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Microbiology and Molecular Biology Reviews, June 2008, p. 317-364, Vol. 72, No. 2
1092-2172/08/$08.00+0 doi:10.1128/MMBR.00031-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Structure, Function, and Evolution of Bacterial ATP-Binding Cassette Systems
Amy L. Davidson,1* Elie Dassa,2 Cedric Orelle,1 and Jue Chen3Department of Chemistry, Purdue University, West Lafayette, Indiana 47907,1 Unité des Membranes Bactériennes, CNRS URA2172, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France,2 Department of Biology, Purdue University, West Lafayette, Indiana 479073
SUMMARY INTRODUCTION Overview of Prokaryotic Transporters Multiple Functions of ABC Transporters Inventory and Classification of ABC Systems and Evolution of the Superfamily Comparative Genomics of ABC Systems STRUCTURE, FUNCTION, AND DYNAMICS OF THE ABC High-Resolution Structures of an ABC Module Conformational Changes Mechanism of ATP Hydrolysis, Still an Open Question Role of Two Nucleotide-Binding Sites IMPORT INTO THE CYTOPLASM (ABC IMPORTERS) Transport across the Outer Membrane BPD Uptake Systems Conformational changes in periplasmic BPs. (i) Bending at the hinge. (ii) Energetics of domain closure. Specificity of substrate-BPs. Substrate recognition by membrane transporters. Coupling of transport to hydrolysis. (i) Interaction of BP with transporter. (ii) BPs stimulate ATP hydrolysis. (iii) Conformational changes associated with transport. BP-independent mutants may mimic the P-open conformation. Structures of BPD importers. BP-Independent ABC Importers Cobalt and nickel transporters (CBU subfamily). Biotin and hydroxymethyl pyrimidine uptake systems (Y179 subfamily). Class I IM-ABC transporters that may be importers. EXPORT OUT OF THE CYTOPLASM BY CLASS 1 TRANSPORTERS Protein Trafficking between and across Membranes Components of a type I protein secretion system. Mechanism of action of type I protein secretion. (i) Signal for protein secretion. (ii) Signal recognition by type I machinery. (iii) Protein substrates are unfolded during secretion. (iv) Interactions between components of the type I machinery. Drug Efflux Pumps in Bacteria Drug resistance in bacteria. Active efflux. Drug transport and specificity: an overview of classical methodologies. LmrA, the first bacterial ABC MDR transporter discovered. LmrCD from Lactococcus lactis. BmrA from Bacillus subtilis. Other class 1 ABC bacterial drug resistance proteins. Structure of the bacterial exporter Sav1866. Drug-binding site: localization and substrate recognition. Translocation (Flipping) of Lipids and Lipid-Linked Oligosaccharides Lipid A flippase MsbA. CLASS 3 ABC SYSTEMS THAT ARE PROBABLY NOT IMPORTERS Systems Involved in Resistance and Immunity to Antibiotics, Drugs, Lantibiotics, and Bacteriocins DrrAB proteins (DRA family) from streptomycetes and their homologues. The DRI family, providing immunity to bacteriocins and lantibiotics. ABC Transporters Mediating Antibiotic Resistance and Lipoprotein Release from the Cytoplasmic Membrane The MacA-MacB-TolC system in Enterobacteriaceae. Lol, an ABC transporter involved in lipoprotein trafficking. Biogenesis of Extracellular Polysaccharides Polysaccharide transporters of the CLS family. Translocation of LPS to the outer membrane. CLASS 2 ABC SYSTEMS INVOLVED IN NONTRANSPORT CELLULAR PROCESSES AND IN ANTIBIOTIC RESISTANCE The UVR Family, Involved in Nucleotide Excision Repair and Drug Resistance The RLI Family, Involved in Ribosome Biogenesis ART Family of Proteins with Diverse Functions The EF-3 subfamily, involved in translation elongation. The REG subfamily. ARE subfamily proteins that confer resistance to MLS antibiotics. CONCLUSION Common Themes Emerging from Studies of ABC Transporters with Diverse Functions The Challenge of Membrane Protein Crystallography Understanding the Mechanism: Areas for Future Investigation ACKNOWLEDGMENTS REFERENCES
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Summary: ATP-binding cassette (ABC) systems are universally distributed among living organisms and function in many different aspects of bacterial physiology. ABC transporters are best known for their role in the import of essential nutrients and the export of toxic molecules, but they can also mediate the transport of many other physiological substrates. In a classical transport reaction, two highly conserved ATP-binding domains or subunits couple the binding/hydrolysis of ATP to the translocation of particular substrates across the membrane, through interactions with membrane-spanning domains of the transporter. Variations on this basic theme involve soluble ABC ATP-binding proteins that couple ATP hydrolysis to nontransport processes, such as DNA repair and gene expression regulation. Insights into the structure, function, and mechanism of action of bacterial ABC proteins are reported, based on phylogenetic comparisons as well as classic biochemical and genetic approaches. The availability of an increasing number of high-resolution structures has provided a valuable framework for interpretation of recent studies, and realistic models have been proposed to explain how these fascinating molecular machines use complex dynamic processes to fulfill their numerous biological functions. These advances are also important for elucidating the mechanism of action of eukaryotic ABC proteins, because functional defects in many of them are responsible for severe human inherited diseases.
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The ATP-binding cassette (ABC) systems constitute one of the largest superfamilies of paralogous sequences. All ABC systems share a highly conserved ATP-hydrolyzing domain or protein (the ABC; also referred to as a nucleotide-binding domain [NBD]) that is unequivocally characterized by three short sequence motifs (Fig. 1): these are the Walker A and Walker B motifs, indicative of the presence of a nucleotide-binding site, and the signature motif, unique to ABC proteins, located upstream of the Walker B motif (426). Other motifs diagnostic of ABC proteins are also indicated in Fig. 1. The biological significance of these motifs is discussed in Structure, Function, and Dynamics of the ABC. ABC systems are widespread among living organisms and have been detected in all genera of the three kingdoms of life, with remarkable conservation in the primary sequence of the cassette and in the organization of the constitutive domains or subunits (203, 420).
