Receptor-transporter interactions of canonical ATP-binding cassette import systems in prokaryotes

ATP-binding cassette (ABC) transport systems mediate the translocation of solutes across biological membranes at the expense of ATP. They share a common modular architecture comprising two pore-forming transmembrane domains and two nucleotide binding domains. In prokaryotes, ABC transporters are involved in the uptake of a large variety of chemicals, including nutrients, osmoprotectants and signal molecules. In pathogenic bacteria, some ABC importers are virulence factors. Canonical ABC import systems require an additional component, a substrate-specific receptor or binding protein for function. Interaction of the liganded receptor with extracytoplasmic loop regions of the transmembrane domains initiate the transport cycle. In this review we summarize the current knowledge on receptor-transporter interplay provided by crystal structures as well as by biochemical and biophysical means. In particular, we focus on the maltose/maltodextrin transporter of enterobacteria and the transporters for positively charged amino acids from the thermophile Geobacillus stearothermophilus and Salmonella enterica serovar Typhimurium.     

Mapping of interdomain interfaces required for the functional architecture of Yor1p, a eukaryotic ATP-binding cassette (ABC) transporter

ATP-binding cassette (ABC) transporters are a large superfamily of proteins that mediate substrate translocation across biological membranes. Our goal was to define the intramolecular interactions that contribute to quaternary assembly of a eukaryotic ABC transporter and determine how the architecture of this protein influences its biogenesis within the secretory pathway. We used chemical cross-linking approaches to map interdomain interactions in the yeast ABC transporter, Yor1p, which functions as a pleiotropic drug pump at the plasma membrane. We have defined interactions between the two nucleotide-binding domains (NBDs) and between the NBDs and specific intracellular loops (ICLs) that are consistent with current structural models of bacterial ABC exporters. Furthermore, we detected relatively weak NBD-NBD and ICL-ICL interactions that may correspond to transient sites of cross-talk between domains required for coupling of ATP hydrolysis with substrate translocation. Mutation of a key residue in ICL2 caused misassembly of the altered protein, leading to increased sensitivity to the mitochondrial poison, oligomycin. We identified intragenic suppressing mutations that rescued the oligomycin resistance associated with this aberrant protein and demonstrated that the suppressing mutations restored multiple interdomain interfaces. Together, our biochemical and genetic approaches contribute to a greater understanding of the architecture of this important class of proteins and provide insight into the quality control surveillance that regulates their biogenesis and deployment within the eukaryotic cell.     

Structure,Function, and Evolution of Bacterial ATP-Binding Cassette Systems

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.     

ABC transporters, mechanisms and biology: an overview

This chapter concentrates mainly on structural and mechanistic aspects of ABC (ATP-binding cassette) transporters and, as an example of the physiological significance of these proteins, on lipid transport, vitally important for human health. The chapter considers those aspects of ABC transporter function that appear reasonably well established, those that remain controversial and what appear to be emerging themes. Although we have seen dramatic progress in ABC protein studies in the last 20 years, we are still far from a detailed molecular understanding of function. Nevertheless two critical steps - capture and release of allocrites (transport substrates) involving a binding cavity in the membrane domain, and hydrolysis of ATP by the NBD (nucleotide-binding domain) dimer - are now described by persuasive and testable models: alternating access, and sequential firing of catalysis sites respectively. However, these need to be tested rigorously by more structural and biochemical studies. Other aspects considered include the level at which ATP binding and dimer activation are controlled, the nature of the power stroke delivering mechanical energy for transport, and some unexpected and intriguing differences between importers and exporters. The chapter also emphasizes that some ABC transporters, although important for elimination of toxic compounds (xenobiotics), are also increasingly seen to play crucial roles in homoeostatic regulation of membrane biogenesis and function through translocation of endogenous allocrites such as cholesterol. Another emerging theme is the identification of accessory domains and partners for ABC proteins, resulting in a corresponding widening of the range of activities. Finally, what are the prospects for translational research and ABC transporters?     

