MBP-MalFGK2相互作用动力学:ABC转运蛋白的模型研究

本文主要描述了麦芽糖结合蛋白(MBP)和属于ATP结合盒式蛋白(ABC)家族的麦芽糖转运蛋白复合物MalFGK2的相互作用。通过基因、结构和生化分析可知,MBP和MalFGK2以不同构象进行相互作用。在这个转运系统中,MBP与麦芽糖结合,并与MalFGK2发生相互作用,从而将麦芽糖从胞外转运至胞内,但由于MBP和MalFGK2都有多种构象,所以它们的相互作用很复杂。相互作用机理模型最重要的特点是结合配体的MBP,通过稳定MalFGK2的高能量构象来启动依赖ATP的麦芽糖转运过程。麦芽糖转运蛋白机理模型表明,ABC型转运系统利用外周结合蛋白,其转运过程基本上是不可逆的。     

Full engagement of liganded maltose‐binding protein stabilizes a semi‐open ATP‐binding cassette dimer in the maltose transporter

MalFGK₂ is an ATP‐binding cassette (ABC) transporter that mediates the uptake of maltose/maltodextrins into Escherichia coli. A periplasmic maltose‐binding protein (MBP) delivers maltose to the transmembrane subunits (MalFG) and stimulates the ATPase activity of the cytoplasmic nucleotide‐binding subunits (MalK dimer). This MBP‐stimulated ATPase activity is independent of maltose for purified transporter in detergent micelles. However, when the transporter is reconstituted in membrane bilayers, only the liganded form of MBP efficiently stimulates its activity. To investigate the mechanism of maltose stimulation, electron paramagnetic resonance spectroscopy was used to study the interactions between the transporter and MBP in nanodiscs and in detergent. We found that full engagement of both lobes of maltose‐bound MBP unto MalFGK₂ is facilitated by nucleotides and stabilizes a semi‐open MalK dimer. Maltose‐bound MBP promotes the transition to the semi‐open state of MalK when the transporter is in the membrane, whereas such regulation does not require maltose in detergent. We suggest that stabilization of the semi‐open MalK₂ conformation by maltose‐bound MBP is key to the coupling of maltose transport to ATP hydrolysis in vivo, because it facilitates the progression of the MalK dimer from the open to the semi‐open conformation, from which it can proceed to hydrolyze ATP.     

Sequential Action of MalE and Maltose Allows Coupling ATP Hydrolysis to Translocation in the MalFGK2 Transporter

ATP-binding cassette (ABC) transporters have evolved an ATP-dependent alternating-access mechanism to transport substrates across membranes. Despite important progress, especially in their structural analysis, it is still unknown how the substrate stimulates ATP hydrolysis, the hallmark of ABC transporters. In this study, we measure the ATP turnover cycle of MalFGK₂ in steady and pre-steady state conditions. We show that (i) the basal ATPase activity of MalFGK₂ is very low because the cleavage of ATP is rate-limiting, (ii) the binding of open-state MalE to the transporter induces ATP cleavage but leaves release of P_i limiting, and (iii) the additional presence of maltose stimulates release of P_i, and therefore increases the overall ATP turnover cycle. We conclude that open-state MalE stabilizes MalFGK₂ in the outward-facing conformation until maltose triggers return to the inward-facing state for substrate and P_i release. This concerted action explains why ATPase activity of MalFGK₂ depends on maltose, and why MalE is essential for transport.     

Nucleotide-free MalK Drives the Transition of the Maltose Transporter to the Inward-facing Conformation

The complex MalFGK₂ hydrolyzes ATP and alternates between inward- and outward-facing conformations during maltose transport. It has been shown that ATP promotes closure of MalK₂ and opening of MalFG toward the periplasm. Yet, why the transporter rests in a conformation facing the cytosol in the absence of nucleotide and how it returns to this state after hydrolysis of ATP is unknown. The membrane domain MalFG may be naturally stable in the inward-facing conformation, or the ABC domain may catalyze the transition. We address this question by analyzing the conformation of MalFG in nanodiscs and in proteoliposomes. We find that MalFG alone exists in an intermediate state until MalK binds and converts the membrane domain to the inward-facing state. We also find that MalK, if overly-bound to MalFG, blocks the transition of the transporter, whereas suppressor mutations that weaken this association restore transport. MalK therefore exploits hydrolysis of ATP to reverse the conformation of MalFG to the inward-facing conformation, a step essential for release of maltose in the cytosol.     

