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.     

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.     

Nucleotide dependent monomer/dimer equilibrium of OpuAA, the nucleotide-binding protein of the osmotically regulated ABC transporter OpuA from Bacillus subtilis

The OpuA system of Bacillus subtilis is a member of the substrate-binding-protein-dependent ABC transporter superfamily and serves for the uptake of the compatible solute glycine betaine under hyperosmotic growth conditions. Here, we have characterized the nucleotide-binding protein (OpuAA) of the B.subtilis OpuA transporter in vitro. OpuAA was overexpressed heterologously in Escherichia coli as a hexahistidine tag fusion protein and purified to homogeneity by affinity and size exclusion chromatography (SEC). Dynamic monomer/dimer equilibrium was observed for OpuAA, and the K(D) value was determined to be 6 microM. Under high ionic strength assay conditions, the monomer/dimer interconversion was diminished, which enabled separation of both species by SEC and separate analysis of both monomeric and dimeric OpuAA. In the presence of 1 M NaCl, monomeric OpuAA showed a basal ATPase activity (K(M)=0.45 mM; k(2)=2.3 min(-1)), whereas dimeric OpuAA showed little ATPase activity under this condition. The addition of nucleotides influenced the monomer/dimer ratio of OpuAA, demonstrating different oligomeric states during its catalytic cycle. The monomer was the preferred species under post-hydrolysis conditions (e.g. ADP/Mg(2+)), whereas the dimer dominated the nucleotide-free and ATP-bound states. The affinity and stoichiometry of monomeric or dimeric OpuAA/ATP complexes were determined by means of the fluorescent ATP-analog TNP-ATP. One molecule of TNP-ATP was bound in the monomeric state and two TNP-ATP molecules were detected in the dimeric state of OpuAA. Binding of TNP-ADP/Mg(2+) to dimeric OpuAA induced a conformational change that led to the decay of the dimer. On the basis of our data, we propose a model that couples changes in the oligomeric state of OpuAA with ATP hydrolysis.     

Functional reconstitution and osmoregulatory properties of the ProU ABC transporter from Escherichia coli

Abstract

The ATP-binding cassette (ABC) transporter ProU from Escherichia coli translocates a wide range of compatible solutes and contributes to the regulation of cell volume, which is particularly important when the osmolality of the environment fluctuates. We have purified the components of ProU, i.e., the substrate-binding protein ProX, the nucleotide-binding protein ProV and the transmembrane protein ProW, and reconstituted the full transporter complex in liposomes. We engineered a lipid anchor to ProX for surface tethering of this protein to ProVW-containing proteoliposomes. We show that glycine betaine binds to ProX with high-affinity and is transported via ProXVW in an ATP-dependent manner. The activity ProU is salt and anionic lipid-dependent and mimics the ionic strength-gating of transport of the homologous OpuA system.     

Interdomain dynamics and ligand binding: molecular dynamics simulations of glutamine binding protein

Periplasmic binding proteins from Gram-negative bacteria possess a common architecture, comprised of two domains linked by a hinge region, a fold which they share with the neurotransmitter-binding domains of ionotropic glutamate receptors (GluRs). Glutamine-binding protein (GlnBP) is one such protein, whose crystal structure has been solved in both open and closed forms. Multi-nanosecond molecular dynamics simulations have been used to explore motions about the hinge region and how they are altered by ligand binding. Glutamine binding is seen to significantly reduce inter-domain motions about the hinge region. Essential dynamics analysis of inter-domain motion revealed the presence of both hinge-bending and twisting motions, as has been reported for a related sugar-binding protein. Significantly, the influence of the ligand on GlnBP dynamics is similar to that previously observed in simulations of rat glutamate receptor (GluR2) ligand-binding domain. The essential dynamics analysis of GlnBP also revealed a third class of motion which suggests a mechanism for signal transmission in GluRs.     

Two FHA domains on an ABC transporter, Rv1747, mediate its phosphorylation by PknF, a Ser/Thr protein kinase from Mycobacterium tuberculosis

Bacterial genomics have revealed the widespread occurrence of eukaryotic-like protein kinases in prokaryotes, but little is known about their regulation, endogenous substrates, and physiological role. The present study concerns one of these enzymes, the serine/threonine protein kinase PknF from Mycobacterium tuberculosis. It is shown that, in addition to its autokinase activity, PknF is able to phosphorylate Rv1747, a newly described ABC transporter. This reaction appears to involve two FHA domains of Rv1747. It is suggested that recruitment and phosphorylation of Rv1747 depend on the interaction between its two non-redundant FHA domains and the autophosphorylated form of PknF.     