The tripartite ATP-independent periplasmic (TRAP) transporters of bacteria and archaea

Until recently, extracytoplasmic solute receptor (ESR)-dependent uptake systems were invariably found to possess a conserved ATP-binding protein (the ATP-binding cassette protein or ABC protein), which couples ATP hydrolysis to the translocation of the solute across the cytoplasmic membrane. While it is clear that this class of ABC transporter is ubiquitous in prokaryotes, it is now firmly established that other, unrelated types of membrane transport systems exist which also have ESR components. These systems have been designated tripartite ATP-independent periplasmic (TRAP) transporters, and they form a distinct class of ESR-dependent secondary transporters where the driving force for solute accumulation is an electrochemical ion gradient and not ATP hydrolysis. Currently, the most well characterised TRAP transporter at the functional and molecular level is the high-affinity C4-dicarboxylate transport (Dct) system from Rhodobacter capsulatus. This consists of three proteins; an ESR (DctP) and small (DctQ) and large (DctM) integral membrane proteins. The characteristics of this system are discussed in detail. Homologues of the R. capsulatus DctPQM proteins are present in a diverse range of prokaryotes, both bacteria and archaea, but not in eukaryotes. The deduced structures and possible functions of these homologous systems are described. In addition to the DctP family, other types of ESRs can be associated with TRAP transporters. A conserved family of immunogenic extracytoplasmic proteins is shown to be invariably associated with TRAP systems that contain a large DctQM fusion protein. All of the currently known archaeal systems are of this type. It is concluded that TRAP transporters are a widespread and ancient type of solute uptake system that transport a potentially diverse range of solutes and most likely evolved by the addition of auxiliary proteins to a single secondary transporter.     

Insight in eukaryotic ABC transporter function by mutation analysis

With regard to structure-function relations of ATP-binding cassette (ABC) transporters several intriguing questions are in the spotlight of active research: Why do functional ABC transporters possess two ATP binding and hydrolysis domains together with two ABC signatures and to what extent are the individual nucleotide-binding domains independent or interacting? Where is the substrate-binding site and how is ATP hydrolysis functionally coupled to the transport process itself? Although much progress has been made in the elucidation of the three-dimensional structures of ABC transporters in the last years by several crystallographic studies including novel models for the nucleotide hydrolysis and translocation catalysis, site-directed mutagenesis as well as the identification of natural mutations is still a major tool to evaluate effects of individual amino acids on the overall function of ABC transporters. Apart from alterations in characteristic sequence such as Walker A, Walker B and the ABC signature other parts of ABC proteins were subject to detailed mutagenesis studies including the substrate-binding site or the regulatory domain of CFTR. In this review, we will give a detailed overview of the mutation analysis reported for selected ABC transporters of the ABCB and ABCC subfamilies, namely HsCFTR/ABCC7, HsSUR/ABCC8,9, HsMRP1/ABCC1, HsMRP2/ABCC2, ScYCF1 and P-glycoprotein (Pgp)/MDR1/ABCB1 and their effects on the function of each protein.     

Simulation of the coupling between nucleotide binding and transmembrane domains in the ATP binding cassette transporter BtuCD

The nucleotide-induced structural rearrangements in ATP binding cassette (ABC) transporters, leading to substrate translocation, are largely unknown. We have modeled nucleotide binding and release in the vitamin B(12) importer BtuCD using perturbed elastic network calculations and biased molecular dynamics simulations. Both models predict that nucleotide release decreases the tilt between the two transmembrane domains and opens the cytoplasmic gate. Nucleotide binding has the opposite effect. The observed coupling may be relevant for all ABC transporters because of the conservation of nucleotide binding domains and the shared role of ATP in ABC transporters. The rearrangements in the cytoplasmic gate region do not provide enough space for B(12) to diffuse from the transporter pore into the cytoplasm, which could suggest that peristaltic forces are needed to exclude B(12) from the transporter pore.     

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

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

Structure of ABC transporters

ABC (ATP-binding cassette) transporters are primary active membrane proteins that translocate solutes (allocrites) across lipid bilayers. The prototypical ABC transporter consists of four domains: two cytoplasmic NBDs (nucleotide-binding domains) and two TMDs (transmembrane domains). The NBDs, whose primary sequence is highly conserved throughout the superfamily, bind and hydrolyse ATP to power the transport cycle. The TMDs, whose primary sequence and protein fold can be quite disparate, form the translocation pathway across the membrane and generally (but not always) determine allocrite specificity. Structure determination of ABC proteins initially took advantage of the relative ease of expression and crystallization of the hydrophilic bacterial NBDs in isolation from the transporter complex, and revealed detailed information on the structural fold of these domains, the amino acids involved in the binding and hydrolysis of nucleotide, and the head-to-tail arrangement of the NBD-NBD dimer interface. More recently, several intact transporters have been crystallized and three types have, so far, been characterized: type I and II ABC importers, and ABC exporters. All three are present in prokaryotes, but only the ABC exporters appear to be present in eukaryotes. Their structural determination has provided insight into the mechanisms of energy and signal transduction between the NBDs and TMDs (i.e. between the ATP- and allocrite-binding sites) and, for some, the nature of the allocrite-binding site(s) within the TMDs. In this chapter, we focus primarily on the ABC exporters and describe the structural, biochemical and biophysical evidence for and against the controversial bellows-like mechanism proposed for allocrite efflux.     