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FIG. 1. Conserved motifs in the ABC. Three characteristic motifs found in all ABC ATPases are represented by hatched red boxes. The Walker A motif and the Walker B motif form the nucleotide-binding fold of the P-loop ATPase family. The signature motif, also called the C loop, is unique to ABC proteins and also interacts with ATP. Other characteristic motifs, including the Q loop and the H loop (also called the switch region), contain just one highly conserved residue and are represented by hatched green boxes. These residues make contacts with the  -phosphate of ATP. In the context of the ABC dimer, the D loop makes contacts with Walker motif A of the other monomer. Sequences between the Q loop and the signature constitute a helical domain, also referred to as a structurally diverse region (SDR), that contains residues important for the interaction of ABC proteins with their membrane partners.
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ABC systems can be divided into three main functional categories, as follows. Importers mediate the uptake of nutrients in prokaryotes. The nature of the substrates that are transported is very wide, including mono- and oligosaccharides, organic and inorganic ions, amino acids, peptides, iron-siderophores, metals, polyamine cations, opines, and vitamins. Exporters are involved in the secretion of various molecules, such as peptides, lipids, hydrophobic drugs, polysaccharides, and proteins, including toxins such as hemolysin. The third category of systems is apparently not involved in transport, with some members being involved in translation of mRNA and in DNA repair.
Despite the large, diverse population of substrates handled and the difference in the polarity of transport, importers and exporters share a common organization made of two hydrophobic membrane-spanning or integral membrane (IM) domains and two hydrophilic domains carrying the ABC peripherally associated with the IM domains on the cytosolic side of the membrane (26). In importers, these four domains are almost always independent polypeptide chains that come together to form a multimeric complex. In most exporters, including the E. coli hemolysin exporter HlyB, the N-terminal IM and the C-terminal ABC domains are fused as a single polypeptide chain (IM-ABC). An inverted organization in which the IM domain is C-terminal with respect to the ABC domain (ABC-IM) exists, such as in the MacB protein, involved in macrolide resistance in E. coli. No IM domain partners have been identified for ABC proteins falling into the third category, and these proteins consist of two ABCs fused together (ABC2). Since more and more proteins are being found to be related to the last category, we propose calling the whole superfamily "ABC systems" rather than ABC transporters, a term that should be restricted to systems containing typical IM domains.
The tertiary structures of many ABC monomers and dimers have been determined, and they display remarkable conservation in their global folding pattern, as expected from sequence homology (268) (see Structure, Function, and Dynamics of the ABC). IM domains are typically comprised of six TM 
-helices, and several structures of intact transporters have been determined (102, 205, 285, 343, 366, 518). Input from structural biology has greatly impacted our understanding of the mechanism of action of ABC systems.
ABC transporters are one of many different types of transporters operating in bacteria and other organisms. Transporters are of critical importance for living organisms, and selective permeability to nutrients and metabolites was probably the first distinctive property of primitive cells. Functionally and structurally different transporters have been identified in living organisms (Fig. 2). It is customary to distinguish channels, primary transporters, and secondary transporters with respect to the source of energy used (411). Channels catalyze the facilitated diffusion of solutes down a concentration gradient, an energy-independent process. In the outer membranes of gram-negative bacteria, porin channels allow diffusion through a TM-spanning aqueous pore (Fig. 2a). Cytoplasmic membrane channels are gated, opening or closing in response to voltage (396) or to membrane tension, as seen for McsS, which plays a role in protecting bacteria from hypo-osmotic shock (22, 276). Primary active transporters, including the ABC transporters, couple transport against a concentration gradient to the hydrolysis of ATP (Fig. 2b, diagrams E and F, and c, diagram H). Secondary active transporters, including uniporters, antiporters, and symporters (Fig. 2a, diagrams C and D), use the energy stored in ion gradients to drive transport (411). A novel family of secondary high-affinity transporters, the TRAP (tripartite ATP-independent periplasmic) transporters (Fig. 2a, diagram B) (159, 224), which primarily catalyze the transport of C4-dicarboxylates (see reference 239 for a review), although other substrates, including sialic acid, have been described (442), was recently reported for prokaryotes. Another family of transporters unique to prokaryotes, the phosphoenolpyruvate:sugar phosphotransferase (PTS) transporters (Fig. 2a, diagram A), catalyze the uptake of sugars. Energy coupling to transport in these systems occurs via a series of phosphoryl transfer reactions (116). Transporters belong to at least 300 different protein families, and the two largest of these are the primary ABC superfamily and the secondary major facilitator superfamily (MFS) (412).
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FIG. 2. Schematic view of the organization of transport systems. In gram-negative bacteria, substrates can cross the outer membrane by facilitated diffusion through porins, which are trimeric channels. Red circles represent transported substrates, small green circles represent cotransported ions, and small blue circles represent phosphates. (a) Group translocators and secondary transporters. (A) PTSs. PTSs consist of a set of cytoplasmic energy-coupling proteins and various integral membrane permeases/sugar phosphotransferases, each specific for a different sugar. The E. coli mannitol permease consists of two cytoplasmic domains (EIIA and EIIB) involved in mannitol phosphorylation and an integral membrane domain (EIIC) which is sufficient to bind mannitol but which transports mannitol at a rate that is dependent on phosphorylation of the EIIA and EIIB domains. The two other components are common to all PTS systems. The soluble enzyme I (EI) autophosphorylates in the presence of Mg2+. The histidine protein (HPr) is the energy-coupling protein and delivers phosphoryl groups from EI to the sugar-specific transporters (EIIs). (B) TRAP transporters. A periplasmic BP, which is unrelated to an ABC BP at the sequence level but similar in secondary structure, functions in association with two membrane components, namely, a large TM subunit involved in the translocation process and a smaller membrane component of unknown function. The driving force for solute accumulation is an electrochemical ion gradient, not ATP hydrolysis. (C) Ion-driven MFS transporters. These transporters typically consist of a single cytoplasmic membrane protein with 12 TM segments that couples transport of small solutes to existing gradients of ions, such as protons or sodium ions. Symporters pump two or more types of solutes in the same direction simultaneously, using the electrochemical gradient of one of the solutes as the driving force. Antiporters (not shown) are driven in a similar way, except that the solutes are transported in opposite directions across the membrane. (D) Uniporters transport one type of solute and are driven directly by the substrate gradient. (b) ABC import systems. (E) Vitamin B12 importer. The vitamin B12 uptake system of E. coli includes a high-affinity OMR, BtuB, that translocates the substrate through the outer membrane in an energy-dependent step that requires an active TonB-ExbB-ExbD complex. Substrates are captured by the periplasmic BP BtuF in the periplasmic space and presented to a cytoplasmic complex made of two copies each of BtuC and BtuD. This complex mediates the ATP hydrolysis-dependent translocation of vitamin B12 into the cytoplasm. (F) Maltose-maltodextrin importer. The transport of maltodextrins larger than maltotriose through the outer membrane requires the trimeric maltoporin LamB. Substrates are captured by the maltose-BP MalE in the periplasmic space and presented to a cytoplasmic complex made of MalF, MalG, and two copies of MalK. (c) Comparison between secondary RND and primary ABC export systems. (G) AcrAB-TolC exporter. This hypothetical model of the RND family AcrA-AcrB-TolC drug efflux pump is based on the trimeric structures determined for TolC and AcrB. TolC is predicted to contact the apex of the AcrB trimer. Two molecules of the MFP AcrA are shown, but it is probable that this protein exists as higher-order oligomers in the complex. Hydrophobic drugs are probably pumped out of the membrane lipid bilayer coupled to the downhill movement of protons across the cytoplasmic membrane. (H) Hemolysin HlyBD-TolC exporter. This hypothetical assembled model consists of a TolC trimer, a dimer of the IM-ABC protein HlyB, and the MFP HlyD. The exact oligomeric state of HlyD is not known accurately, though it may be trimeric. The TM and ABC domains of HlyB are represented by red rectangles and green circles, respectively. Hemolysin is translocated through the envelope by an ATP hydrolysis-dependent process.
