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FEMS Microbiology Reviews 22 (1998) 1^20 ATP-binding-cassette (ABC) transport systems: Functional and structural aspects of the ATP-hydrolyzing subunits/domains Erwin Schneider *, Sabine Hunke Humboldt-Universitaët zu Berlin, Institut fuër Biologie/Bakterienphysiologie, Unter den Linden 6, D-10099 Berlin, Germany Received 6 October 1997; accepted 28 November 1997 Abstract Members of the superfamily of adenosine triphosphate (ATP)-binding-cassette (ABC) transport systems couple the hydrolysis of ATP to the translocation of solutes across a biological membrane. Recognized by their common modular organization and two sequence motifs that constitute a nucleotide binding fold, ABC transporters are widespread among all living organisms. They accomplish not only the uptake of nutrients in bacteria but are involved in diverse processes, such as signal transduction, protein secretion, drug and antibiotic resistance, antigen presentation, bacterial pathogenesis and sporulation. Moreover, some human inheritable diseases, like cystic fibrosis, adrenoleukodystrophy and Stargardt's disease are caused by defective ABC transport systems. Thus, albeit of major significance, details of the molecular mechanism by which these systems exert their functions are still poorly understood. In this review, recent data concerning the properties and putative role of the ATP-hydrolyzing subunits/domains are summarized and compared between bacterial and eukaryotic systems. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. Keywords : Adenosine triphosphate-binding-cassette transport system ; Bacterial binding protein-dependent system ; Bacterial exporter; P-glycoprotein; Cystic ¢brosis transmembrane regulator protein Contents 1. 2. 3. 4. 5. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . Secondary structural models of the ABC domain Enzymatic properties . . . . . . . . . . . . . . . . . . . . . Sensitivity to inhibitors . . . . . . . . . . . . . . . . . . . Function of conserved amino acid residues . . . . . 5.1. Walker sites A and B . . . . . . . . . . . . . . . . . 5.2. Catalytic carboxylate . . . . . . . . . . . . . . . . . 5.3. Helical domain . . . . . . . . . . . . . . . . . . . . . 5.4. Linker peptide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * Corresponding author. Humboldt-Universitaët zu Berlin, Institut fuër Biologie/Bakterienphysiologie, Chausseestr. 117, D-10115 Berlin, Germany. Tel.: +49 (30) 2093 8121; Fax: +49 (30) 2093 8126; E-mail: [email protected] 0168-6445 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 1 6 8 - 6 4 4 5 ( 9 8 ) 0 0 0 0 2 - 3 FEMSRE 605 29-5-98 . . . . . . . . . . . . . . . . . . 2 5 6 7 8 8 8 9 9 2 E. Schneider, S. Hunke / FEMS Microbiology Reviews 22 (1998) 1^20 5.5. Switch region . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Recognition of the transported solute by the ABC protein/domain . . . . . . . . . Functional necessity of both ABC domains . . . . . . . . . . . . . . . . . . . . . . . . . . . Interaction with the membrane-spanning components/domains . . . . . . . . . . . . . Transmembrane orientation? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tentative models on the role of the ABC domains in the translocation process 10.1. KpsMT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2. Relevance to binding protein-dependent import systems? . . . . . . . . . . . . . 10.3. P-Glycoprotein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Summary and concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12. Note added in proof . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. 7. 8. 9. 10. 1. Introduction The rapidly growing family of ABC (`ATP-Binding Cassette') transport systems [1] (or Tra¤c ATPases, [2]) comprise an extremely diverse class of membrane transport proteins that couple the energy of ATP hydrolysis to the translocation of solutes across biological membranes. Members of this family not only accomplish the uptake of nutrients but are involved in a large variety of processes, such as signal transduction, protein secretion, drug and antibiotic resistance, antigen presentation, bacterial pathogenesis and sporulation, to name just a few [3]. ABC trans- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 10 11 12 13 14 14 14 15 15 16 16 16 porters have been identi¢ed in organisms belonging to each of the three major domains (bacteria, archaea and eukarya, including man), and might thus be considered as an ancient proteinaceous device for solutes to pass a lipid bilayer against a concentration gradient. Typically, an ABC transporter is composed of four parts: two membrane-integral domains each of which spans the membrane six times, and two ATP-hydrolyzing domains (referred to as ABC subunits/domains from hereon) [3]. In eukaryotic systems, the modules are mostly fused to yield a single polypeptide chain, while bacterial ABC transporters are built up from individual subunits [3] (Fig. 1). Fig. 1. Organization of the four structural domains of ABC transporters. The domains can basically be expressed as separate polypeptides or can be fused in a variety of con¢gurations. A is mainly represented by binding protein-dependent bacterial import systems. In some cases either the membrane-integral domains or the ABC domains are fused. In others, two copies of one membrane-integral subunit or of one ABC subunit are present. The extracellular substrate-binding protein that is unique to this subclass is also shown. Examples for con¢guration B include various bacterial export systems and the mammalian TAP/TAP2 peptide transporter. C is veri¢ed in eukaryotic systems, such as the P-glycoprotein and in the cystic ¢brosis transmembrane regulator protein (CFTR). In these proteins, the N- and C-terminal ABC domains are also referred to as nucleotide binding folds (NBF) 1 and 2, respectively. In CFTR, an additional regulatory domain is located between NBF1 and the following transmembrane domain, which is not shown here. See text for more details. FEMSRE 605 29-5-98 E. Schneider, S. Hunke / FEMS Microbiology Reviews 22 (1998) 1^20 3 Fig. 2. Linear representation of a prototype ABC domain, with assignment of the main functional sites. See text for details. However, examples with one hydrophobic domain fused to one ATPase domain that function as homo- or heterodimers have also been reported [4^ 6]. The ATP-hydrolyzing domains are characterized by two short sequence motifs in their primary structure (`Walker' site A: GXXGXGKS/T, X can be varied; `Walker' site B: hhhhD, h stands for hydrophobic) that are supposed to constitute a nucleotide binding fold [7,8]. The Walker B-site is immediately preceded by a highly conserved sequence motif (`linker peptide', LSGGQQ/R/KQR) that is unique to the ABC transport family (`signature sequence') [1,2] and has proven to be a useful tool in identifying putative new members of the family (Fig. 2). By these criteria, a few proteins have also been recognized as typical ABC proteins that clearly serve other functions than transport, such as UvrA from E. coli and elongation factor EF-3 of yeast (summarized in [3]). Bacterial binding protein-dependent transport systems that mediate the uptake of a large variety of nutrients, such as sugars, amino acids, peptides, inorganic ions and vitamins represent the best-characterized subclass of the family [9]. They are uniquely equipped with an additional extracellular (periplasmic) protein component that is designed to act as a scavenger for substrate