Energy Coupling Factor-Type ABC Transporters for Vitamin Uptake in Prokaryotes

Energy coupling factor (ECF) transporters are a subgroup of ATP-binding cassette (ABC) transporters involved in the uptake of vitamins and micronutrients in prokaryotes. In contrast to classical ABC importers, ECF transporters do not make use of water-soluble substrate binding proteins or domains but instead employ integral membrane proteins for substrate binding (named S-components). S-components form active translocation complexes with the ECF module, an assembly of two nucleotide-binding domains (NBDs, or EcfA) and a second transmembrane protein. In some cases, the ECF module is dedicated to a single S-component, but in many cases, the ECF module can interact with several different S-components that are unrelated in sequence and bind diverse substrates. The modular organization with exchangeable S-components on a single ECF module allows the transport of chemically different substrates via a common route. The recent determination of the crystal structures of the S-components that recognize thiamin and riboflavin has provided a first clue about the mechanism of S-component exchange. This review describes recent advances and the current views of the mechanism of transport by ECF transporters.     

Conformational changes in MetNI: steered molecular dynamic studies of the methionine ABC transporter with and without substrates

ATP-binding cassette (ABC) transporters are integral membrane proteins that utilised energy from ATP hydrolysis to translocate substrates across the membrane. In addition to the common nucleotide-binding domains (NBDs) and transmembrane domains (TMDs), the methionine ABC transporter has C-terminal regulatory domains (C2 domains) that belong to ACT protein family. When the amount of methionine in the cell is high, the transport stops. This phenomenon is called trans-inhibition. To understand how a trans-inhibited protein returns to an uninhibited, resting state, we performed steered molecular dynamic simulations with and without the substrates. From the simulations, we observed some important conformational changes in the whole ABC transporter, including the constriction in the translocation pathway in the TMDs and approach of the NBDs. However, the C2 domains behaved differently in two types of the simulations. These findings might help to explain the relationship of the conformational changes of the C2 domains with the rearrangements of the NBDs or TMDs, and provide a way to understand the trans-inhibition from an opposite direction.     

ABC transporters and immunity: mechanism of self-defense

The transporter associated with antigen processing (TAP) is a prototype of an asymmetric ATP-binding cassette (ABC) transporter, which uses ATP binding and hydrolysis to translocate peptides from the cytosol to the lumen of the endoplasmic reticulum (ER). Here, we review molecular details of peptide binding and ATP binding and hydrolysis as well as the resulting allosteric cross-talk between the nucleotide-binding domains and the transmembrane domains that drive translocation of the solute across the ER membrane. We also discuss the general molecular architecture of ABC transporters and demonstrate the importance of structural and functional studies for a better understanding of the role of the noncanonical site of asymmetric ABC transporters. Several aspects of peptide binding and specificity illustrate details of peptide translocation by TAP. Furthermore, this ABC transporter forms the central part of the major histocompatibility complex class I (MHC I) peptide-loading machinery. Hence, TAP is confronted with a number of viral factors, which prevent antigen translocation and MHC I loading in virally infected cells. We review how these viral factors have been used as molecular tools to decipher mechanistic aspects of solute translocation and discuss how they can help in the structural analysis of TAP.     

Structure and mechanism of ABC transporters

ATP-binding cassette (ABC) transporters facilitate unidirectional translocation of chemically diverse substrates across cell or organelle membranes. The recently determined crystal structures of the vitamin B(12) importer BtuCD and its cognate binding protein BtuF have revealed critical architectural features that are probably shared by other ABC transporters. For example, the arrangement of the ABC domains and their interface with the membrane-spanning domains are probably conserved, whereas the number of transmembrane helices and their arrangement are not. Two distinct mechanistic schemes for how ABC engines couple ATP hydrolysis to substrate transport have been proposed recently and are being explored.     