Transmembrane Signaling in the Maltose ABC Transporter MalFGK2-E: PERIPLASMIC MalF-P2 LOOP COMMUNICATES SUBSTRATE AVAILABILITY TO THE ATP-BOUND MalK DIMER*

ABC transporters are ubiquitous membrane proteins that translocate solutes across biological membranes at the expense of ATP. In prokaryotic ABC importers, the extracytoplasmic anchoring of the substrate-binding protein (receptor) is emerging as a key determinant for the structural rearrangements in the cytoplasmically exposed ATP-binding cassette domains and in the transmembrane gates during the nucleotide cycle. Here the molecular mechanism of such signaling events was addressed by electron paramagnetic resonance spectroscopy of spin-labeled ATP-binding cassette maltose transporter variants (MalFGK₂-E). A series of doubly spin-labeled mutants in the MalF-P2 domain involving positions 92, 205, 239, 252, and 273 and one triple mutant labeled at positions 205/252 in P2 and 83 in the Q-loop of MalK were assayed. The EPR data revealed that the substrate-binding protein MalE is bound to the transporter throughout the transport cycle. Concomitantly with the three conformations of the ATP-binding cassette MalK₂, three functionally relevant conformations are found also in the periplasmic MalF-P2 loop, strictly dependent on cytoplasmic nucleotide binding and periplasmic docking of liganded MalE to MalFG. The reciprocal communication across the membrane unveiled here gives first insights into the stimulatory effect of MalE on the ATPase activity, and it is suggested to be an important mechanistic feature of receptor-coupled ABC transporters.     

Large-scale purification, dissociation and functional reassembly of the maltose ATP-binding cassette transporter (MalFGK2) of Salmonella typhimurium

The maltose ATP-binding cassette (ABC) transporter of Salmonella typhimurium is composed of a membrane-associated complex (MalFGK₂) and a periplasmic substrate binding protein. To further elucidate protein-protein interactions between the subunits, we have studied the dissociation and reassembly of the MalFGK₂ complex at the level of purified components in proteoliposomes. First, we optimized the yield in purified complex protein by taking advantage of a newly constructed expression plasmid that carries the malK, malF and malG genes in tandem orientation. Incorporated in proteoliposomes, the complex exhibited maltose binding protein/maltose-dependent ATPase activity with a V_max of 1.25 μmol P_i/min/mg and a K_m of 0.1 mM. ATPase activity was sensitive to vanadate and enzyme IIA^Glc, a component of the enterobacterial glucose transport system. The proteoliposomes displayed maltose transport activity with an initial rate of 61 nmol/min/mg. Treatment of proteoliposomes with 6.6 M urea resulted in the release of medium-exposed MalK subunits concomitant with the complete loss of ATPase activity. By adding increasing amounts of purified MalK to urea-treated proteoliposomes, about 50% of vanadate-sensitive ATPase activity relative to the control could be recovered. Furthermore, the phenotype of MalKQ140K that exhibits ATPase activity in solution but not when associated with MalFG was confirmed by reassembly with MalK-depleted proteoliposomes.     

The mechanism of nucleotide‐binding domain dimerization in the intact maltose transporter as studied by all‐atom molecular dynamics simulations

The Escherichia coli maltose transporter MalFGK₂‐E belongs to the protein superfamily of ATP‐binding cassette (ABC) transporters. This protein is composed of heterodimeric transmembrane domains (TMDs) MalF and MalG, and the homodimeric nucleotide‐binding domains (NBDs) MalK₂. In addition to the TMDs and NBDs, the periplasmic maltose binding protein MalE captures maltose and shuttle it to the transporter. In this study, we performed all‐atom molecular dynamics (MD) simulations on the maltose transporter and found that both the binding of MalE to the periplasmic side of the TMDs and binding of ATP to the MalK₂ are necessary to facilitate the conformational change from the inward‐facing state to the occluded state, in which MalK₂ is completely dimerized. MalE binding suppressed the fluctuation of the TMDs and MalF periplasmic region (MalF‐P2), and thus prevented the incorrect arrangement of the MalF C‐terminal (TM8) helix. Without MalE binding, the MalF TM8 helix showed a tendency to intrude into the substrate translocation pathway, hindering the closure of the MalK₂. This observation is consistent with previous mutagenesis experimental results on MalF and provides a new point of view regarding the understanding of the substrate translocation mechanism of the maltose transporter.     