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ABC transporters have a great impact on bacterial physiology, and their dysfunction can have strong deleterious effects. While some ABC transporters are clearly dedicated to the export of virulence factors, under appropriate conditions many other bacterial ABC transporters can become important for viability, virulence, and pathogenicity. An example is provided by iron ABC uptake systems, which have long been recognized as important effectors of virulence (191). Because iron exists primarily in the insoluble Fe3+ form under aerobic conditions, biologically available iron in the body is found chelated by high-affinity iron-binding proteins (BPs) (e.g., transferrins, lactoferrins, and ferritins) or as a component of erythrocytes (such as heme, hemoglobin, or hemopexin) (255). Pathogens are able to scavenge iron from these sources by secreting high-affinity iron-complexing molecules called siderophores and reabsorbing them as iron-siderophore complexes (see reference 516 for a review). Another example of involvement in virulence is the chvE-gguAB operon in Agrobacterium tumefaciens, which encodes a glucose and galactose importer (61, 241). Sugar binding to ChvE triggers a signaling response that results in virulence gene expression. Finally, a potentially lethal upshift in osmotic strength is counterbalanced by activation of osmosensing ABC transporters that mediate uptake of compatible solutes (375).
In addition to their importance in transport, ABC systems are involved in the regulation of several physiological processes. In this context, direct regulatory roles need to be distinguished clearly from indirect effects mediated through the transported substrate. Well-documented examples of cases where additional regulatory domains (RDs) in the transporter itself are involved are the C-terminal domain of MalK of the maltose-maltodextrin transporter from E. coli and S. enterica serovar Typhimurium, the tandem cystathionine-synthase domains of OpuA from Lactococcus lactis and of other members of the OTCN family, and the R domain of the human cystic fibrosis protein CFTR (reviewed in reference 26).
Computer-assisted methods have been applied to understand the complexity and the diversity of the ABC superfamily. The use of multiple sequence alignments was instrumental in the first definition of the superfamily (196). In most cases, ABC proteins of a given organism (44, 280, 385) or ABC systems with clear functional similarity (144, 211, 259) were compared. However, the presence of the highly conserved ATPase domain also allows more global comparisons (359, 420). The last publication (420), which constitutes the first global study specifically devoted to the ABC superfamily, was updated to include about 600 ABCs (87). These sequences segregate into 29 clusters or families (Table 1). A phylogenetic tree derived from the comparison of these sequences is given in Fig. 3.
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TABLE 1. Classes, families, and subfamilies of ABC systemsa
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FIG. 3. Simplified neighbor-joining tree of ABC domains. For clarity, only the main branches that point to ABC families are drawn. The major subdivisions of the tree correspond to the three classes of ABC systems, whose schematic structural representations are given in the right part of the figure. ABC domains are shown with green circles, and IM domains are shown with differently colored rectangles. For the sake of simplicity, accessory proteins (BPs, MFPs, and OMFs) are omitted. Class 1 (red branches) systems have fused ABC and IM domains, corresponding mainly to exporters. IM domains shown in purple represent ABC transporters with the IM domain at the N terminus, corresponding to IM-ABC and (IM-ABC)2 topologies. N- and C-terminal domains are symbolized by N and C, respectively. IM domains shown in orange represent ABC transporters with the IM domain at the C terminus, corresponding to ABC-IM and (ABC-IM)2 topologies. Class 1 contains two atypical families of systems, CCM and MCM, with a different structural organization. Class 2 (blue branches) systems have tandemly repeated ABC domains and no known TM domains (ABC2 topology), corresponding to proteins involved in nontransport processes. The UVR family was omitted during the generation of the tree because large domain insertions within the ABC domains prevent the establishment of the multiple sequence alignment. However, binary comparisons established the relationship between UVR proteins and class 2 systems. Class 3 (green branches) systems have IM (red rectangles) and ABC (green circles) domains carried by independent polypeptide chains, corresponding mainly to importers. For class 3, systems that could be exporters are shown in boxes. Family names are abbreviated according to the conventions used in Table 1 and throughout the text. See Table 1 for the abbreviations of family names and for functional descriptions. OPN-D, OPN-F, HAA-F, and HAA-G correspond to the two different ABC subunits of the OPN and HAA systems, respectively. MOS-N and MOS-C correspond to the N- and C-terminal ABC domains of MOS family ATPases. The scale at the top of the figure corresponds to 5% divergence per site between sequences.
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Similar studies were performed on the less conserved IM domains and BPs of ABC importers. It is notoriously difficult, if not impossible, to build multiple alignments of these proteins or domains, due to the lack of overall homology. However, clustering methods were applied to the scores of binary alignments to generate families of IM domains and BPs (421, 470). There is good agreement between families derived from the classifications of BPs, IM domains, and ABCs, suggesting that components of ABC transporters coevolve with minimal shuffling of their components (42, 259). A good correlation exists between the sequences of the ABCs and the global substrate specificity of ABC systems. This apparent relationship between sequence and function could reflect constraints imposed by the interaction of ABC proteins with their IM partners, which are thought to carry substrate recognition sites (488).