molecules and, in some cases, also as chemoreceptor [10]. The structural organization of the four membrane-associated domains is the most variable in this subfamily (Fig. 1). In the majority of transporters two di¡erent hydrophobic proteins are assembled with a homodimer of one ABC subunit. However, complexes comprising either fused hydrophobic or ABC subunits as well as transporters that have all four domains encoded by individual genes have also been described [9]. The transported substrates of bacterial ABC exporters are diverse but many of them are virulence factors [3,6] or confer resistance to antibiotics [11] or cellular defence factors on pathogenic bacteria [12], thereby contributing to a growing threat to public health [13]. Prototype bacterial ABC exporters include systems that translocate extracellular polypeptides across the cell envelope of Gram-negative bacteria, such as haemolysin in certain E. coli strains (HlyBD) [6] and proteases in the plant pathogen Erwinia chrysanthemi (PrtED) [14]. They represent examples for the Type I secretion pathway that functions in a sec (general secretion system)-independent manner [15,16]. These systems require, in addition to the ABC protein an accessory component that belongs to a recently identi¢ed, novel class of export proteins, designated the membrane fusion protein (MFP) family [17]. Members of another subfamily of bacterial ABC exporters are involved in the secretion of drugs and carbohydrates in both Gram-positive and Gram-negative bacteria [18]. Examples are the KpsMT system that secretes the capsular polysialic acid in E. coli K1 [19], and the NodIJ proteins involved in nodulation of the nitrogen-¢xing plant symbiont Rhizobium leguminosarum [20]. Prominent and well-studied eukaryotic members of the family include the STE6 protein that secretes the a-mating factor in yeast [21] and several medically important mammalian proteins, like the TAP1- FEMSRE 605 29-5-98 4 E. Schneider, S. Hunke / FEMS Microbiology Reviews 22 (1998) 1^20 FEMSRE 605 29-5-98 E. Schneider, S. Hunke / FEMS Microbiology Reviews 22 (1998) 1^20 TAP2 peptide transporter, associated with major histocompatibility complex (MHC) class I antigen presentation [4], the P-glycoprotein that, when ampli¢ed, enables certain cancer cells to extrude chemotherapeutic drugs [22], and the cystic ¢brosis transmembrane regulator protein (CFTR) which is mutated in patients a¡ected by the most common hereditary disease cystic ¢brosis [23]. Most recently, adrenoleukodystrophy, an error in peroxisomal L-oxidation of long fatty acids [24], and Stargardt's disease, the most common recessive macular dystrophy [25,26] were identi¢ed as further examples of inheritable diseases that are caused by defective ABC transport proteins. Thus, elucidating the molecular mechanism by which ABC-transport systems exert their functions has become a major scienti¢c challenge. In particular, the structure and precise role of the ABC subunits/ domains as the key players need to be unravelled. ABC transport systems have been the subject of a number of reviews covering either the family as a whole [3,27] or summarizing the knowledge on subgroups under various aspects [2,6,9,19,22,28,29]. It is the scope of this review to focus on recent new insights concerning the structure and function of the ABC subunits/domains from di¡erent systems and to discuss current models on their putative role during the transport process. 2. Secondary structural models of the ABC domain To date, structural data at high resolution are not available for any ABC transport system. Thus, we rely on two slightly di¡ering secondary structural models that were proposed for the ABC domain on the basis of the known structures of adenylate kinase 5 and two GTP-binding proteins (p21ras , elongation factor Tu) [30], and that of adenylate kinase only [1], respectively. According to these predictions, the consensus fragment of all ABC domains (approx. 250 residues) is folded in an alternating series of six K-helical subdomains and ¢ve L-strands. The Walker motifs A (K-helical, preceded by a glycinerich loop) and B (L-strand) are linked by an extended peptide fragment of approx. 100 residues that largely folds into K-helical conformation (`helical domain or loop') and has no equivalent in adenylate kinase. The primary structure of this fragment is less conserved and considerably variable in length when compared between di¡erent proteins. At the joining point between the helical domain and the Walker B site, a well conserved peptide fragment, rich in glutamine and glycine residues is located (`linker peptide'), that has been suggested by Ames and Lecar [31] to resemble peptide linkers. Based on sequence similarities with the RecA protein of E. coli, the socalled `switch region' was also recognized at the end of the L4-strand in ABC proteins [32] (Figs. 2 and 3). Recently, two alternative models were proposed for the N-terminal nucleotide-binding fold of CFTR (NBF1) that are based on a more detailed comparison with the K-subunits of G-proteins [33] and with the X-ray structure of bovine mitochondrial F1 -ATPase [34]. Manavalan et al. concluded from their analysis that the core of the linker peptide is in fact part of the nucleotide binding pocket [33], while Annereau et al. suggested a L-sheet-like rather than helical conformation of that part of the helical domain that encompasses phenylalanine 508 in CFTR [34]. A deletion of the latter is found in 68^ 70% of all patients a¡ected by cystic ¢brosis and is thought to cause misfolding and subsequent proteolytic degradation of the protein [35]. The authors 6 Fig. 3. Sequence alignment of ABC domains from bacterial and eukaryotic ABC transporters. Sequences were retrieved from the SWISSPROT database and aligned using the DNASIS software package (Hitachi). The proteins considered are: MDR1NBF1, P-glycoprotein (human, N-terminal domain); MDR1NBF2, P-glycoprotein (human, C-terminal domain); STE6NBF1, mating a factor secretion protein (yeast, N-terminal domain); STE6NBF2, mating a factor secretion protein (yeast, C-terminal domain); CFTRNBF1, cystic ¢brosis transmembrane regulator protein (human, N-terminal domain); CFTRNBF2, cystic ¢brosis transmembrane regulator protein (human, C-terminal domain); TAP1, TAP2, transporters associated with antigen processing (human); HLYBEC, hemolysin A secretion protein (E. coli); KPSTEC, ABC subunit of capsular polysialic acid secretion system (E. coli); HISPST, ABC subunit of histidine transport system (S. typhimurium); POTAEC, ABC subunit of polyamine transport system (E. coli) ; LACKAR, ABC subunit of lactose transport system (Agrobacterium radiobacter) ; MALKST, ABC subunit of maltose transport system (S. typhimurium). FEMSRE 605 29-5-98 6 E. Schneider, S. Hunke / FEMS Microbiology Reviews 22 (1998) 1^20 predict that a L-structural conformation of this fragment would result in a more buried position of F508 which could better account for the drastic consequences seen when deleted. 