A network of dynamically conserved residues deciphers the motions of maltose transporter

The maltose transporter of Escherichia coli is a member of the ATP‐binding cassette (ABC) transporter superfamily. The crystal structures of maltose transporter MalK have been determined for distinct conformations in the presence and absence of the ligand ATP, and other interacting proteins. Using the distinct MalK structures, normal mode analysis was performed to understand the dynamics behavior of the system. A network of dynamically important residues was obtained from the normal mode analysis and the analysis of point mutation on the normal modes. Our results suggest that the intradomain rotation occurs earlier than the interdomain rotation during the maltose‐binding protein (MBP)‐driven conformational changes of MalK. We inquire if protein motion and functional‐driven evolutionary conservation are related. The sequence conservation of MalK was analyzed to derive a network of evolutionarily important residues. There are highly significant correlations between protein sequence and protein dynamics in many regions on the maltose transporter MalK, suggesting a link between the protein evolution and dynamics. The significant overlaps between the network of dynamically important residues and the network of evolutionarily important residues form a network of dynamically conserved residues. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

A comparative electron paramagnetic resonance study of the nucleotide-binding domains' catalytic cycle in the assembled maltose ATP-binding cassette importer

We present a quantitative analysis of conformational changes of the nucleotide-binding subunits, MalK₂, of the maltose ATP-binding cassette importer MalFGK₂ during the transport cycle. Distance changes occurring between selected residues were monitored in the full transporter by site-directed spin-labeling electron paramagnetic resonance spectroscopy and site-directed chemical cross-linking. We considered S83C and A85C from the conserved Q-loop and V117C located on the outer surface of MalK. Additionally, two native cysteines (C350, C360) were included in the study. On ATP binding, small rearrangements between the native sites, and no distance changes between positions 117 were detected. In contrast, positions 85 come closer together in the ATP-bound state and in the vanadate-trapped intermediate and move back toward the apo-state after ATP hydrolysis. The distance between positions 83 is shown to slightly decrease on ATP binding, and to further decrease after ATP hydrolysis. Results from cross-linking experiments are in agreement with these findings. The data are compared with in silico spin-labeled x-ray structures from both isolated MalK₂ and the MalFGK₂-E complex. Our results are consistent with a slightly modified “tweezers-like” model of closure and reopening of MalK₂ during the catalytic cycle, and show an unforeseen potential interaction between MalK and the transmembrane subunit MalG.     

Molecular dynamics simulations reveal that apo‐HisJ can sample a closed conformation

The Escherichia coli histidine binding protein HisJ is a type II periplasmic binding protein (PBP) that preferentially binds histidine and interacts with its cytoplasmic membrane ABC transporter, HisQMP₂, to initiate histidine transport. HisJ is a bilobal protein where the larger Domain 1 is connected to the smaller Domain 2 via two linking strands. Type II PBPs are thought to undergo “Venus flytrap” movements where the protein is able to reversibly transition from an open to a closed conformation. To explore the accessibility of the closed conformation to the apo state of the protein, we performed a set of all‐atom molecular dynamics simulations of HisJ starting from four different conformations: apo‐open, apo‐closed, apo‐semiopen, and holo‐closed. The simulations reveal that the closed conformation is less dynamic than the open one. HisJ experienced closing motions and explored semiopen conformations that reverted to closed forms resembling those found in the holo‐closed state. Essential dynamics analysis of the simulations identified domain closing/opening and twisting as main motions. The formation of specific inter‐hinge strand and interdomain polar interactions contributed to the adoption of the closed apo‐conformations although they are up to 2.5‐fold less prevalent compared with the holo‐closed simulations. The overall sampling of the closed form by apo‐HisJ provides a rationale for the binding of unliganded PBPs with their cytoplasmic membrane ABC transporters. Proteins 2014; 82:386–398. © 2013 Wiley Periodicals, Inc.     

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.     