The clustering of ABCs into three classes that correspond to the topology of ABC systems is intriguing. The ABCs segregate mostly according to sequence differences in a region that lies between Walker motifs A and B and includes the helical domain (Fig. 1) (426). This region has been termed the structurally diverse region (425). Using the maltose transporter, we have demonstrated experimentally that this region is critical for the interaction between the ABC and IM domains (216, 319). Recent insight from complete structural determinations of ABC importers and exporters sheds further light on this point. In class 1 transporters, whose prototype is Sav1866, it is clear that the transmission interface between IM domains and the ABC is dual, involving both IM intracellular loops 1 and 2 (ICL1 and ICL2), which make contact with the Q loop and with a newly discovered TEVGERV motif in the helical domain, respectively (102). This new motif is conserved only in exporters. In contrast, in class 3 importers, exemplified by the Archeoglobus fulgidus ModABC transporter and the E. coli MalFGK2 transporter, a single intracellular loop, known as the EAA loop of the IM component, interacts with the Q loop of the ABC as well as with helices 3 and 5 within the helical domain (96, 205, 343). Class 2 proteins appear not to interact with a TM domain, and as exemplified by the structure of RLI (232), there is no cleft at the corresponding position to accommodate an EAA loop from an IM subunit. These differences might account, at least in part, for the differential clustering of ABC domains as importers, exporters, and others.
Proteins from all three kingdoms of life, i.e., Bacteria, Archaea, and Eukarya, are found in each class of ABC systems, and species-specific differences are observed at the very ends of the branches of the tree. These observations suggest that ABC systems began to specialize very early, probably before the separation of the three kingdoms, and that functional constraints on the ABC domain are responsible for the global conservation of sequences.
From this analysis, we propose a hypothetical scenario for the evolution of ABC systems (87, 420) (Fig. 4). The ancestor "progenote" cell or last universal common ancestor (LUCA) already had all classes of ABC systems. Prokaryotes inherited all ABC classes. However, archaea are apparently devoid of transporters of the ABC-IM type, though they have IM-ABC transporters. An alternative scenario to account for the absence of ABC-IM systems in archaea is that these systems arose in bacteria only. Eukaryotes probably acquired most IM-ABC and ABC-IM (class 1) systems and ABC2 (class 2) systems from the symbiotic bacteria that are the putative ancestors of organelles. It is noteworthy that most eukaryotic IM-ABC transporters are specifically targeted to organelle membranes, which probably descend from a prokaryotic ancestor. For instance, the mammalian TAP protein, an IM-ABC exporter involved in the presentation of antigenic peptides to the class I major histocompatibility complex, is inserted into the endoplasmic reticulum (192). The ALD protein, putatively involved in the export of very-long-chain fatty acids from the cytosol into peroxisomes, is targeted to the peroxisomal membrane (515). From genes encoding IM-ABC or ABC-IM transporters, eukaryotes developed specific systems by several independent duplication-fusion events, including those that led to the formation of the PDR (plant and fungal pleiotropic drug resistance) and P-glycoprotein-like families of proteins. Class 3 systems, particularly BPD transporters, are virtually absent from eukaryotic genomes, though there is evidence that they were acquired first and lost subsequently (see the next paragraph). The reason for the absence of class 3 transporters in eukaryotes is not clear, but it may be related to different bioenergetic requirements and environmental constraints.
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FIG. 4. Hypothetical scenario for the evolution of ABC systems. The figure uses the same topological representations and color coding as those used in Fig. 3. The hypothetical LUCA is predicted to possess all classes of ABC systems. The cell membrane is represented as a thick black line surrounding the intracellular medium (blue). The eukaryotic cell nucleus is a gray oval. The eukaryotic organelle is represented as a bacterium to symbolize its endosymbiotic origin. The arrows symbolize the evolutionary relationships between archaea, bacteria, and eukaryotes. The question mark under the arrow joining archaea and eukaryotes recalls the hypothesis that the ancestral eukaryotic cell arose by a unique endosymbiotic event involving engulfment of an archaebacterium by a gram-negative eubacterial host prior to the other endosymbiotic events leading to the appearance of organelles. Alternatively, eukaryotes may have arisen directly from the LUCA via an unidentified transient archaebacterium. A class 3 transporter, represented by a hatched pattern, recalls the hypothesis that these systems were acquired by eukaryotes and subsequently lost. See details of the scenario in the text.
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The complete nucleotide sequences of more than 500 genomes are presently available, and much effort has been put forth to build complete inventories of ABC proteins in Saccharomyces cerevisiae (110), Escherichia coli (88, 280), Bacillus subtilis (385), Mycobacterium tuberculosis (44), Arabidopsis thaliana (417), Caenorhabditis elegans (449), Oryza sativa (165), and many other organisms. Global comparisons of the ABC protein contents of several genomes have also been made (161, 219, 356, 359, 485). In the course of the constitution of ABCISSE, our database of ABC systems, we have analyzed the compositions of more than 250 completely sequenced genomes.
When the total number of ABC systems is plotted against the size of prokaryotic genomes (Fig. 5), a linear relationship is seen, in agreement with the observation that the number of transporters of all categories (ion gradient-driven, PTS, ABC, and facilitator proteins) is approximately proportional to genome size (359). Bacteria with genomes in the range of 0.5 to 1.5 Mb have about 15 ABC systems. Most bacteria of this size are intracellular parasites, and the availability of intracellular metabolites or the presence of homologous host genes may have rendered some ABC genes inessential, leading to their subsequent disruption or deletion. It is therefore possible that the subset of ABC systems that are common to these species constitute the minimal requirement of ABC systems for life. The relatively small, 1.86-Mb genome of Thermotoga maritima has a very large number of ABC systems (68 systems) compared with that of species of similar genome size. This is partly due to the extensive amplification of operons encoding ABC systems putatively involved in the uptake of oligosaccharides (11 systems) and oligopeptides (12 systems). Escherichia coli (4.6 Mb) and Bacillus subtilis (4.2 Mb) have 78 and 84 ABC systems, respectively, which are typical numbers for this size of genome. In contrast, the genome of Mycobacterium tuberculosis (4.4 Mb) has only 38 systems. The lack of high-affinity import systems might be related to the intracellular lifestyle of this bacterium. This number is also significantly lower than that found in soil bacteria, such as Agrobacterium tumefaciens and Mesorhizobium loti (5.67 and 7.6 Mb, respectively), which have more than 200 ABC systems. The large number of ABC systems in this instance is probably due to highly competitive environmental conditions in the soil or within plant nodules. Eukaryotes display a smaller number of ABC proteins with respect to genome size than do prokaryotes, and this is particularly evident in the case of Saccharomyces cerevisiae, a free-living microorganism which shares with bacteria almost the same ecological niches. Indeed, ABC importers are lacking in eukaryotes.