3. Enzymatic properties The hydrolysis of ATP has been demonstrated for members of most subclasses of ABC transport systems, either with puri¢ed complete protein complexes or separated ABC subunits and domains. The Michaelis constants are generally in the micro- molar range while the reported Vmax values lie between 0.01^20 Wmoles per min and mg protein (Table 1). Puri¢ed ABC subunits of bacterial binding protein-dependent import systems exhibit a spontaneous ATPase activity in the absence of the membrane-integral components [36^41]. In contrast, complete transport systems, when reconstituted into liposomes, catalyzed the hydrolysis of ATP only concomitantly with translocation of the substrate, that is, in the presence of the substrate-loaded binding protein [42^45]. Thus, the soluble component is thought to function as a signal transducer that ini- Table 1 Enzymatic properties of puri¢ed complete ABC transport systems and isolated ABC proteins/domains Protein(s) Substrate(s) Bacterial binding protein-dependent MalFGK2 maltose, maltodextrines MalK maltose, maltodextrines RbsA ribose MglECA galactose MglA galactose PotA polyamines HisQMP2 histidine, arginine Bacterial export systems HlyB-ABC hemolysin (HlyA) PrtD metalloproteases OleB (N-term.)oleandomycin Eucaryotic transporters P-glycoprotein cationic drugs P-gly-NBD1 P-gly-NBD1 P-gly-NBD2 CFTR chloride ions CFTR-NBD1 CFTR-NBD2 Organism ATPase activity (Wmol min31 mg31 ) import systems E. coli 0.86 (Vmax ) S. typhimurium 0.7^1.3 (Vmax ) E. S. S. E. S. 0.010 0.02 0.14 0.4 0.35 coli typhimurium typhimurium coli typhimurium E. coli Km (ATP) Inhibitors (WM) 74 70^80 Other features References vanadate in liposomes [42,43] NEM insensitive to vanadate [37,38] stimulated by galactose inhibited by spermidine in liposomes [40] [44] [39] [41] [45] fusion to GST [47] 140 nd 60 385 8000 1.0 (Vmax ) E. chrysanthemi 0.025 (Vmax ) S. antibioticus 19.4 (Vmax ) vanadate vanadate NEM 200 vanadate 12 1 vanadate azide chinese hamster 0.3 (Vmax ) 940 human 1.65 400 chinese hamster 3.9 (Vmax ) 800 human 0.11 nd vanadate NEM vanadate ba¢lomycin vanadate NEM vanadate mouse human chinese hamster human 0.025 (Vmax ) 0.180 0.024 (Vmax ) 0.05 (Vmax ) 0.03 (Vmax ) 0.006 2100 nd 20 000 303 110 86 NEM vanadate nd azide azide Ap5A fusion to MBP, insensitive to vanadate in detergent, stimulated by drugs in detergent, stimulated by drugs in liposomes stimulated by drugs in detergent stimulated by drugs fusion to L-Gal fusion to MBP in liposomes fusion to MBP fusion to MBP kinase activity [46] [48] [50] [49] [51] [52] [54] [55] [56] [57] [58] [59] Ap5A, P1 , P5 -di(adenosine-5P)pentaphosphate ; GST, glutathione-S-transferase; MBP, maltose binding protein; NBD, nucleotide binding domain; NEM, N-ethylmaleimide. FEMSRE 605 29-5-98 E. Schneider, S. Hunke / FEMS Microbiology Reviews 22 (1998) 1^20 tiates transport by a speci¢c interaction with the membrane-spanning domains at the external surface of the lipid bilayer [2,42]. In the case of the maltose transport system, the enzymatic activities compared well between the reconstituted complex from E. coli and the puri¢ed ATPase subunit from S. typhimurium (see Table 1). In the case of bacterial ABC exporters, relatively low ATPase activity was reported for the puri¢ed integral membrane protein PrtD of E. chrysanthemi in detergent solution [46], while the soluble ABC domain of HlyB, puri¢ed as a fusion to glutathione-S-transferase exhibited an ATPase activity comparable to that of import systems [47]. Unusually high activity was reported for a fusion to maltose-binding protein of the N-terminal nucleotidebinding fold of OleB involved in oleandomycin resistance of S. antibioticus [48]. Highly puri¢ed preparations of mammalian P-glycoprotein displayed ATPase activities in detergent solution [49,50] and/or reconstituted into liposomes [49^52] with rather low a¤nity as compared to bacterial proteins (Table 1). In liposomes only, the rate of hydrolysis is stimulated several-fold by drugs that are expelled by the protein. Separately expressed Nor C-terminal half-molecules also exhibited basal levels of ATPase activity, that however were not enhanced by drugs [53]. Moreover, low intrinsic ATPase activity was also demonstrated for both N- and C-terminal nucleotide binding domains of P-glycoprotein when analyzed as individual polypeptides [54^56]. However, the kinetic parameters are di¡erent from those of intact P-glycoprotein (Table 1). These ¢ndings indicate that both domains are catalytically active. Puri¢ed CFTR protein that functions as a gated chloride channel, was only recently demonstrated to hydrolyze ATP when reconstituted into liposomes. The enzymatic activity is modulated by kinase-dependent phosphorylation of a speci¢c peptide fragment (R-domain), which connects both half molecules [57]. Both the ¢rst and second nucleotidebinding fold exhibit ATPase activity as fusions to maltose binding protein with surprisingly similar kinetic properties as the mature protein [58,59]. Strikingly, the C-terminal domain was reported to additionally display adenylate kinase activity [59]. In general, ATP is the preferred substrate of ABC 7 subunits/domains or puri¢ed transporters but other nucleotides, such as guanosine triphosphate (GTP) [37,39,47^49,51,59], cytidine triphosphate (CTP) [37,47,49,51], uridine triphosphate (UTP) [47,49,51] and inosine triphosphate (ITP) [47,51] are also accepted, albeit with much lower a¤nity and substantially reduced rates of hydrolysis. The interaction with nucleotides was frequently found to result in a global conformational change of the protein, as assessed by various biophysical means [34,60^62] and by limited proteolysis [47,60]. In the case of the bacterial MalK protein, MgATP induced a speci¢c alteration in the structure that resulted in protection of the helical domain against proteolytic attack [60]. In contrast to these ¢ndings, the separated ¢rst ABC domain of CFTR fused to maltose binding protein could not be protected by ATP against trypsin, although the protein exhibited nucleotide binding activity [63]. 4. Sensitivity to inhibitors ABC transporters are a¡ected to di¡erent extents by a variety of inhibitors typically used as tools to distinguish P-, V- and F-type ATPases [64], thus indicating that they represent a unique family of ATPases [37,41,47,48,51]. The ATPase activity of most ABC transporters was reported to be impaired by ortho-vanadate in the micromolar range (see Table 1), a speci¢c inhibitor of P-type ATPases [64]. The reaction mechanism of the latter involves an aspartyl phosphate intermediate, the formation of which is blocked by vanadate [65]. In the case of P-glycoprotein, Senior and colleagues [66] have presented convincing evidence that vanadate inhibits the enzymatic activity by trapping ADP in a catalytic site, thereby preventing the release of the nucleotide from the protein. The authors further demonstrated that trapping of nucleotides at either of the two ATPase domains completely blocked activity which indicated that both sites cannot function independently (see also Section 7). The reconstituted bacterial binding protein-dependent ABC transport system for maltose (MalFGK2 ) also exhibits sensitivity to vanadate [43,67]. Strikingly, the enzymatic activity of the isolated ABC subunit MalK remains una¡ected [37,67]. FEMSRE 605 29-5-98 8 E. Schneider, S. Hunke / FEMS Microbiology Reviews 22 (1998) 1^20 These results indicated that functional coupling of the ABC domain to the membrane-integral parts of an ABC transporter is a prerequisite for vanadate action [67]. Thus, the data might be taken as additional evidence for the view that in fully assembled (complete) ABC transport system, ATP is hydrolyzed cooperatively [43]. Thus far, studies that compare the e¡ect of vanadate on both the mature transport system and a separated ABC domain have not been performed with other ABC transporters. However, the above notion is supported by the observation that the ATPase activity of the N-terminal nucleotide binding domain of OleB from