain open. In the presence of ATP, the transporter binds both substrate-free and liganded MalE, both inducing the outward-facing conformation trapped in the crystal with open MalE at the periplasmic side and NBDs tightly closed. In contrast to ATP, ADP–Mg²⁺ alone is sufficient to induce a semiopen conformation in the NBDs. In this nucleotide-driven state, the transporter binds both open and closed MalE with slightly different periplasmic configurations. We also found that dissociation of MalE is not a required step for substrate translocation since a supercomplex with MalE cross-linked to MalG retains the ability to hydrolyze ATP and to transport maltose. These features of MalE–MalFGK₂ interactions highlight the conformational plasticity of the maltose importer, providing insights into the ATPase stimulation by unliganded MalE.ATP-binding cassette (ABC) systems are found in all kingdoms of life, forming one of the largest protein superfamilies (1–4). ABC transporters comprise two transmembrane domains (TMDs) that form the translocation pathway and two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP. Based on biochemical and structural evidence, all ABC transporters are thought to function by an “alternate-access” mode, with the translocation path shuttling between an inward-facing and outward-facing conformation in response to substrate and ATP binding, the latter causing the NBD dimer to close (5).Canonical ABC importers are subdivided into type I and type II based on structural and biochemical evidence (6) and are dependent on extracellular (or periplasmic) substrate binding proteins (SBPs) (4), which play a crucial role in initial steps of the transport cycle (7–10). SBPs generally consist of two symmetrical lobes that rotate toward each other upon substrate binding (11).The type I maltose transporter of Escherichia coli/Salmonella is probably the best understood ABC transporter to date (12). It is composed of the periplasmic maltose binding protein, MalE, the membrane-integral subunits, MalF and MalG, and the nucleotide-binding subunits (NBDs), MalK₂. The available crystal structures in the pretranslocation, ATP-, and vanadate-trapped states (13–16) have largely contributed to the understanding of the details of the inward- to outward-facing mechanism. The posthydrolytic state has not yet been crystallized, but data exist proposing this state to have a distinct structure from the other three known crystal snapshots (8, 9). MalE interacts with the transporter throughout the nucleotide cycle (15–18), and the X-ray structures revealed the switch of the binding protein from the liganded (closed) to the substrate-free (open) conformation concomitantly with ATP binding to the NBDs. Despite all these structural insights, the response of the transporter to substrate availability is poorly understood. Furthermore, the mechanism behind the stimulation of the ATPase activity of the transporter by unliganded MalE (10, 19) is still elusive (20, 21).Our results show that the apo- and ADP-states of the transporter bind both open and closed MalE, but the complex adopts different periplasmic configurations. The ATP-state of the transporter can bind either closed MalE, inducing its opening and release of substrate to MalF or directly unliganded MalE, which generates a futile cycle. In both cases the NBDs are triggered into a fully closed dimer primed for ATP hydrolysis. Furthermore, we demonstrate that dissociation of the binding protein is not a prerequisite for substrate translocation. These features highlight the conformational plasticity of a type I ABC importer.     

Regulation of the nucleotide dependence of the cardiac sarcolemma Ca(2+)-ATPase.

The nucleotide dependence of the Ca(2+)-ATPase purified from cardiac sarcolemma by calmodulin-affinity chromatography was investigated for preparations either in the basal state or activated by three procedures: (i) addition of calmodulin; (ii) addition of phosphatidylserine and (iii) controlled proteolysis. Upon activation, the maximal velocity of ATP hydrolysis increases by a factor of 4-5, while the curves of ATP dependence of ATP hydrolysis change from hyperbolic to biphasic, revealing the presence of two Kmapp for ATP. A tight coupling between Ca2+ and ATP binding sites was also observed. At high ATP concentration, the ATPase activity of the basal state shows a complex dependence on Ca2+ concentration, increasing sharply at millimolar Ca2+. Our results indicate that this increase in ATPase activity is paralleled by the appearance of a second, low affinity Kmapp for ATP. When only the high affinity site for ATP is occupied the ATPase activity of the basal state displays a simple, hyperbolic dependence on the Ca2+ concentration. In addition, increasing Ca2+ concentration appears to decrease the ATP binding at the low affinity site of the enzyme. The effect of ADP on ATP hydrolysis was also examined. The finding that ADP is a potent inhibitor of the purified Ca(2+)-ATPase from heart suggests that the stimulatory action of ADP observed in cardiac sarcolemmal vesicles is not an intrinsic property of the enzyme.     