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FIG. 5. Genomic distribution of ABC systems in living organisms. The plot shows the number of ABC ATPases versus the number of total genes in completely sequenced genomes. The number of ABC ATPases per genome (which roughly reflects the number of ABC systems) is plotted against the total number of genes (purple dots, archaea; blue dots, bacteria; green dots, eukaryotes). Selected genomes with exceptionally large or small numbers of ABC proteins are indicated with circles on the graph and are discussed in the text.
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Very few families of ABC systems appear to be species or kingdom specific (Table 1). For example, the MCM family of proteins with unknown function is found only in methanogenic archaea, and the PDR subfamily is found only in plants and fungi. In general, most families have been identified in more than one kingdom. Several families have been recognized from sequence analysis but have not been characterized at the functional level. All of the observations reported above establish that the ABC constitutes an ancient and universal molecular motor with a fascinating range of diverse applications.
In this review, we aim to discuss the most recent advances in the understanding of structural and functional aspects of model ABC transporters and to provide a wide overview of the diversity and versatility of bacterial ABC systems. We offer our apologies to those whose work we overlooked or were not able to include.
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Crystal structures of isolated ABC modules from many members of the ABC superfamily are now available (reviewed in reference 349). In addition, the structures of six full-length ABC transporters have been reported (102, 205, 285, 343, 366, 518). Since many of these structures have recently been reviewed (96, 349), we focus here on key characteristics and their implications in function.
All ABCs contain two domains (167), a larger domain similar to the core structure found in many RecA-like motor ATPases and a smaller, predominantly helical domain that is unique to ABC transporters (Fig. 6). The RecA-like domain typically consists of two β-sheets and six -helices and includes the Walker A motif (GxxGxGKS/T, where x is any amino acid) and the Walker B motif (
D, where is a hydrophobic residue). The helical domain consists of either three or four helices and a signature motif, which is also known as the LSGGQ motif, the linker peptide, or the C motif. The two domains are joined by two flexible loops, one of which contains a highly conserved glutamine residue and is known as the Q loop. In the structures of intact ABC transporters, the Q loop mediates interactions between the ABC subunits and the TM subunits (102, 205, 285, 366).
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FIG. 6. Structure of an ATP-bound ABC dimer. The structure of a MalK homodimer with two ATPs bound (PDB accession no. 1Q12) is shown. Each NBD consists of two subdomains, a RecA-like subdomain (green) and a helical subdomain (blue). A C-terminal RD (not present in all ABC proteins) is shown in yellow. Corresponding domains in the second MalK subunit follow the same color scheme but are rendered in lighter colors. Two ATPs, represented as a ball-and-stick model, are bound between the NBDs. The Walker A motif is shown in red.
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- and -phosphates, thereby holding both phosphates in a defined orientation. A Mg2+ ion is coordinated by O atoms from the β- and -phosphates and residues in the Walker A motif (167, 508). A highly conserved histidine residue located in the H loop forms a hydrogen bond with the -phosphate and is required for hydrolysis (68, 545). The side chain of the serine and the backbone amide groups of the glycine residues in the LSGGQ motif coordinate the -phosphate. It was suggested that the LSGGQ motif is reminiscent of the "arginine finger" of other RecA-like ATPases, where an arginine residue provided by one subunit extends into the nucleotide-binding site of the other (540). In addition to nucleotide binding, the conserved histidine residue also contacts residues across the dimer interface in the Walker A motif and the D loop, a conserved sequence following the Walker B motif, suggestive of a tight coupling between ATP binding and formation of the dimer (68, 206, 457, 545). The sharing of nucleotide-binding sites between subunits explains why all ABC proteins have two NBDs or subunits even though one site has sometimes deviated from the consensus. Moreover, the observation of positive cooperativity in ATP hydrolysis in some systems (97, 176, 281, 463, 546) is consistent with a requirement that ATP must bind to both sites before the NBDs can assume the closed, catalytically active conformation. One of the essential tasks for ABC transporters is to harness the energy of ATP binding and/or hydrolysis for mechanical work. This task appears to be achieved through conformational changes of the transporter. The dynamic nature of the ABCs has been revealed by comparison of structures of isolated ABC modules in apo and ATP- and ADP-bound forms (96, 349) and is supported by biochemical data sensing nucleotide-dependent changes in parameters such as protease sensitivity, fluorescence, and rates of hydrogen/deuterium exchange (427, 510). In the absence of nucleotides and TM subunits, the ABC structures show high degrees of intrinsic flexibility. When the structures of different ABCs are compared, the helical domain shows rigid-body motion relative to that of the RecA-like domain (reviewed in reference 96). In addition, two different conformations of the same protein in the apo form are reported for both Sulfolobus solfataricus GlcV (508) and E. coli MalK (68), and differences in domain orientations account for these structural differences also.
In comparison with the apo form, the ATP-bound structures in the monomeric form show that the helical domain rotates toward the RecA-like domain upon ATP binding, moving the LSGGQ loop into a position where it contacts the nucleotide across the dimer interface (68, 457, 545). It was suggested that, upon hydrolysis, the helical domain rotates away from the active site to facilitate nucleotide exchange (234). Since the ABC protein functions as a dimer, it is important to analyze the conformational changes of the dimeric structures in a transporter cycle. The nature of these changes is illustrated by the crystal structures of MalK in the resting, ATP-bound, and ADP-bound states (68, 292). While other NBDs or nucleotide-binding subunits display low affinity for each other in the absence of the TM segments, the MalK dimer is stabilized through interactions of an additional C-terminal RD, and its propensity to form a dimer in the cytoplasm was demonstrated quite early (243). A tweezer-like motion, in which the two monomers of MalK are held together at the base by the RDs and the NBDs open and close like the tips of a pair of tweezers, is revealed by crystal structures in three different dimeric configurations (Fig. 7). In nucleotide-free structures, the N-terminal NBDs are separated and the dimer is maintained solely through contacts with the C-terminal RDs. In the ATP-bound form, the NBDs make contact and two ATPs lie buried along the dimer interface. The structure of MalK in a posthydrolysis state shows that the two NBDs of the ADP-bound form are separated, similar to the case for the resting form, indicating that ADP, unlike ATP, cannot stabilize the closed form. The three different conformations are achieved principally by a rotation of the entire NBD relative to the RD about a hinge region located in the loop connecting the NBD and RD. In addition, a second rigid-body rotation within the NBDs, between the RecA-like and helical subdomains, results in inward movement of the helical subdomain toward the nucleotide-binding site on the same subunit. As we have already discussed, this second rotation is also observed by comparison of different monomer structures. In contrast to the changes at the NBD-NBD interface, the interaction between the RDs in all three homodimer structures was essentially unchanged.