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.     

ABC transporter architecture and mechanism: implications from the crystal structures of BtuCD and BtuF

ABC transporters are ubiquitous membrane proteins that facilitate unidirectional substrate translocation across the lipid bilayer. Over the past five years, new crystal structures have advanced our understanding of how ABC transporters couple adenosine triphosphate (ATP) hydrolysis to substrate transport. In the following, we will briefly review the results of these structural investigations and outline their mechanistic implications.     

Validation of inhibitors of an ABC transporter required to transport lipopolysaccharide to the cell surface in Escherichia coli

The presence of lipopolysaccharide (LPS) in the outer leaflet of the outer membrane (OM) of Gram-negative bacteria creates a permeability barrier that prevents the entry of most currently available antibiotics. The seven lipopolysaccharide transport (Lpt) proteins involved in transporting and assembling this glycolipid are essential for growth and division in Escherichia coli; therefore, inhibiting their functions leads to cell death. LptB, the ATPase that provides energy for LPS transport and assembly, forms a complex with three other inner membrane (IM) components, LptC, F, and G. We demonstrate that inhibitors of pure LptB can also inhibit the full IM complex, LptBFGC, purified in detergent. We also compare inhibition of LptB and the LptBFGC complex with the antibiotic activity of these compounds. Our long-term goal is to develop tools to study inhibitors of LPS biogenesis that could serve as potentiators by disrupting the OM permeability barrier, facilitating entry of clinically used antibiotics not normally used to treat Gram-negative infections, or that can serve as antibiotics themselves.     

An important role of a "probable ATP-binding component of ABC transporter" during the process of Pseudomonas aeruginosa resistance to fluoroquinolone

In order to find new drug target to eliminate the fluoroquinolone resistance, the in vitro progress of Pseudomonas aeruginosa fluoroquinolone resistance was mimicked, and then proteomic analysis was applied to comparing different protein profiles during the resistant process. The results show that the expression of a "probable ATP-binding component of ATP binding cassette (ABC) transporter" existed in ciprofloxacin-intermediate and -resistant strains, but not in sensitive strain. In addition, the ciprofloxacin concentrations in P. aeruginosa strains, which were obtained from the progress of P. aeruginosa fluoroquinolone resistance, were determined by means of HPLC; the results show that the decrease of the intracellular concentration of drug and the expression of this new protein nearly take place simultaneously. The changes of mRNA levels of the probable ATP-binding component of ABC transporter were detected by virtue of RT-PCR and showed that this protein did not express in the sensitive strains but expressed increasingly in the intermediate and resistant strains. In order to determine the relationships between the development of antibiotic resistance and this protein further, a DNAzyme was designed to aim at the mRNA of the probable ATP-binding component of ABC transporter directly; the ciprofloxacin resistance of P. aeruginosa was partially reduced in vivo by inhibiting the expression of this protein. This DNAzyme has no effect on sensitive strain. And the comparison of drug intracellular concentrations between DNAzyme-treated strains and its control strains shows that this protein may be included in the course of active drug efflux.     

Arg149 is involved in switching the low affinity, open state of the binding protein AfProX into its high affinity, closed state

The substrate binding protein AfProX from the Archaeoglobus fulgidus ProU ATP binding cassette transporter is highly selective for the compatible solutes glycine betaine (GB) and proline betaine, which confer thermoprotection to this hyperthermophilic archaeon. A detailed mutational analysis of the substrate binding site revealed the contribution of individual amino acids for ligand binding. Replacement of Arg149 by an Ala residue displayed the largest impact on substrate binding. The structure of a mutant AfProX protein (substitution of Tyr111 with Ala) in complex with GB was solved in the open liganded conformation to gain further insight into ligand binding. In this crystal structure, GB is bound differently compared to the GB closed liganded structure of the wild-type AfProX protein. We found that a network of amino acid side chains communicates the presence of GB toward Arg149, which increases ligand affinity and induces domain closure of AfProX. These results were corroborated by molecular dynamics studies and support the view that Arg149 finalizes the high-affinity state of the AfProX substrate binding protein.     