A mutation of the H-loop selectively affects rhodamine transport by the yeast multidrug ABC transporter Pdr5

The yeast ABC transporter Pdr5 plays a major role in drug resistance against a large number of structurally unrelated compounds. Although Pdr5 has been extensively studied, many important aspects regarding its molecular mechanisms remain unresolved. For example, a striking degeneration of conserved amino acid residues exists in the nucleotide binding domains (NBDs), but their functional relevance is unknown. Here, we performed in vivo and in vitro experiments to address the functional asymmetry of NBDs. It became evident by ATPase activity and drug transport studies that catalysis at only one of the two NBD composite sites is crucial for protein function. Furthermore, mutations of the proposed "catalytic carboxylate" (E1036) and the "catalytic dyad histidine" (H1068) were characterized. Although a mutation of the glutamate abolished ATPase activity and substrate transport, mutation of H1068 had no influence on ATP consumption. However, the H1068A mutation abolished rhodamine transport in vivo and in vitro, while leaving the transport of other substrates unaffected. By contrast to mammalian P-glycoprotein (P-gp), the ATPase activity of yeast Pdr5 is not stimulated by the addition of substrates, indicating that Pdr5 is an uncoupled ABC transporter that constantly hydrolyses ATP to ensure active substrate transport. Taken together, our data provide important insights into the molecular mechanism of Pdr5 and suggest that not solely the transmembrane domains dictate substrate selection.     

Mechanism of regulation of Hsp70 chaperones by DnaJ cochaperones

Hsp70 chaperones assist a large variety of protein folding processes within the entire lifespan of proteins. Central to these activities is the regulation of Hsp70 by DnaJ cochaperones. DnaJ stimulates Hsp70 to hydrolyze ATP, a key step that closes its substrate-binding cavity and thus allows stable binding of substrate. We show that DnaJ stimulates ATP hydrolysis by Escherichia coli Hsp70, DnaK, very efficiently to >1000-fold, but only if present at high (micromolar) concentration. In contrast, the chaperone activity of DnaK in luciferase refolding was maximal at several hundredfold lower concentration of DnaJ. However, DnaJ was capable of maximally stimulating the DnaK ATPase even at this low concentration, provided that protein substrate was present, indicating synergistic action of DnaJ and substrate. Peptide substrates were poorly effective in this synergistic action. DnaJ action required binding of protein substrates to the central hydrophobic pocket of the substrate-binding cavity of DnaK, as evidenced by the reduced ability of DnaJ to stimulate ATP hydrolysis by a DnaK mutant with defects in substrate binding. At high concentrations, DnaJ itself served as substrate for DnaK in a process considered to be unphysiological. Mutant analysis furthermore revealed that DnaJ-mediated stimulation of ATP hydrolysis requires communication between the ATPase and substrate-binding domains of DnaK. This mechanism thus allows DnaJ to tightly couple ATP hydrolysis by DnaK with substrate binding and to avoid jamming of the DnaK chaperone with peptides. It probably is conserved among Hsp70 family members and is proposed to account for their functional diversity.     

