Download ATP-binding-cassette (ABC) transport systems: Functional and

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 . . . . . . . . . . . . . . . . . . . . . .
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* 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
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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-
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9
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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
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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
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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].
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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
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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-
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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
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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
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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
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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
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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)
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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. The work from the authors' laboratory was supported by the Deutsche Forschungsgemeinschaft (SFB171, TP C12; SCHN274/6-1) and by
the Fonds der Chemischen Industrie.
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