Insights into structural features determining odorant affinities to honey bee odorant binding protein 14

Molecular interactions between odorants and odorant binding proteins (OBPs) are of major importance for understanding the principles of selectivity of OBPs towards the wide range of semiochemicals. It is largely unknown on a structural basis, how an OBP binds and discriminates between odorant molecules. Here we examine this aspect in greater detail by comparing the C-minus OBP14 of the honey bee (Apis mellifera L.) to a mutant form of the protein that comprises the third disulfide bond lacking in C-minus OBPs. Affinities of structurally analogous odorants featuring an aromatic phenol group with different side chains were assessed based on changes of the thermal stability of the protein upon odorant binding monitored by circular dichroism spectroscopy. Our results indicate a tendency that odorants show higher affinity to the wild-type OBP suggesting that the introduced rigidity in the mutant protein has a negative effect on odorant binding. Furthermore, we show that OBP14 stability is very sensitive to the position and type of functional groups in the odorant.     

Emerging computational approaches for the study of protein allostery

Allosteric regulation of protein function is key in controlling cellular processes so its underlying mechanisms are of primary concern to research in areas spanning protein engineering and drug design. However, due to the complex nature of allosteric mechanisms, a clear and predictive understanding of the relationship between protein structure and allosteric function remains elusive. Well established experimental approaches are available to offer a limited degree of characterization of mechanical properties within proteins, but the analytical capabilities of computational methods are evolving rapidly in their ability to accurately define the subtle and concerted structural dynamics that comprise allostery. This review includes a brief overview of allostery in proteins and an exploration of relevant experimental methods. An explanation of the transition from experimental toward computational methods for allostery is discussed, followed by a review of existing and emerging methods.     

Characterization of the ABCA transporter subfamily: identification of prokaryotic and eukaryotic members,phylogeny and topology

An alignment of the mammalian ABCA transporters enabled the identification of sequence segments, specific to the ABCA subfamily, which were used as queries to search for eukaryotic and prokaryotic homologues. Thirty-seven eukaryotic half and full-length transporters were found, and a close relationship with prokaryotic subfamily 7 transporters was detected. Each half of the ABCA full-transporters is predicted to comprise a membrane-spanning domain (MSD) composed of six helices and a large extracellular loop, followed by a nucleotide-binding domain (NBD) and a conserved cytoplasmic 80-residue sequence, which might have a regulatory function. The topology predicted for the ABCA transporters was compared to the crystal structures of the MsbA and BtuCD bacterial transporters. The alignment of the MSD and NBD domains provided an estimate of the degree of residue conservation in the cytoplasmic, extracellular and transmembrane domains of the ABCA transporter subfamily. The phylogenic tree of eukaryotic ABCA transporters based upon the NBD sequences, consists of three major clades, corresponding to the half-transporter single NBDs and to the full-transporter NBDls and NBD2s. A phylogenic tree of prokaryotic transporters and the eukaryotic ABCA transporters confirmed the evolutionary relationship between prokaryotic subfamily 7 transporters and eukaryotic ABCA half and full-transporters.     

Crystal structure of AlgQ2, a macromolecule (alginate)-binding protein of Sphingomonas sp. A1 at 2.0A resolution

Sphingomonas sp. A1 possesses a high molecular mass (average 25,700 Da) alginate uptake system mediated by a novel pit-dependent ABC transporter. The X-ray crystallographic structure of AlgQ2 (57,200 Da), an alginate-binding protein in the system, was determined by the multiple isomorphous replacement method and refined at 2.0 A resolution with a final R-factor of 18.3% for 15 to 2.0 A resolution data. The refined structure of AlgQ2 was comprised of 492 amino acid residues, 172 water molecules, and one calcium ion. AlgQ2 was composed of two globular domains with a deep cleft between them, which is expected to be the alginate-binding site. The overall structure is basically similar to that of maltose/maltodextrin-binding protein, except for the presence of an N2-subdomain. The entire calcium ion-binding site is similar to the site in the EF-hand motif, but comprises a ten residue loop. This calcium ion-binding site is about 40 A away from the alginate-binding site.     