KATP channel interaction with adenine nucleotides

ATP-sensitive potassium (K(ATP)) channels are regulated by adenine nucleotides to convert changes in cellular metabolic levels into membrane excitability. Hence, elucidation of interaction of SUR and Kir6.x with adenine nucleotides is an important issue to understand the molecular mechanisms underlying the metabolic regulation of the K(ATP) channels. We analyzed direct interactions with adenine nucleotides of each subunit of K(ATP) channels. Kir6.2 binds adenine nucleotides in a Mg(2+)-independent manner. SUR has two NBFs which are not equivalent: NBF1 is a Mg(2+)-independent high affinity nucleotide binding site, whereas NBF2 is a Mg-dependent low affinity site. Although SUR has ATPase activity at NBF2, it is not used to transport substrates against the concentration gradient unlike other ABC proteins. The ATPase cycle at NBF2 serves as a sensor of cellular metabolism. This may explain the low ATP hydrolysis rate compared to other ABC proteins. Based on studies of photoaffinity labeling, a model of K(ATP) channel regulation is proposed, in which K(ATP) channel activity is regulated by SUR via monitoring the intracellular MgADP concentration. K(ATP) channel activation is expected to be induced by the cooperative interaction of ATP binding at NBF1 and MgADP binding at NBF2.     

In vitro disassembly and reassembly of an ABC transporter, the histidine permease

The membrane-bound complex of the Salmonella typhimurium periplasmic 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. The complex hydrolyzes ATP upon induction of the activity by the liganded soluble receptor, the periplasmic histidine-binding protein, HisJ. Here we take advantage of the modular organization of this system to show that the nucleotide-binding component can be stripped off the integral membrane components, HisQ and HisM. The complex can be reconstituted by using the HisP-depleted membranes containing HisQ and HisM and pure soluble HisP. We show that HisP has high affinity for the HisP-depleted complex, HisQM, and that two HisP molecules are recruited independently of each other for each HisQM unit. The in vitro reassembled complex has entirely normal properties, responding to HisJ and ATPase inhibitors with the same characteristics as the original complex and in contrast to those of soluble HisP. These results show that HisP is absolutely required for ATP hydrolysis, that HisQM cannot hydrolyze ATP, that HisP depends on HisQM to relay the inducing signal from the soluble receptor, HisJ, and that HisQM regulates the ATPase activity of HisP. We also show that HisP changes conformation upon exposure to phospholipids.     

Functional characterization of the adrenoleukodystrophy protein (ALDP) and disease pathogenesis

X-linked adrenoleukodystrophy (X-ALD) is the most common peroxisomal disorder characterized by abnormal accumulation of saturated very long chain fatty acids in tissues and body fluids with predominance in brain white matter and adrenal cortex. The clinical phenotype is highly variable ranging from the severe childhood cerebral form to asymptomatic persons. The responsible ALD gene encodes the adrenoleukodystrophy protein (ALDP), a peroxisomal integral membrane protein that is a member of the ATP-binding cassette (ABC) transporter protein family. The patient gene mutations are heterogeneously distributed over the functional domains of ALDP. The extreme variability in clinical phenotype, even within one affected family, indicates that besides the ALD gene mutations other factors strongly influence the clinical phenotype. To understand the cell biology and function of mammalian peroxisomal ABC transporters and to determine their role in the pathogenesis of X-ALD we developed a system for expressing functional ABC protein domains in fusion with the maltose binding protein. Wild type and mutant fusion proteins of the nucleotide-binding fold were overexpressed, purified, and characterized by photoaffinity labeling with 8-azido ATP or 8-azido GTP and a coupled ATP regenerating enzyme assay for ATPase activity. Our studies provide evidence that peroxisomal ABC transporters utilize ATP to become a functional transporter and that ALD gene mutations alter peroxisomal transport function. The established disease model will be used further to study the influence of possible disease modifier proteins on ALDP function.     