S. antibioticus [48] was also found to be insensitive to vanadate. In contrast, ATP hydrolysis by MglA, the ABC protein of the galactose transport system from S. typhimurium was reported to be inhibited by vanadate [39]. Since the enzymatic activity of this preparation was also (unexpectedly) stimulated by the substrate galactose (see also Section 6), this result should be interpreted with caution. Several ABC subunits or intact transport proteins are a¡ected by N-ethylmaleimide (Table 1). The molecular basis for this inhibition was shown in P-glycoprotein [51,54,68,69] and others [37,39,41] to be the covalent modi¢cation of a cysteine residue that is frequently found within the Walker A motif. The proteins are rendered insensitive to the reactant by preincubation with ATP [37,54,69] or by replacing the respective cysteine residues [41,68]. Ba¢lomycin A1 , a macrolide antibiotic that inhibits V-ATPases at nanomolar concentrations and PATPases in the micromolar range [70] also impaired the ATPase activity of ABC transporters, including P-glycoprotein [49] and the binding protein-dependent transport system for maltose from S. typhimurium [67]. The mode of action of ba¢lomycin is currently unknown in any of the above cases. The ATPase activity of most ABC proteins and transporters was also found to be sensitive to ADP [37,49,51,69] and non-hydrolyzable nucleotide analogs, such as AMP-PNP [51,58,69] and ATPQS [37,51]. Generally, rather high (millimolar) concentrations were required for half-maximal inhibition. In contrast, the enzymatic activity of the ABC domain of HlyB fused to glutathione-S-transferase was reported to be una¡ected by these inhibitors [47]. 5. Function of conserved amino acid residues 5.1. Walker sites A and B In analogy to those nucleotide-binding and -hydrolyzing proteins from which structural data are available [71^74], the invariant lysine residue in the Walker A motif of ABC transporters has been suggested to be crucial for the binding of the L- and Qphosphates of the nucleotides while the conserved aspartate in site B is thought to play a role in liganding the Mg2 ion that accompanies the nucleotide. Detailed mutational analyses performed with numerous ABC transporters con¢rmed that amino acid changes in the Walker A and B motifs are generally not tolerated with respect to ATPase activity [41,46,48,52,60,75]. Conservative substitution of the invariant lysine by arginine abolished ATP hydrolysis [46,60] but nucleotide-binding activity was retained [60,76]. From nuclear magnetic resonance (NMR) analyses carried out with adenylate kinase of E. coli it was suggested that the corresponding (Walker A) lysine residue functions in orienting the triphosphate chain of MgATP to a proper conformation required for catalysis and, in turn, ensures interaction of the substrate with the active site residues. A mutation to arginine interferes with these activities due to a localized conformational change [77]. While other replacements of the lysine residues still allowed interaction with nucleotides as demonstrated by photocross-linking with the analog 8-azido-ATP [78^80] or enhancement of £uorescence of trinitrophenyl-ATP [75], a change to asparagine also eliminated the nucleotide-binding activity ([78,81]; Wilken, Schmees and Schneider, unpublished). Similarly, substitutions of the conserved aspartate residue in site B were generally shown to abolish ATPase activity and nucleotide binding [75,78,81]. 5.2. Catalytic carboxylate The crystal structures of the E. coli RecA protein [72] and of the F1 -ATPase from bovine mitochondria [74] have led to the proposal that the attack of the Qphosphate of ATP by a water molecule requires activation by a (`catalytic') carboxylate that is rather equidistantly positioned between the Walker A and FEMSRE 605 29-5-98 E. Schneider, S. Hunke / FEMS Microbiology Reviews 22 (1998) 1^20 B motifs. In a recent hypothesis based on sequence alignments, Yoshida and Amano [32] identi¢ed two glutamic acid residues as putative candidates for such a function in ABC transporters. However, results from our laboratory clearly showed that in the case of the bacterial MalK protein, neither of the two residues (Glu64 , Glu94 ) were required for activity [82]. Thus, the identi¢cation of a catalytic carboxylate in ABC transporters has likely to await structural information. 5.3. Helical domain Only a few conserved residues are located within this predicted peptide loop connecting the Walker A and B motifs. A detailed mutational analysis of the HisP protein from S. typhimurium revealed that in most cases amino acid substitutions, while a¡ecting transport, did not alter nucleotide binding activity [81]. In line with these ¢ndings was the observation that mutation of a highly conserved glutamine residue in MalK (Q82) did not signi¢cantly reduce the ATPase activity of the puri¢ed protein [83]. In CFTR, F508 which is deleted in 70% of the patients a¡ected by cystic ¢brosis is positioned adjacent to the respective glutamine residue. In vitro, the mutant protein is functional with respect to nucleotide-induced channel activity [63], indicating again that mutations in the helical domain do not interfere with the catalytic activity. As discussed in Section 8, this region might be involved in interactions with the membrane-integral components rather than with the catalytic process. 5.4. Linker peptide Residues within the linker peptide have been the subject of intensive research [57,75,78^80,84^87] due to its unique presence in ABC transporters and because natural mutations in CFTR that cause cystic ¢brosis (G551D, G551S) have been localized to this motif [35]. Most mutations abolished ATP hydrolysis, thus resulting in a transport-negative phenotype while nucleotide binding remained largely una¡ected. The CFTR G551D protein was recently shown to exhibit altered channel gating [57]. In KpsT, a protein that is involved in the export of capsular polysaccharides in E. coli K1, a mutation in the linker 9 peptide (S126F) suppressed in cis the dominant negative phenotype of a mutation adjacent to the Walker B motif [80]. Thus, the available data all indicate an essential role of the linker peptide in the transport process. Ames and Lecar [31] proposed that this peptide might enable the helical domain to change conformation upon ATP hydrolysis and subsequently put the membrane spanning components into motion. This notion, however, is not fully consistent with the observation that mutations also affected ATPase function. Moreover, and as already discussed, an alternative view was recently presented on the basis of sequence comparison of ABC proteins with GTP-binding proteins. According to these authors the core motif of the linker peptide forms part of the nucleotide binding pocket [33]. 