Molecular characterisation of ABC transporter type FtsE and FtsX proteins of Mycobacterium tuberculosis

Elicitation of drug resistance and various survival strategies inside host macrophages have been the hallmarks of Mycobacterium tuberculosis as a successful pathogen. ATP Binding Cassette (ABC) transporter type proteins are known to be involved in the efflux of drugs in bacterial and mammalian systems. FtsE, an ABC transporter type protein, in association with the integral membrane protein FtsX, is involved in the assembly of potassium ion transport proteins and probably of cell division proteins as well, both of which being relevant to tubercle bacillus. In this study, we cloned ftsE gene of M. tuberculosis, overexpressed and purified. The recombinant MtFtsE-6xHis protein and the native MtFtsE protein were found localized on the membrane of E. coli and M. tuberculosis cells, respectively. MtFtsE-6xHis protein showed ATP binding in vitro, for which the K42 residue in the Walker A motif was found essential. While MtFtsE-6xHis protein could partially complement growth defect of E. coli ftsE temperature-sensitive strain MFT1181, co-expression of MtFtsE and MtFtsX efficiently complemented the growth defect, indicating that the MtFtsE and MtFtsX proteins might be performing an associated function. MtFtsE and MtFtsX-6xHis proteins were found to exist as a complex on the membrane of E. coli cells co-expressing the two proteins.     

Structural stability of GlcV, the nucleotide binding domain of the glucose ABC transporter of Sulfolobus solfataricus

GlcV is the nucleotide binding domain of the ABC-type glucose transporter of the hyperthermoacidophile Sulfolobus solfataricus. GlcV consists of two domains, an N-terminal domain containing the typical nucleotide binding-fold and a C-terminal β-barrel domain with unknown function. The unfolding and structural stability of the wild-type (wt) protein and three mutants that are blocked at different steps in the ATP hydrolytic cycle were studied. The G144A mutant is unable to dimerize, while the E166A and E166Q mutants are defective in ATP hydrolysis and dimer dissociation. Unfolding of the wt GlcV and G144A GlcV occurred with a single transition, whereas the E166A and E166Q mutants showed a second transition at a higher melting temperature indicating an increased stability of the ABCα/β subdomain. The structural stability of GlcV was increased in the presence of nucleotides suggesting that the transition corresponds to the unfolding of the NBD domain. Unfolding of the C-terminal domain appears to occur at temperatures above the unfolding of the NBD which coincides with the aggregation of the protein. Analysis of the domain organization of GlcV by trypsin digestion demonstrates cleavage of the NBD domain into three fragments, while nucleotides protect against proteolysis. The cleaved GlcV protein retained the ability to bind nucleotides and to dimerize. These data indicate that the wt GlcV NBD domain unfolds as a single domain protein, and that its stability is modified by mutations in the glutamate after the Walker B motif and by nucleotide binding.     

A comparative study of the backbone dynamics of two closely related lipid binding proteins: Bovine heart fatty acid binding protein and porcine ileal lipid binding protein

The backbone dynamics of bovine heart fatty acid binding protein (H-FABP) and porcine ileal lipid binding protein (ILBP) were studied by 15N NMR relaxation (T1 and T2) and steady state heteronuclear 15N{1H} NOE measurements. The microdynamic parameters characterizing the backbone mobility were determined using the model-free approach. For H-FABP, the non-terminal backbone amide groups display a rather compact protein structure of low flexibility. In contrast, for ILBP an increased number of backbone amide groups display unusually high internal mobility. Furthermore, the data indicate a higher degree of conformational exchange processes in the sec-msec time range for ILBP compared to H-FABP. These results suggest significant differences in the conformational stability for these two structurally highly homologous members of the fatty acid binding protein family.     

The pH-dependent unfolding mechanism of P2 myelin protein: an experimental and computational study

The P2 protein is a small, extrinsic protein of the myelin membrane in the peripheral nervous system that structurally belongs to the fatty acid binding proteins (FABPs) family, sharing with them a 10 strands beta-barrel structure. FABPs appear to be involved in cellular fatty acid transport, but very little is known about the role of P2 in the metabolism of peripheral myelin lipids. Study of protein conformation at different pHs is a useful tool for the characterization of the unfolding mechanisms and the intrinsic conformational properties of the protein, and may give insight into factors that guide protein folding pathways. In particular, low pH conditions have been shown to induce partially folded states in several proteins. In this paper, the acidic unfolding of purified P2 protein was studied with both spectroscopic techniques and molecular dynamics simulation. Both experimental and computational results indicate the presence of a partly folded state at low pH, which shows structural changes mainly involving the lid that is formed by the helix-turn-helix domain. The opening of the lid, together with a barrel relaxation, could regulate the ligand exchanges near the cell membrane, supporting the hypothesis that the P2 protein may transport fatty acids between Schwann cells and peripheral myelin.