Increased ATPase activity produced by mutations at arginine-1380 in nucleotide-binding domain 2 of ABCC8 causes neonatal diabetes

Gain-of-function mutations in the genes encoding the ATP-sensitive potassium (K(ATP)) channel subunits Kir6.2 (KCNJ11) and SUR1 (ABCC8) are a common cause of neonatal diabetes mellitus. Here we investigate the molecular mechanism by which two heterozygous mutations in the second nucleotide-binding domain (NBD2) of SUR1 (R1380L and R1380C) separately cause neonatal diabetes. SUR1 is a channel regulator that modulates the gating of the pore formed by Kir6.2. K(ATP) channel activity is inhibited by ATP binding to Kir6.2 but is stimulated by MgADP binding, or by MgATP binding and hydrolysis, at the NBDs of SUR1. Functional analysis of purified NBD2 showed that each mutation enhances MgATP hydrolysis by purified isolated fusion proteins of maltose-binding protein and NBD2. Inhibition of ATP hydrolysis by MgADP was unaffected by mutation of R1380, but inhibition by beryllium fluoride (which traps the ATPase cycle in the prehydrolytic state) was reduced. MgADP-dependent activation of K(ATP) channel activity was unaffected. These data suggest that the R1380L and R1380C mutations enhance the off-rate of P(i), thereby enhancing the hydrolytic rate. Molecular modeling studies supported this idea. Because mutant channels were inhibited less strongly by MgATP, this would increase K(ATP) currents in pancreatic beta cells, thus reducing insulin secretion and producing diabetes.     

Adenosine 5'-triphosphate-dependent vitamin D sterol binding to heat shock protein-70 chaperones

Chaperone proteins in the heat shock protein-70 family possess endogenous ATP binding and ATPase activity and interact with intracellular protein substrates in an ATP-dependent manner; the hydrolysis of ATP to ADP results in an increase in the affinity of the chaperone for protein substrates. Heat shock protein-70s can also specifically interact with 25-hydroxylated vitamin D metabolites. Using constitutively expressed heat shock protein-70 (hsc70) as chaperone, here we demonstrate that vitamin D metabolite binding to hsc70 is also ATP dependent. Transient overexpression of an hsc70-green fluorescent protein chimeric construct in primate kidney cells resulted in a 6-fold increase in specific, extractable 25-hydroxyvitamin D(3) binding. When ATPase capability of hsc70 was disabled, this increase was completely blocked. In solution, the binding of 25-hydroxylated vitamin D metabolites to hsc70 was significantly increased (P < 0.01) in the presence of ATP and a nonmetabolizable ATP analog. The ATP-directed increase in specific binding resulted from an increase in the abundance of relatively high-affinity hormone-binding sites (K(d), approximately 0.24 nM). These results suggest that ATP hydrolysis to ADP would favor the release of vitamin D from a donor hsc70 molecule at a time when an hsc70-bound acceptor protein substrate is anchored to the chaperone with relative avidity. We theorize that the endogenous ATPase activity of hsc70 promotes the transfer of vitamin D sterols to other intracellular vitamin D binding proteins, such as the vitamin D receptor and vitamin D hydroxylases, to which hsc70 is known to bind.     

Properties of the duplex DNA-dependent ATPase activity of Escherichia coli RecA protein and its role in branch migration.

We have investigated the double-stranded DNA (dsDNA)-dependent ATPase activity of recA protein. This activity is distinguished from the single-stranded DNA (ssDNA)-dependent ATPase activity by the presence of a pronounced lag time before the onset of steady-state ATP hydrolysis. During the lag phase there is little ATP hydrolysis. The duration of the lag phase, referred to as the lag time, is found to increase with the thermal stability of the dsDNA substrate. Increasing either the MgCl2 or NaCl concentration increases the lag time, whereas increasing the temperature decreases the lag time. The lag time shows little dependence on recA protein concentration but is strongly dependent on ATP concentration. After the lag phase, a steady-state ATP hydrolysis rate is achieved that approaches the rate observed with ssDNA. The steady-state phase of the reaction is proportional to the concentration of recA protein-DNA complex and shows saturation behavior at approximately equal to 5 +/- 1 base pairs per recA protein monomer. These results suggest that the lag phase represents a rate-limiting step in the dsDNA-dependent ATP hydrolysis reaction that requires a structural transition in the dsDNA and that involves a ternary complex of ATP, recA protein, and DNA. We propose that this transition involves the transient denaturation of the dsDNA to form regions of ssDNA. Elsewhere we demonstrate that the dsDNA-dependent ATPase activity is proportional to the rate of recA protein-catalyzed branch migration. We suggest that this activity is responsible for a polar polymerization that drives the branch migration reaction.     