Phosphatidylglycerol Directs Binding and Inhibitory Action of EIIAGlc Protein on the Maltose Transporter

The signal-transducing protein EIIA^Glc belongs to the phosphoenolpyruvate carbohydrate phosphotransferase system. In its dephosphorylated state, EIIA^Glc is a negative regulator for several permeases, including the maltose transporter MalFGK₂. How EIIA^Glc is targeted to the membrane, how it interacts with the transporter, and how it inhibits sugar uptake remain obscure. We show here that acidic phospholipids together with the N-terminal tail of EIIA^Glc are essential for the high affinity binding of the protein to the transporter. Using protein docking prediction and chemical cross-linking, we demonstrate that EIIA^Glc binds to the MalK dimer, interacting with both the nucleotide-binding and the C-terminal regulatory domains. Dissection of the ATPase cycle reveals that EIIA^Glc does not affect the binding of ATP but rather inhibits the capacity of MalK to cleave ATP. We propose a mechanism of maltose transport inhibition by this central amphitropic regulatory protein.     

Biochemical evidence for the presence of two alpha-glucoside ABC-transport systems in the hyperthermophilic archaeon Pyrococcus furiosus

The hyperthermophilic archaeon Pyrococcus furiosus can utilize different carbohydrates, such as starch, maltose and trehalose. Uptake of alpha-glucosides is mediated by two different, binding protein-dependent, ATP-binding cassette (ABC)-type transport systems. The maltose transporter also transports trehalose, whereas the maltodextrin transport system mediates the uptake of maltotriose and higher malto-oligosaccharides, but not maltose. Both transport systems are induced during growth on their respective substrates.     

NMR assignments of the periplasmic loop P2 of the MalF subunit of the maltose ATP binding cassette transporter

We have assigned the ¹H, ¹⁵N, ¹³C backbone resonances of the second periplasmic loop P2 of the MalF subunit of the maltose ATP binding cassette transporter of Escherichia coli/Salmonella which is important for the recognition of the maltose binding protein MalE.     

Maltose-binding protein is open in the catalytic transition state for ATP hydrolysis during maltose transport

The maltose transport complex of Escherichia coli, a member of the ATP-binding cassette superfamily, mediates the high affinity uptake of maltose at the expense of ATP. The membrane-associated transporter consists of two transmembrane subunits, MalF and MalG, and two copies of the cytoplasmic ATP-binding cassette subunit, MalK. Maltose-binding protein (MBP), a soluble periplasmic protein, delivers maltose to the MalFGK(2) transporter and stimulates hydrolysis by the transporter. Site-directed spin labeling electron paramagnetic resonance spectroscopy is used to monitor binding of MBP to MalFGK(2) and conformational changes in MBP as it interacts with MalFGK(2). Cysteine residues and spin labels have been introduced into the two lobes of MBP so that spin-spin interaction will report on ligand-induced closure of the protein (Hall, J. A., Thorgeirsson, T. E., Liu, J., Shin, Y. K., and Nikaido, H. (1997) J. Biol. Chem. 272, 17610-17614). At least two different modes of interaction between MBP and MalFGK(2) were detected. Binding of MBP to MalFGK(2) in the absence of ATP resulted in a decrease in motion of spin label at position 41 in the C-terminal domain of MBP. In a vanadate-trapped transition state intermediate, all free MBP became tightly bound to MalFGK(2), spin label in both lobes became completely immobilized, and spin-spin interactions were lost, suggesting that MBP was in an open conformation. Binding of non-hydrolyzable MgATP analogs or ATP in the absence of Mg is sufficient to stabilize a complex of open MBP and MalFGK(2). Taken together, these data suggest that closure of the MalK dimer interface coincides with opening of MBP and maltose release to the transporter.     

<Emphasis Type="Italic">Escherichia coli tat</Emphasis> mutant strains are able to transport maltose in the absence of an active <Emphasis Type="Italic">malE</Emphasis> gene

The twin-arginine transport (Tat) system is a prokaryotic protein transport system. Escherichia coli mutants in this pathway show a defect in cell separation during cell division, resulting in destabilization and permeability of the outer membrane. Maltose uptake is catalysed by a membrane-bound transporter of the ATP binding cassette (ABC) superfamily, where MalE is the essential periplasmic binding protein component. Here, we report that tat mutants are unexpectedly able to transport maltose in the absence of malE. This observation is specific to the MalE component since co-inactivation of malF, which encodes one of the channel components of the transporter, completely abolishes maltose transport even when the Tat system is inactivated. Genetic repair of the outer membrane leaky phenotype of the tat mutant strain re-established the absolute requirement for MalE in maltose uptake. In addition, we demonstrate that phenotypic repair of the outer membrane defect of the tat strain can also be achieved chemically by the inclusion of high concentrations of calcium or magnesium in the growth medium.     