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FIG. 7. Three structures of the MalK dimer. In the absence of nucleotide, the two NBDs of MalK are separated from each other, held as a dimer primarily through contacts between the C-terminal RDs. In the presence of ATP, the NBDs are closed, permitting ATP hydrolysis to occur. In the ADP-bound state, the NBDs are again separated, suggesting a possible cycle for hydrolysis-driven conformational change. Coloring is the same as that in Fig. 6. (Reprinted from reference 292 with permission of the publisher. Copyright 2005 National Academy of Sciences, U.S.A.)
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Molecular dynamics simulations based on structures of the isolated HisP (59, 227), MalK dimer (344), BtuD (222), and MJ0796 (60, 228) proteins largely recapitulate the conformational changes suggested by the structures of nucleotide-free and ATP-bound subunits. These studies also offer insight into the function of a "complete" ATPase active site, as H2O, Mg2+, and/or a key catalytic residue lacking in the structure can be reinstated (545).
In vivo, the MalK dimer is stably assembled into a complex with TM proteins MalF and MalG, and several lines of evidence suggest that the conformational changes seen in the crystal structures of isolated MalK subunits also occur in the intact MalFGK2 transporter. When a cysteine substitution is introduced for Ala85 in the Q loop region, a cross-linked MalK dimer is observed only upon addition of ATP (216). The failure of ADP to induce cross-linking suggests that ATP, not ADP, induces closure of the NBDs in MalFGK2 (216). Photocleavage experiments using vanadate as a transition state analogue indicated that ATP hydrolysis occurs via the closed dimer (156), and the solvent accessibility of a fluorescent probe covalently attached to an amino acid in the Walker A motif is reduced in the catalytic transition state compared to that in the resting state (299), consistent with the closure of the interface between the NBDs. We discuss how nucleotide-associated conformational changes in the ABC modules may be coupled to conformational changes in the TM domains in a later section [see "Coupling of transport to hydrolysis. (iii) Conformational changes associated with transport"].
The structures of ABCs in isolation and in the context of intact transporters show a highly conserved fold, suggesting that they hydrolyze ATP by a common mechanism. However, the precise molecular mechanism of ATP hydrolysis is still controversial. An acidic residue at the end of the Walker B motif, a glutamate in most ABC transporters, has been proposed to act as a general base polarizing the attacking water molecule (168, 318). In the crystal structures, this residue extends into the active site and makes a hydrogen bond with the putative hydrolytic water. In several ABCs, replacement of this acidic residue with the corresponding amide abolishes the ATPase activity and triggers the formation of a trapped, ATP-bound dimer (318, 347, 378). Since high-resolution structures show that the attacking water is still well positioned in the mutants (378, 457), an appealing explanation for the lack of activity is that the substituted amide cannot act as a catalytic base for hydrolysis. A very similar mechanism, by which general acids coordinate the
-phosphate of ATP and a general base polarizes the attacking water, has been suggested for other RecA-like ATPases (540). However, some data in the literature stand in opposition to the general base mechanism. Mutation of the Walker B glutamate (E/Q) in Pgp, HlyB, and GlcV leaves some residual ATPase activity (483, 493, 508, 545), leading some to question its role as a general base. In addition, if the Walker B glutamate is the catalytic base, one would expect a change in the pH dependence of ATPase activity upon substitution of glutamine for glutamate, as glutamine is a poor base, but the pH profiles of the wild-type HlyB ABC module and the E/Q mutant both show maximal activity near pH 7.0 (545). Thus, Schmitt and colleagues proposed an alternative mechanism, namely, substrate-assisted catalysis (545), in which the H loop histidine residue plays a relatively greater role in catalysis, forming part of a catalytic dyad with the Glu following the Walker B motif. In the substrate-assisted catalysis model, the histidine acts as a "linchpin" to hold the -phosphate of ATP, the attacking water, Mg2+, and other catalytically important amino acids together to support hydrolysis. In contrast to its function in the general base model, the function of the Walker B glutamate proposed in this model is to restrict the flexibility of the H loop histidine so that it adopts a catalytically competent conformation (141, 349, 545). It should be noted that in a different ABC module, mutation of Asp to Glu in the catalytic dyad does alter the pH profile of ATPase activity (141), though this result does not rule out either model, as local changes in the environment at the active site can alter the pKa of a side chain (141). While substitution of the His eliminates ATP hydrolysis in the HlyB ABC module, as demonstrated clearly by the ability to obtain crystals of a Mg·ATP-bound dimer (545), 2% of the wild-type ATPase activity is retained in the intact maltose transporter with substitution of arginine for histidine (99), suggesting subtle differences in architecture of the active sites of different ABC modules. Further research is needed before we can conclude that substrate-assisted catalysis or general base catalysis is a universal mechanism underlying ATP hydrolysis of the ABC transporter family, or even if there is a uniform mechanism. A question of great interest for all ABC transporters is the role that the two nucleotide-binding sites play in the energization of the translocation process. ATP is hydrolyzed with positive cooperativity in both the maltose and histidine transporters (97, 283), indicating that the two sites interact. Since both sites lie along the dimer interface of the ABCs, the simplest explanation for the presence of cooperativity is that both sites must be occupied before the NBDs can close and hydrolyze ATP. Structural data so far are consistent with this hypothesis, since nucleotide is bound at both sites in each of the structures of ATP-bound ABC dimers (68, 457, 545). It is not yet clear, however, whether one or both ATPs are hydrolyzed during a single cycle of conformational change. In some ABC transporters, such as the ribose transporter (57), only one of the two nucleotide-binding sites retains all of the highly conserved residues thought to be essential for ATP hydrolysis, suggesting that hydrolysis at just one site is sufficient for transport. In support of this hypothesis, mutation of the catalytically important histidine residue in the nucleotide-binding site of just one of the two HisP subunits in the intact histidine transporter was relatively well tolerated (335). However, the same substitution in a single site of the maltose transporter severely impaired both transport and ATPase activity, suggesting that hydrolysis at both sites is important for function (99). Interestingly, even the glutamate substitutions that promote stable formation of the NBD dimer, when present in a single site, inactivate the intact P-glycoprotein transporter (482), though it should be mentioned that the isolated NBDs of GlcV and HlyB (509, 546), containing a similar single-site mutation, retain ATPase activity. This seeming contradiction suggests that there may be functionally important cooperative interactions between nucleotide-binding sites that are lost once the NBDs are stripped from the IM domains, although noone has compared the effects of a single-site mutation in both intact and isolated ABC subunits of the same transporter.