Evidence for a requirement for ATP hydrolysis at two distinct steps during a single turnover of the catalytic cycle of human P-glycoprotein

P-glycoprotein (Pgp) is an ATP-dependent hydrophobic natural product anticancer drug efflux pump whose overexpression confers multidrug resistance to tumor cells. The work reported here deals with the elucidation of the energy requirement for substrate interaction with Pgp during the catalytic cycle. We show that the K(d) (412 nM) of the substrate analogue [(125)I]iodoarylazidoprazoin for Pgp is not altered by the presence of the nonhydrolyzable nucleotide 5'-adenylylimididiphosphate and vanadate (K(d) = 403 nM). Though binding of nucleotide per se does not affect interactions with the substrate, ATP hydrolysis results in a dramatic conformational change where the affinity of [(125)I]iodoarylazidoprazoin for Pgp trapped in transition-state conformation (Pgp x ADP x vanadate) is reduced >30-fold. To transform Pgp from this intermediate state of low affinity for substrate to the next catalytic cycle, i.e., a conformation that binds substrate with high affinity, requires conditions that permit ATP hydrolysis. Additionally, there is an inverse correlation (R(2) = 0.96) between 8AzidoADP (or ADP) release and the recovery of substrate binding. These results suggest that the release of nucleotide is necessary for reactivation but not sufficient. The hydrolysis of additional molecule(s) of ATP (or 8AzidoATP) is obligatory for the catalytic cycle to advance to completion. These data are consistent with the observed stoichiometry of two ATP molecules hydrolyzed for the transport of every substrate molecule. Our data demonstrate two distinct roles for ATP hydrolysis in a single turnover of the catalytic cycle of Pgp, one in the transport of substrate and the other in effecting conformational changes to reset the pump for the next catalytic cycle.     

Assembly and mechanism of a group II ECF transporter

Energy-coupling factor (ECF) transporters are a recently discovered family of primary active transporters for micronutrients and vitamins, such as biotin, thiamine, and riboflavin. Found exclusively in archaea and bacteria, including the human pathogens Listeria, Streptococcus, and Staphylococcus, ECF transporters may be the only means of vitamin acquisition in these organisms. The subunit composition of ECF transporters is similar to that of ATP binding cassette (ABC) importers, whereby both systems share two homologous ATPase subunits (A and A′), a high affinity substrate-binding subunit (S), and a transmembrane coupling subunit (T). However, the S subunit of ECF transporters is an integral membrane protein, and the transmembrane coupling subunits do not share an obvious sequence homology between the two transporter families. Moreover, the subunit stoichiometry of ECF transporters is controversial, and the detailed molecular interactions between subunits and the conformational changes during substrate translocation are unknown. We have characterized the ECF transporters from Thermotoga maritima and Streptococcus thermophilus. Our data suggests a subunit stoichiometry of 2S:2T:1A:1A′ and that S subunits for different substrates can be incorporated into the same transporter complex simultaneously. In the first crystal structure of the A–A′ heterodimer, each subunit contains a novel motif called the Q-helix that plays a key role in subunit coupling with the T subunits. Taken together, these findings suggest a mechanism for coupling ATP binding and hydrolysis to transmembrane transport by ECF transporters.