5.5. Switch region In the RecA protein a short motif that is located carboxy-terminal to the Walker B site at the end of the L5 strand was proposed to play a crucial role in propagating conformational changes triggered by ATP hydrolysis [32]. In the homologous region of ABC transport proteins (end of L4 strand), an almost invariant histidine residue is found that appears to be essential for the transport function. Mutations of this residue in the bacterial ABC proteins HisP [81], MalK [83], and KpsT [79] abolished the respective transport activities. However, nucleotide binding to the mutated histidine transport complex in membrane vesicles [81] and ATPase activity of the puri¢ed variant of MalK [83] were largely unaltered. Thus, the residues might be crucial for a subsequent step. E¤cient nucleotide-binding activity was also shown for another variant of HisP carrying a mutation of the moderately conserved threonine residue (T205) within the same region [81]. Moreover, suppressor mutations that restore histidine uptake in an otherwise de¢cient transport complex lacking the binding protein in S. typhimurium also map in this motif of the HisP protein [88]. Similarly, a MalK mutant protein that, when overproduced, restored growth of an E. coli strain with missense mutations in two conserved cytoplasmic loops of the membrane-integral components MalF and MalG carried a substitution (M187I) in that region [89]. In P-glycoprotein (Mdr3 from mouse), muta- FEMSRE 605 29-5-98 10 E. Schneider, S. Hunke / FEMS Microbiology Reviews 22 (1998) 1^20 tion of a threonine residue (T578C, corresponding to T582 of human MDR1 in Fig. 3) in the equivalent segment of the ¢rst nucleotide-binding fold altered the drug resistance pro¢le of the protein [90]. Thus, the data from di¡erent proteins add to the view that this particular region may be implicated in signal transduction to the ABC domain by sensing conformational changes in the membrane-spanning domains upon substrate binding [88]. 6. Recognition of the transported solute by the ABC protein/domain Binding protein-dependent uptake systems initially recognize their substrate (liganded to the respective binding protein), and ATP at opposite sides of the membrane. Consequently, this implies that the ABC subunits do not necessarily participate directly in substrate recognition. Consistent with this notion are the ¢ndings that MalK and UgpC from E. coli, involved in the transport of chemically distinct substrates, such as maltose and glycerol-3-phosphate, respectively, are exchangeable [91], and that the LacK protein of Agrobacterium radiobacter, participating in the uptake of lactose can substitute for MalK in S. typhimurium and E. coli [92]. Moreover, the ATPase activity of the puri¢ed MalK protein was neither stimulated nor inhibited by maltose [37]. Other groups reported di¡ering results. While the ATPase activity of the puri¢ed PotA protein from E. coli was inhibited by millimolar concentrations of the transported substrate spermidine [41], galactose stimulated the enzymatic activity of the MglA protein from E. coli [39]. These e¡ects might, however, not be speci¢c and rather due to intrinsic properties of polyamines or to an inhomogenous protein preparation in the latter case. In contrast, in bacterial or eukaryotic exporters, both the substrate to be translocated and ATP are targeted to the same (cytoplasmic) side of the transport system. Genetic and biochemical approaches provided some evidence supporting the notion that the ABC domains of bacterial polypeptide secretion systems might participate in substrate recognition. The ATPase activity of PrtD from E. crysanthemi was strongly inhibited by the C-terminal secretion signal of the secreted protease PrtG, while the cor- responding fragment of HasA that is not a substrate of the Prt system exhibited only a minor e¡ect [46]. Further analysis of the same system revealed an initial interaction of the ATPase subunit with the substrate as a prerequisite for the assembly of the transport complex [93]. By suppressor analysis, Sheps et al. [94] found a single mutation in the ABC domain of HlyB, involved in the secretion of K-hemolysin (HlyA) in E. coli, that corrected the transport defect of a C-terminally truncated variant of HlyA. This ¢nding was interpreted in support of a direct interaction with either the substrate or the membranespanning domains (see also Section 8). On the other hand, and in contrast to the result obtained with PrtG, a peptide encompassing the C-terminal 200 amino acid residues of HlyA caused only marginal inhibition of the ATPase activity of the ABC domain from HlyB fused glutathione-S-transferase [47]. Biochemical evidence obtained with export systems that mediate resistance to the antibiotic oleandomycin in Streptomyces antibioticus [95] or secretion of capsular polysaccharide in E. coli [96] was also taken as evidence in favor of the ABC domains to be involved in binding of the substrate. However, in both cases a note of caution seems appropriate. Oleandomycin, at a concentration of 2 mM, changed the intrinsic £uorescence of a fusion of the N-terminal ABC domain of OleB to maltose binding protein by 20% [95], which might indicate a conformational change upon interaction with the substrate. Since the exact boundaries of an ABC domain are di¤cult to de¢ne, the results do not rule out the participation of residues not strictly belonging to the ABC domain in substrate binding. Bliss and Silver showed that KpsT, involved in the secretion of polysialic acid in E. coli can be co-immunoprecipitated with the substrate, but only to a minor extent and in the context of the complete system. Again, these results do not exclude the interaction of the substrate with other transport components. Taken together, further experimental data are required until a more uniform picture on how bacterial export systems recognize their substrates might emerge. In mammalian systems, the observation that the ATPase activity of P-glycoprotein is regulated by drugs that are transported (see Section 3 and Table 1) is not conclusive since substrate binding sites have FEMSRE 605 29-5-98 E. Schneider, S. Hunke / FEMS Microbiology Reviews 22 (1998) 1^20 been demonstrated on the membrane-spanning domains but thus far not on the nucleotide-binding folds [22,97]. 7. Functional necessity of both ABC domains The structural organization of eukaryotic ABC transporters has provided an experimental advantage for studies on the functional necessity of both nucleotide binding domains. Analyses of mutations in either one of the two Walker A motifs in P-glycoprotein [76], or the STE6 protein from yeast [21] unanimously revealed that two nucleotide binding folds matching the consensus sequence are indispensable for function. This conclusion was supported by results from bacterial uptake systems. In the oligopeptide permease of S. typhimurium, which comprises two ABC proteins encoded by individual genes, both were shown to be required for activity [98]. The functional analysis of MalFGK2 complexes from E. coli containing a single mutated copy of MalK [99], and of covalently coupled wild-type and mutant MalK fusion proteins from S. typhimurium (Wilken, Schmees and Schneider, unpublished) also indicated that two intact nucleotide-binding folds are indispensable for transport activity. These ¢ndings also correlate with a stoichiometry of nearly two ATP hydrolyzed per substrate as the most realistic number measured for bacterial binding protein-dependent systems (reviewed in [9]) and the reconstituted P-glycoprotein [100]. Moreover, studies from several groups that used di¡erent experimental approaches suggested that both nucleotide binding sites of P-glycoprotein must interact (reviewed in [101]), although the failure to detect cooperativity between the two ATP sites with a puri¢ed preparation of Pglycoprotein in detergent solution was also reported [49]. In contrast, cooperative interaction was also observed with the reconstituted maltose transport system of E. coli [43]. Thus, the functional interplay of both sites might require a membrane-bound state of the protein. Members of a subgroup of bacterial ABC proteins involved in the uptake of certain sugars, such as ribose [102], arabinose [103], and