Subunit interactions in ABC transporters: towards a functional architecture

The ABC superfamily is a diverse group of integral membrane proteins involved in the ATP-dependent transport of solutes across biological membranes in both prokaryotes and eukaryotes. Although ABC transporters have been studied for over 30 years, very little is known about the mechanism by which the energy of ATP hydrolysis is used to transport substrate across the membrane. The recent report of the high resolution crystal structure of HisP, the nucleotide-binding subunit of the histidine permease complex of Salmonella typhimurium, represents a significant breakthrough toward the elucidation of the mechanism of solute translocation by ABC transporters. In this review, we use data from the crystallographic structures of HisP and other nucleotide-binding proteins, combined with sequence analysis of a subset of atypical ABC transporters, to argue a new model for the dimerisation of the nucleotide-binding domains that embraces the notion that the C motif from one subunit forms part of the ATP-binding site in the opposite subunit. We incorporate this dimerisation of the ATP-binding domains into our recently reported beta-barrel model for P-glycoprotein and present a general model for the cooperative interaction of the two nucleotide-binding domains and the translocation of mechanical energy to the transmembrane domains in ABC transporters.     

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.     

ATP-binding cassette transporters in Escherichia coli

ATP-binding cassette (ABC) transporters are integral membrane proteins that actively transport molecules across cell membranes. In Escherichia coli they consist primarily of import systems that involve in addition to the ABC transporter itself a substrate binding protein and outer membrane receptors or porins, and a number of transporters with varied functions. Recent crystal structures of a number of ATPase domains, substrate binding proteins, and full-length transporters have given new insight in the molecular basis of transport. Bioinformatics approaches allow an approximate identification of all ABC transporters in E. coli and their relation to other known transporters. Computational approaches involving modeling and simulation are beginning to yield insight into the dynamics of the transporters. We summarize the function of the known ABC transporters in E. coli and mechanistic insights from structural and computational studies.     

The ATP switch model for ABC transporters

ABC transporters mediate active translocation of a diverse range of molecules across all cell membranes. They comprise two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs). Recent biochemical, structural and genetic studies have led to the ATP-switch model in which ATP binding and ATP hydrolysis, respectively, induce formation and dissociation of an NBD dimer. This provides an exquisitely regulated switch that induces conformational changes in the TMDs to mediate membrane transport.     

Cysteine-scanning mutagenesis provides no evidence for the extracellular accessibility of the nucleotide-binding domains of the multidrug resistance transporter P-glycoprotein

Multidrug resistance of cancer cells is, at least in part, conferred by overexpression of P-glycoprotein (P-gp), a member of the ATP-binding cassette (ABC) superfamily of active transporters. P-gp actively extrudes chemotherapeutic drugs from cells, thus reducing their efficacy. As a typical ABC transporter, P-gp has four domains: two transmembrane domains, which form a pathway through the membrane through which substrates are transported, and two hydrophilic nucleotide-binding domains (NBDs), located on the cytoplasmic side of the membrane, which couple the energy of ATP hydrolysis to substrate translocation. It has been proposed that the NBDs of ABC transporters, including the histidine permease of Salmonella typhimurium and the cystic fibrosis transmembrane conductance regulator, are accessible from the extracellular surface of the cell, spanning the membrane directly or potentially contributing to the transmembrane pore. Such organization would have significant implications for the transport mechanism. We determined to establish whether the NBDs of P-gp are exposed extracellularly and which amino acids are accessible, using cysteine-scanning mutagenesis and limited proteolysis. In contrast to other transporters, the data provided no evidence that the P-gp NBDs are exposed to the cell surface. The implications for the structure and mechanism of P-gp and other ABC transporters are discussed.     

ATP-binding cassette transporters in Escherichia coli

ATP-binding cassette (ABC) transporters are integral membrane proteins that actively transport molecules across cell membranes. In Escherichia coli they consist primarily of import systems that involve in addition to the ABC transporter itself a substrate binding protein and outer membrane receptors or porins, and a number of transporters with varied functions. Recent crystal structures of a number of ATPase domains, substrate binding proteins, and full-length transporters have given new insight in the molecular basis of transport. Bioinformatics approaches allow an approximate identification of all ABC transporters in E. coli and their relation to other known transporters. Computational approaches involving modeling and simulation are beginning to yield insight into the dynamics of the transporters. We summarize the function of the known ABC transporters in E. coli and mechanistic insights from structural and computational studies.     

The role of ATP-binding cassette transporters in bacterial pathogenicity

The ATP-binding cassette transporter superfamily is present in all three domains of life. This ubiquitous class of integral membrane proteins have diverse biological functions, but their fundamental role involves the unidirectional translocation of compounds across cellular membranes in an ATP coupled process. The importance of this class of proteins in eukaryotic systems is well established as typified by their association with genetic diseases and roles in the multi-drug resistance of cancer. In stark contrast, the ABC transporters of prokaryotes have not been exhaustively investigated due to the sheer number of different roles and organisms in which they function. In this review, we examine the breadth of functions associated with microbial ABC transporters in the context of their contribution to bacterial pathogenicity and virulence.