Stability of Ligand-induced Protein Conformation Influences Affinity in Maltose-binding Protein

Our understanding of what determines ligand affinity of proteins is poor, even with high-resolution structures available. Both the non-covalent ligand–protein interactions and the relative free energies of available conformations contribute to the affinity of a protein for a ligand. Distant, non-binding site residues can influence the ligand affinity by altering the free energy difference between a ligand-free and ligand-bound conformation. Our hypothesis is that when different ligands induce distinct ligand-bound conformations, it should be possible to tweak their affinities by changing the free energies of the available conformations. We tested this idea for the maltose-binding protein (MBP) from Escherichia coli. We used single-molecule Förster resonance energy transfer (smFRET) to distinguish several unique ligand-bound conformations of MBP. We engineered mutations, distant from the binding site, to affect the stabilities of different ligand-bound conformations. We show that ligand affinity can indeed be altered in a conformation-dependent manner. Our studies provide a framework for the tuning of ligand affinity, apart from modifying binding site residues.     

Functional reconstitution of a maltose ATP-binding cassette transporter from the thermoacidophilic gram-positive bacterium Alicyclobacillus acidocaldarius

The thermoacidophilic gram-positive bacterium Alicyclobacillus acidocaldarius grows at 60 °C and pH 2-3. The organism can utilize maltose and maltodextrins as energy source that are taken up by an ATP-binding cassette (ABC) import system. Genes encoding a maltose binding protein, MalE, and two membrane-integral subunits, MalF and MalG, are clustered on the chromosome but a malK gene translating into a cognate ATPase subunit is lacking. Here we report the cloning of malK from genomic DNA by using the msiK gene of Streptomyces lividans as a probe. Purified MalK exhibited a spontaneous ATPase activity with a V_max of 0.13 μmol P_i/min/mg and a K_m of 330 μM that was optimal at the growth temperature of the organism. Coexpression of malK, malF and malG in Escherichia coli resulted in the formation of a complex that could be coeluted from an affinity matrix after solubilization of membranes with dodecylmaltoside. Proteoliposomes prepared from the MalFGK complex and preformed phospholipid vesicles of A. acidocaldarius displayed a low intrinsic ATPase activity that was stimulated sevenfold by maltose-loaded MalE, thereby indicating coupling of ATP hydrolysis to substrate translocation. These results provide evidence for MalK being the physiological ATPase subunit of the A. acidocaldarius maltose transporter. Moreover, to our knowledge, this is the first report on the functional reconstitution of an ABC transport system from a thermophilic microorganism.     

A salt-bridge motif involved in ligand binding and large-scale domain motions of the maltose-binding protein

The uptake of nutrients is essential for the survival of bacterial cells. Many specialized systems have evolved, such as the maltose-dependent ABC transport system that transfers oligosaccharides through the cytoplasmic membrane. The maltose/maltodextrin-binding protein (MBP) serves as an initial high-affinity binding component in the periplasm that delivers the bound sugar into the cognate ABC transporter MalFGK(2). We have investigated the domain motions induced by the binding of the ligand maltotriose into the binding cleft using molecular dynamics simulations. We find that MBP is predominantly in the open state without ligand and in the closed state with ligand bound. Oligosaccharide binding induces a closure motion (30.0 degrees rotation), whereas ligand removal leads to domain opening (32.6 degrees rotation) around a well-defined hinge affecting key areas relevant for chemotaxis and transport. Our simulations suggest that a "hook-and-eye" motif is involved in the binding. A salt bridge between Glu-111 and Lys-15 forms that effectively locks the protein-ligand complex in a semiclosed conformation inhibiting any further opening and promoting complete closure. This previously unrecognized feature seems to secure the ligand in the binding site and keeps MBP in the closed conformation and suggests a role in the initial steps of substrate transport.