L-methylgalactoside [104], respectively, from E. coli and ribose from B. subtilis [105], appear to be natural dimers 11 as deduced from their primary structures. Computational analysis suggested that these proteins are phylogenetically more related to each other than to other members of the family [106]. Strikingly however, while in their N-termini a consensus Walker A motif is present, the carboxy-terminal A-sites either contain an arginine in place of the invariant lysine [102,103,105] or are even more degenerated [104]. Since other residues (discussed in Section 5) cannot substitute for the conserved lysine without loss of ATPase activity, the C-terminal nucleotide binding folds are most likely catalytically inactive. Consequently, a functional transport complex would have to contain a homodimer of these proteins. However, no experimental evidence in support of this view is available at present. Interestingly, several P-glycoprotein-like ABC exporters from yeast and fungi carry a cysteine for lysine substitution in the ¢rst nucleotide binding fold [29,107,108]. Again, the functionality of this site as well as the oligomeric state of the transport systems in the membrane are currently unknown. The question whether both nucleotide binding folds in an ABC transport protein are functionally equivalent or energize distinct steps in the translocation process has been addressed in studies involving the CFTR protein. CFTR displays a gated Cl3 channel activity that can be monitored by electrophysiological means in membranes or planar lipid bilayers, thereby allowing the elucidation of individual steps of the translocation process. From such studies, most authors agree on a model that suggests distinct functions of both nucleotide-binding folds in channel gating, but otherwise the individual steps that are powered by ATP hydrolysis are discussed controversely. Hwang et al., studying the e¡ects of non-hydrolyzable ATP analogs [109], and Carson et al., using variants which contained mutations in the conserved Walker A lysine residue in either one or both nucleotide-binding sites [81], proposed that ATP hydrolysis at the N-terminal ABC domain is required to open the channel while closing is catalyzed by hydrolysis at the C-terminal site. Furthermore, Carson and collaborators deduced from biochemical studies that ATP binding at both sites might occur simultaneously [81]. In contrast, Hwang et al. interpreted their data by proposing the necessity of ATP hydrolysis at one site for the interaction FEMSRE 605 29-5-98 12 E. Schneider, S. Hunke / FEMS Microbiology Reviews 22 (1998) 1^20 of ATP with the second site [109] which would be consistent with the alternate site model of P-glycoprotein [101]. Wilkinson et al., also based on studies with mutants, suggested that ATP hydrolysis at the N-terminal nucleotide binding fold (NBF1; see Fig. 1) is critical for entering the active state, but ATP binding is thought to contribute to this process [84]. A somewhat di¡erent view, again based on mutational analyses, was presented by Gunderson and Kopito [110] who proposed a central role of the Cterminal ABC domain in opening the channel. In sharp contrast, Schultz et al. questioned the necessity of ATP hydrolysis for channel gating due to their failure to monitor e¡ects of non-hydrolyzable ATP analogs on ATP-dependent regulation of CFTR activity [111]. However, the recent ¢nding that puri¢ed CFTR exhibits signi¢cant ATPase activity [57] provides a strong argument against this view. 8. Interaction with the membrane-spanning components/domains The ABC subunits/domains must interact with the membrane-spanning domains in order to transmit conformational changes resulting from the hydrolysis of ATP. While in eukaryotic systems fusion of the domains into one polypeptide chain assures their physical proximity, assembly of the multicomponent bacterial transporters requires speci¢c recognition sites for the ABC proteins on the membrane-integral subunits. At the molecular level, such interactions are still poorly understood. Since relatively enriched in hydrophobic amino acids and considerably less well conserved, the extended helical domain that connects the Walker A motif with the linker peptide has been suggested as a candidate to direct the ABC protein to the membrane components [1^3,30]. Recently, for the ¢rst time, several groups using mainly genetic approaches provided evidence in favor of this view. Suppressor analyses led to the identi¢cation of amino acid residues within the helical domains of several bacterial ABC proteins that might participate in subunit interactions [89,92,94]. In this laboratory, we took advantage of the unusually high sequence identity shared between the ABC proteins MalK of S. typhimurium, and LacK of Agrobacterium radiobacter, involved in the uptake of maltose and lactose, respectively. Based on the initial observation that LacK partially substituted for MalK in maltose transport, we showed that by exchanging the N-terminal 137 amino acids of LacK with the corresponding MalK fragment the performance of LacK was substantially improved [92]. Together with previous ¢ndings from a HisP-MalK chimera [112] the fragment crucial for subunit interactions was narrowed down to the C-terminal two thirds of the helical domain. The same phenotype was observed with suppressors carrying single missense mutations (V114M, L123F) within that segment of the helical domain of LacK. In each case, and in agreement with most other studies (see below), residues with bulkier side chains were put in place but otherwise, the hydrophobic character of the wild-type residues was maintained [92]. Strikingly, full `MalK-like' activity was achieved only by a LacK mutant protein that carried an additional replacement in the poorly conserved C-terminal domain [92]. This argues at least for a structural role of the latter in domain interactions and implies that the helical domain is crucial but probably not su¤cient to assure speci¢c association of the ABC proteins with the respective membrane components. Mourez et al., studying the maltose transport system from E. coli, ¢rst introduced mutations in a conserved motif (referred to as `EAA loop') of the membrane-spanning subunits MalF and/or MalG [89]. According to topological models, this motif is located in a cytoplasmic loop at a distance of approx. 90 residues from the C-terminus. It is shared by the membrane-integral components of the subclass of bacterial binding protein-dependent transporters which otherwise greatly di¡er in their primary structures (reviewed in [9,113]). Mutational analyses have revealed that a central and invariant glycine residue is crucial for transport activity [9,89,114] and genetic evidence suggested that the EAA loop might interact with other transport components [114]. Suppressor mutations in malK that restored transport of these mutants were found to map mainly in the helical domain of the MalK protein (A85M, V117M) [89], close to those residues which when mutated allowed LacK to substitute more e¤ciently for MalK in maltose transport [92]. By using a similar approach, a mutation in the helical domain of HlyB (V599I) was isolated that FEMSRE 605 29-5-98 E. Schneider, S. Hunke / FEMS Microbiology Reviews 22 (1998) 1^20 compensated for a deletion in the substrate molecule HlyA. Since the majority of suppressors had been localized to transmembrane segments of HlyB, the authors concluded that this region of the ABC domain might be involved in interaction with transmembrane domains [112]. Evidence from studies on the functional exchangeability of the N- and C-terminal ABC domains of Pglycoprotein also suggested to the authors that residues in the helical domain might participate in signal transduction between the nucleotide-binding site and the transmembrane domains or, alternatively, might directly be involved in substrate recognition. This conclusion was based on the analyses of chimeras and mutants which demonstrated that most of the amino-terminal ABC domain could be replaced by the corresponding carboxy-terminal residues without dramatic e¡ects on protein function. In contrast, replacement of a short sequence motif within the helical domain of the N-terminal half of the protein by the corresponding C-terminal residues caused alterations of the drug resistance pro¢le [90]. However, it should be emphasized that in none of the above examples the physical proximity of amino acid residues from ABC and membrane-spanning domains has thus far been demonstrated. The ¢nding that mutations in the conserved EAA loop of MalF and MalG, the membrane-integral components of the maltose transport system of E. coli, can be suppressed by second site mutations in MalK also sheds some light on the possible role of this motif [89]. Only recently, the implication of the EAA loop became even more evident by the identi¢cation of an EAA-like sequence in certain eukaryotic ABC transporters. Strikingly, mutations in a conserved glutamic acid residue within this motif have been reported in patients su¡ering from adrenoleukodystrophy [115], and a deletion in the corresponding fragment of the CFTR protein altered Cl3 -channel stability [116]. Thus, the EAA motif appears to play an important and more general role in ABC transporters. In prokaryotic transport complexes, it might confer speci¢city by providing a unique site of interaction for the ABC protein of the respective system. How this interaction is achieved in bacterial systems that comprise two distinct membrane proteins but two copies of the same ABC protein remains unclear. The observation that substitutions at the same position in MalF and MalG had di¡erent 13 phenotypes is in favor of individual roles of both EAA motifs [89]. However, whether interaction of the helical domain with the EAA motifs assures only correct assembly of the transport complex or might be crucial for energy coupling remains to be elucidated. 9. Transmembrane orientation? Albeit overall hydrophilic, bacterial ABC proteins were found to be tightly associated with the membrane components, indicating that the transport complex is stabilized by strong interactions between the individual subunits [41,78,117]. This view was supported by reports demonstrating that HisP and MalK are accessible to protease [118,119] and biotinylation [118] in right-side-out membrane vesicles. Thus, a (hydrophilic) peptide segment of ABC proteins, while shielded from the lipid bilayer by the hydrophobic components, might traverse the membrane. KpsT, the ATP-binding component of a bacterial export system was also shown to exhibit sensitivity to trypsin in right-side-out vesicles but only when carrying a mutation that exerts a dominant negative e¡ect [96] (see also chapter 9). Physical interactions between the nucleotide binding folds and the membrane-spanning domains have also been demonstrated by means of immunoprecipitation assays for both the P-glycoprotein [120] and CFTR [121]. Moreover, by using a membrane impermeable probe, exposure to the medium was reported for both the complete CFTR [122] and, most suprisingly, the ¢rst nucleotide-binding site of CFTR fused to maltose-binding protein [123]. Thus, a transmembraneous loop of the ABC domain might represent a characteristic feature of ABC transport proteins. Remarkably, protease accessibility from the medium side was also discovered in the case of SecA, the ATPase component of the E. coli preprotein translocase that also lacks a classic, membrane-spanning apolar sequence [124]. Subsequent studies led to the proposal that the catalytic process of preprotein translocation might require cycling of SecA between a soluble and a membrane-embedded state. While insertion of a carboxy-terminal 30-kDa fragment of SecA is driven by ATP binding to the N-terminal nucleotide binding site, hydrolysis of ATP is necessary for de-insertion [125^128]. However, it should FEMSRE 605 29-5-98 14 E. Schneider, S. Hunke / FEMS Microbiology Reviews 22 (1998) 1^20 be noted that the functional signi¢cance of such a scenario has been questioned [129] and, to make matters even more complicated, an amino-terminal fragment of SecA was recently also found to be protected from proteolysis during translocation [130]. In contrast to SecA, proteolytic fragments have thus far not been identi¢ed in the case of ABC proteins. Thus, the chemical nature of the putative transmembrane domain is currently unknown. In this respect, the recently presented low resolution î ) provides the structure of P-glycoprotein (at 25 A basis for an alternative explanation of the protease data [131]. If one assumes that ABC transporters share the same overall size and structure, then the transmembrane pore constituted by the hydrophobic domains and as seen for the P-glycoprotein by electron microscopy might be su¤ciently large to allow proteases access to the nucleotide-binding domains even in the absence of an exposed fragment. Since these data were obtained in the absence of ATP, it remains to be established whether the pore size is altered in the nucleotide-bound state. Clearly, further e¡orts in order to distinguish between these alternative views are required. 10. Tentative models on the role of the ABC domains in the translocation process 10.1. KpsMT The above data gave rise to the speculation that ABC transporters and the preprotein translocase might share similar steps in their catalytic cycles. In a recent model describing the export of polysialic acid in E. coli K1 by the KpsMT system, Bliss and Silver [19] viewed ATP-dependent transmembrane movement of KpsT as a central aspect. According to their hypothesis, initial interaction of polysialic acid with KpsT results in the binding of ATP by which the insertion of a substrate-associated domain of KpsT into the membrane through KpsM is triggered. Consistent with the `sewing model' for preprotein translocation which is also a secretion process [128], subsequent hydrolysis of ATP drives the retraction of the KpsT domain and the release of the substrate. Experimentally, the model is largely based on the observation that membrane-inserted wild-type KpsT was found to be inaccessible to external protease (discussed in the preceding chapter) and thus, may represent the de-inserted state of the protein. In contrast, a KpsT mutant (E150G) still capable of binding nucleotides but proposed to be impaired in ATP hydrolysis displayed sensitivity to proteolytic attack [96]. Moreover, this mutant also exerted a transdominant e¡ect over wild type [80], indicating strong interaction with KpsM. From this the authors concluded that the failure of KpsTE150G to hydrolyze ATP might cause the protein to be permanently locked in the membrane-embedded state, thereby jamming the translocation pore. The model also accounts for experimental evidence from bacterial exporters suggesting that the ABC protein might be directly involved in the initial recognition of the substrate [94,95] (but see also Section 6). 