As first shown with P-glycoprotein, a mammalian multidrug exporter (495), trapping of Mg·ADP and vanadate in the maltose transporter occurs in just one of the two nucleotide-binding sites (447). Vanadate replaces the Pi that is formed during ATP hydrolysis and prevents the dissociation of ADP from the active site (94, 440). The Mg·ADP·Vi complex is very stable and mimics the transition state for the hydrolysis of ATP. The observation that trapping occurs at just one site suggests that hydrolysis can occur at only one site at a time. Given that both sites can hydrolyze ATP in P-glycoprotein, it is suggested that the sites alternate in catalysis and that just one ATP is hydrolyzed each time a drug is transported (440, 494). Implicit in this model is the assumption that the transporter "remembers" which site hydrolyzed last. One way to envision such a possibility is to suggest that ATP hydrolysis results in the opening and release of ADP and Pi from a single site, while the second site remains closed, with ATP bound. Alternatively, one ATP could still be hydrolyzed per catalytic cycle, but following hydrolysis at one site, ATP at the remaining site is not bound strongly enough to sustain the closed conformation during exchange of ADP for ATP in the other site. In this model, catalysis would not be ordered strictly. Finally, if both ATPs are hydrolyzed sequentially before the NBDs dissociate (the progressive clamp model [226]), then two ATPs would be expended for the transport of a single molecule. To date, no single experiment is able to convincingly rule out any one of these models. The sequential model was put forth to explain the appearance of both trapped ATP and trapped ADP in an NBD dimer stabilized by mutation of the Glu residue following the Walker B motif, which is important for catalysis (226). It is suggested that this dimer does not dissociate until ATP is hydrolyzed at both sites, suggestive of sequential hydrolysis. It is unfortunate, however, that this type of experiment can be performed only in the presence of a mutation that greatly stabilizes the ATP-bound dimer and hence may not recapitulate what occurs in the wild type.
Evidence in support of asymmetric behavior of ABC dimers whose cassettes have identical sequences has been reported. The structure of the Mg·ATP-bound HlyB ABC dimer, stabilized through mutation of His in the catalytic dyad, has a 4.4-Å tunnel in one subunit, blocked by a conserved salt bridge in the second subunit, that could allow Pi to diffuse out of the binding site prior to opening of the dimer interface (547). Sequential hydrolysis and Pi release may allow the energy of ATP hydrolysis to be used in distinct steps, possibly for separate purposes (547). Intriguingly, disruption of this salt bridge results in the loss of cooperativity in ATP hydrolysis in the isolated ABC (547).
In intact transporters, evidence for asymmetry has also been detected. Thiol-specific reagents react more readily with a Cys in one ABC in the histidine transporter than with a Cys in the other, suggesting that structural asymmetry may exist before the binding of nucleotide (257). In the maltose transporter, asymmetries are seen in the pattern of cross-linking between cysteines placed in the helical domains of the ABC MalK and the IM proteins MalF and MalG (92, 216). In both cases, the IM region is heterodimeric, which could contribute to differences. The Lol system, involved in export of lipoproteins, contains two unique IM subunits, LolC and LolE, and a dimer of the LolD ATPase. Suppressors of a dominant negative mutation in a conserved motif of LolD map to LolC and LolE, suggesting that this motif is involved in subunit interaction in the Lol system (221). Interestingly, the suppressors are in a cytoplasmic loop of LolE and a periplasmic loop of LolC, raising the possibility of asymmetries in the way that LolD interacts with the IM subunits.
In proteins that are essentially homodimers, including the exporters MsbA and BmrA and the importer BtuCD, the presence of two identical ABCs would suggest that both are able to hydrolyze ATP, even though asymmetries that restrict ATP hydrolysis to just one of the two sites at a time may arise during the catalytic cycle, as suggested by vanadate-trapping experiments (347, 447, 495). BPD importers have a second potential source of asymmetry because the BPs themselves are asymmetric (388) and could impose functional asymmetries at the nucleotide-binding sites, a possibility that has not been explored. Interestingly, the addition of the BP BtuF did induce asymmetries in the most recent structure of BtuCD (217).
Molecular dynamics simulations and normal mode analysis have also been used to investigate asymmetries in ABC transporters. A simulation beginning with a Mg·ATP/Mg·ADP-bound MJ0796 dimer, designed to mimic ATP hydrolysis at a single site, revealed movement within one helical domain that might loosen the interaction between the ADP-bound monomer and the IM domain (228). Two different groups modeled ATP into both sites in the structure of BtuCD and found that only one ATP-binding site undergoes closure (222, 345), although the idea that one site might close independently of the other runs counter to the traditional interpretation of cooperativity in nucleotide binding and hydrolysis observed in ABC transporters. The work of a third group simulating BtuCD suggests that only simultaneous opening of both ATP-binding sites triggers appropriate conformational changes in the membrane (520).
Efforts have been made to measure the stoichiometric ratio of substrate transport to ATP hydrolysis in vivo and in vitro for several ABC transporters in an effort to determine whether one or both ATPs are hydrolyzed, but no one answer is universally accepted as yet. Often, substantial levels of uncoupled ATP hydrolysis, or possibly leakage of substrate from membrane vesicles, complicate the determination. In the maltose transporter, rates of 1.4 to 17 ATPs per transported sugar are reported, varying with the concentration of ATP trapped inside (98). Stoichiometries ranging from 5 to 25 ATPs per histidine have been reported for the histidine transporter (30). In the oligopeptide transporter, where the BP OpuA is tethered to the membrane via a lipid moiety, ATP hydrolysis is tightly coupled to transport and ratios approaching 2 ATPs per peptide transporter are seen (354). In studies of maltose and glycine-betaine transport in vivo, stoichiometries approaching 1 to 2 have been reported (316). Finally, a stoichiometric ratio of 1 was reported for the maltose transporter, based on comparison of growth yields of bacteria grown on different sugars under anaerobic conditions (321). Larger substrates may offer other complications; long linear maltodextrins are transported more slowly than maltose, leading to the suggestion that maltodextrins may be fed through the maltose transporter via a ratchet-like mechanism that expends more ATP per sugar transported (118).
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Most ABC importers rely on the presence of a high-affinity extracytoplasmic BP for function and are also called BPD transport systems. BPs are soluble proteins located in the periplasmic space between the inner and outer membranes of gram-negative bacteria. In gram-positive organisms, which lack a periplasm, they are often lipoproteins bound to the external face of the cytoplasmic membrane by N-terminal acyl-glyceryl cysteines. BPs are also found fused to the membrane transporter itself in some gram-positive organisms (497). In archaea, BPs either are lipoproteins or display a type III signal sequence. The latter BPs are proposed to constitute an extracellular multioligomeric organelle, the bindosome (553). All BPD transporters belong to class 3 and have ABC and IM domains on independent polypeptide chains. Some BP-independent class 3 importers have been characterized, as well as a few class 1 importers with a fused IM-ABC organization. Typically, the genes encoding a given transporter are carried in an operon, but in some gram-positive organisms, a single ABC subunit is shared by multiple transporters (385).
To be transported efficiently into the cytoplasm in gram-negative bacteria, substrates first pass through the outer membrane, using one of three different pathways (see reference 333 for a comprehensive review).
Most small substrates, with molecular masses below 650 Da, cross the outer membrane through the nonspecific (generalized) porins, such as the OmpF or OmpC porins of Enterobacteriaceae (Fig. 2a) (333). These trimeric porins vary in their preferences for solutes of different sizes and charges. Their importance in transport is highlighted by the observation that mutants lacking these proteins are pleiotropically affected in the utilization of several different substrates (23). Crystallographic analyses reveal that porins adopt a β-barrel conformation with 16 β-strands spanning the outer membrane. A large loop folds back into the barrel, forming a constriction zone about halfway through the channel that contributes to the exclusion limit and ion selectivity of the pore (251).
When the size of the substrate exceeds the size handled by generalized porins, a specific or specialized porin is used. The best example known so far is maltoporin, the lamB gene product, which is essential for the transport of maltodextrins of more than three glucose residues (Fig. 2b, diagram F) (466). In contrast to the case for general porins, the genes coding for specialized porins are often linked genetically to the regions encoding the rest of the transporter, and their expression is tightly coregulated (466). The crystal structure of maltoporin reveals an 18-stranded β-barrel with three inwardly folded loops that constrict the diameter of the channel. Aromatic residues lining the channel constitute part of a diffusion pathway for maltodextrins through the channel, which appears to be designed to facilitate translocation rather than to bind sugars tightly (133, 424). LamB also functions as a general glycoporin, whose increased expression facilitates growth in carbohydrate-limited chemostats, with glucose, lactose, arabinose, or glycerol as the carbon source (108). ScrY, the sucrose porin of Klebsiella pneumoniae, and OprB, the D-glucose and D-xylose porin of Pseudomonas aeruginosa, constitute examples of other specific porins in gram-negative bacteria (429, 490). Pseudomonas aeruginosa actually lacks general porins, and most nutrients are taken in through specific porins of the OprD family, with OprD itself mediating the uptake of basic amino acids (471, 489). The structure of OprD reveals a β-barrel with a ladder of positively charged residues that funnel into a constriction site lined by residues which are not conserved in the family (31). It is suggested that substrate specificity is mediated by variation of residues at the site of constriction in the channel.
Because the size and scarcity of certain nutrients, including vitamin B12 and the Fe3+-siderophore complexes, exceed the size limit of porins (516), these compounds are bound by high-affinity outer membrane receptors (OMRs) that also function as transporters to move the compounds into the periplasmic space (Fig. 2b, diagram E) (523). An expenditure of energy is required to release these compounds from the OMRs so they can be transported, and studies with the E. coli ferrichrome (Fhu) receptor indicate that translocation of the substrate is dependent on the cytoplasmic membrane electrochemical gradient (46). The transduction of energy from the cytoplasmic membrane to the OMR is achieved by a set of three cytoplasmic membrane proteins, ExbB, ExbD, and TonB, that form a complex in the inner membrane (377). TonB and ExbD are proteins with single TM segments and large hydrophilic periplasmic domains. It is postulated that these domains interact with a conserved region of the OMR known as the TonB box to trigger the release of the substrate and its diffusion through a channel within the OMR. The OMRs FhuE, FhuA, and IutA are highly specific for their individual siderophore substrates, i.e., coprogen, ferrichrome, and aerobactin, respectively, though all are transported into the cytoplasm by the same BPD ABC transporter, FhuBDC (143, 255). It is also remarkable that these OMRs display higher affinities for their substrates than does the periplasmic BP FhuD (256). There are currently crystal structures of the following six OMRs: FhuA (150, 286), FepA (55), FecA (149), BtuB (72), FpvA (77), and FptA (78). They show a remarkably conserved organization composed of two domains, a 22-stranded β-barrel and an N-terminal globular domain called a plug that lies within the barrel.
Some OMRs are also involved in the regulation of transcription, signaling from the cell surface to the cytoplasm via a signaling cascade (32, 186). A cascade signals the presence of the inducer in the culture medium to the cytoplasm, where gene transcription occurs. For example, when the OMR FecA binds its substrate, iron dicitrate, signaling is mediated through interaction between the N-terminal domain of FecA and the cytoplasmic membrane signaling protein FecR. Activated FecR interacts with the FecI sigma factor, leading to initiation of transcription of the fecABCDE transport genes (47).
New results are expanding the range of potential substrates that can be transported by OMRs. A very peculiar system, described for a Sphingomonas sp., was found to be expressed in alginate-induced cells. Alginate is a high-molecular-mass polysaccharide (25,000 Da) which is thought to enter the cell intact, since alginate-degrading enzymes are located exclusively in the cytoplasm (188). A specific outer membrane organelle, called the "pit," is thought to mediate alginate uptake through the outer membrane. Alginate-induced outer membrane proteins are similar to TonB-dependent receptors, raising the possibility that some members of this family might be involved in polysaccharide uptake (187). In addition, the outer membrane protein SusC, similar to TonB-dependent receptors, is maltose inducible and essential for maltose and starch uptake in Bacteroides thetaiotaomicron (395). More recently, transport of maltose in Caulobacter crescentus was found to be