10.2. Relevance to binding protein-dependent import systems? Would such a model be relevant to binding protein-dependent ABC transporters that are designed to mediate the uptake of solutes? Although undoubtedly attractive since it assigns a key role to a putative transmembraneous fragment of the ATP-binding domain that was ¢rst postulated for the HisP protein [118], several features by which im- and export systems di¡er must be considered. First, the substrate molecules are transported to opposite directions. As a consequence, binding protein-dependent uptake systems initially recognize their substrate (liganded to the respective external binding protein), and ATP at opposite sites of the membrane. Moreover, and as discussed in Section 6, experimental evidence strongly suggested that their ABC proteins, unlike the homologous components of exporters, do not directly participate in substrate recognition [37,91,92]. Second, the results from proteolysis experiments also di¡er fundamentally. While in the Kps system sensitivity to trypsin was only observed with vesicles containing a KpsT mutant protein (E150G), the wild-type forms of both HisP and MalK were demonstrated to be externally accessible to proteinase K. Accordingly, and by following the argumentation by Bliss and Silver [19], mutations in HisP or MalK that impair ATP hydrolysis but do not a¡ect binding (as the E150G mutation in KpsT) FEMSRE 605 29-5-98 E. Schneider, S. Hunke / FEMS Microbiology Reviews 22 (1998) 1^20 should render the proteins resistant to external protease rather than cause a sensitive phenotype. Thus, one might speculate that in contrast to export systems, in bacterial importers the ground state is represented by the inserted form of the ABC proteins. Consequently, substrate binding forces would drive the peptide fragments back out towards the cytoplasm, followed by translocation of the substrate molecule. Eventually, ATP hydrolysis would catalyze re-insertion of the peptide fragment into the pore. Thus far, experimental evidence in favor of such a view is lacking. However, preliminary protease digestion experiments with right-side-out membrane vesicles from a malK mutant expressing the MalKE159G protein (homologous to KpsTE150G) did not reveal a change in sensitivity as compared to wild type (Hunke, Stein and Schneider, unpublished results). 10.3. P-Glycoprotein Senior and colleagues [66,101] have put forth the hypothesis that in P-glycoprotein both ABC domains might hydrolyze ATP alternately. This is mainly based on vanadate-trapping experiments indicating that inactivation of one domain abolished ATP hydrolysis at both sites. According to this proposal, in the ground state only one nucleotide-binding site is occupied by ATP. Upon interaction with substrate, hydrolysis triggers binding of ATP to the second site while ADP and Pi are retained in a high chemical potential state. Relaxation of this site then is coupled to drug translocation. Hydrolysis of ATP at the second site is prevented by a speci¢c conformational change. Eventually, a new catalytic cycle is initiated by drug binding that is now energized by hydrolysis of ATP at the alternate site. This model accounts for the observed stimulation of the ATPase activity of Pglycoprotein by drugs and also agrees with the above bacterial model in that ATP hydrolysis powers the same step in the transport process. However, due to the lack of experimental data in the case of P-glycoprotein, the model does not consider putative transmembrane fragments of the ABC domains as being involved in the reaction cycle. It should be emphasized that the above hypothesis includes elements of the `binding change mechanism' originally postulated by Boyer to describe the mode 15 of ATP hydrolysis catalyzed by the F1 moiety of the ATP synthase (F1 F0 ) [132]. According to this proposal, the structures of the three catalytic sites on the L-subunits of F1 are always di¡erent, but each passes constantly through a cycle of `empty', `ADP-bound' and `ATP-bound' state. The mechanism also suggested an asymmetric assembly of the protein. These predictions were con¢rmed by Walker and colleagues who recently elucidated the atomic structure of beef heart F1 [74]. It remains to be seen whether this attractive proposal will provide a model for the function of ABC transporters in general. 11. Summary and concluding remarks Within the last couple of years, a huge body of experimental evidence on ABC transport systems from various organisms has been accumulated by numerous laboratories. Despite the diversity of substrates that are translocated by di¡erent ABC transporters, ranging from inorganic ions to proteins, a unifying picture of how these systems might operate is now beginning to emerge from these data. In particular, results from studies concerning the ABC domains contributed substantially to our current knowledge on these systems: the ABC domains are capable of hydrolyzing ATP, their enzymatic activity is sensitive to the same set of inhibitors and in the complete transport protein both sites need to cooperate. With the possible exception of CFTR and closely related proteins, both ABC sites also seem to function identically. Moreover, the functional importance of a strong membrane association, perhaps via a transmembraneous loop, and the role of the putative helical domain in protein^protein interactions are becoming more evident. However, in spite of this progress, at the molecular level the mechanism by which ABC transporters exert their functions is still far from being understood. This is mainly due to the fact that structural data are still lacking. Unfortunately, and regardless of all e¡orts made in recent years, the crystallization of membrane proteins is still in its infancy. Thus, the date by which the tertiary structure of an ABC transport system at high resolution will be available cannot be predicted. However, recent achievements to FEMSRE 605 29-5-98 16 E. Schneider, S. Hunke / FEMS Microbiology Reviews 22 (1998) 1^20 obtain structural information, such as an electron microscopic image of P-glycoprotein at low resolution [131] and the preparation of 3D-crystals of the ABC protein MalK from S. typhimurium that difî (Schmees, Hoëner zu fract to a resolution of 3.2 A Bentrup, Schneider, Vinzenz and Ermler, submitted) are promising and will certainly help to pave the way for understanding more precisely the structure-function relationships in this protein family. This would, for example, be of special importance with respect to the role of conserved amino acid residues in the ABC domain that cause diseases when mutated. Nevertheless, even with a structure at our disposal, to unravel the dynamics of the transport process by applying biophysical means will require highly puri¢ed, reconstituted systems. Those are becoming more frequently available now. Moreover, mapping of protein^protein interaction sites within the membrane and identifying the chemical nature of the putative exposed fragments from ABC proteins/domains are among the most obvious studies to be performed in the near future. [2] [3] [4] [5] [6] [7] [8] [9] 12. Note added in proof The ABC subunit HisP of the histidine permease from S. typhimurium was recently also puri¢ed and demonstrated to exhibit an intrinsic ATPase activity that is insensitive to vanadate (Nikaido, K., Liu, P.-Q. and Ames, G.F.-L. (1997) J. Biol. Chem. 272, 27745^27752). [10] [11] [12] Acknowledgments The authors would like to express their gratitude to M. Mourez and E. Dassa for providing data prior to publication. 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