Báo cáo khoa học: Functional characterization of the maltose ATP-binding-cassette transporter of Salmonella typhimurium by means of monoclonal antibodies directed against the MalK subunit pot

4B3 and 2F9 exhibit reduced binding to the MalFGK2complex in the presence of MalT and enzyme IIAGlc, respectively, thereby providing the first direct evidence for the C-terminal domain of

Functional characterization of the maltose ATP-binding-cassette

antibodies directed against the MalK subunit

Anke Stein1, Martina Seifert2, Rudolf Volkmer-Engert2, Jo¨rg Siepelmeyer3, Knut Jahreis3

and Erwin Schneider1

1

Humboldt Universita¨t zu Berlin, Institut fu¨r Biologie, Berlin, Germany;2Humboldt Universita¨t zu Berlin, Institut fu¨r Medizinische Immunologie, Berlin;3Universita¨t Osnabru¨ck, Fachbereich Biologie/Chemie, Germany

The maltose ATP-binding cassette transporter of Salmonella

typhimuriumis composed of a membrane-associated

com-plex (MalFGK2) and a periplasmic receptor (MalE) In

addition to its role in transport, the complex acts as a

repressor of maltose-regulated gene expression and is subject

to inhibition in the process of inducer exclusion These

activities are thought to be mediated by interactions of the

ATPase subunit, MalK, with the transcriptional activator,

MalT, and nonphosphorylated enzyme IIA of the glucose

phosphotransferase system, respectively To gain further

insight in protein regions that are critical for these functions,

we have generated nine MalK-specific monoclonal

anti-bodies These bind to four nonoverlapping linear epitopes:

60-LFig-63 (5B5), 113-RVNQVAEVLQL-123 (represented

by 4H12), 309-GHETQI-314 (2F9) and 352-LFREDG

SACR-361 (represented by 4B3) All mAbs recognize their

epitopes in soluble MalK and in the MalFGK2complex with

Kdvalues ranging from 10)6to 10)8M ATP reduced the affinity of the mAbs for soluble MalK, indicating a confor-mational change that renders the epitopes less accessible 4H12 and 5B5 inhibit the ATPase activity of MalK and the MalE/maltose-stimulated ATPase activity of proteolipo-somes, while their Fab fragments displayed no significant effect The results suggest a similar solvent-exposed position

of helix 3 in the MalK dimer and in the intact complex and might argue against a direct role in the catalytic process 4B3 and 2F9 exhibit reduced binding to the MalFGK2complex

in the presence of MalT and enzyme IIAGlc, respectively, thereby providing the first direct evidence for the C-terminal domain of MalK being the site of interaction with the reg-ulatory proteins

Keywords: ABC transporter; MalFGK2; enzyme IIAGlc; MalT; monoclonal antibodies

The family of ATP-binding-cassette (ABC) transport

sys-tems comprises an extremely diverse class of membrane

proteins that couple the energy of ATP hydrolysis to the

translocation of solutes across biological membranes [1,2]

A prototype ABC transporter is composed of four

entities: two membrane-integral domains, which

presuma-bly constitute a translocation pore, and two ATPase

domains (also referred to as ABC subunits/domains), that

provide the energy for the transport process The ABC

domains are characterized by a set of canonical Walker A

and B motifs, required for nucleotide binding and by a

unique signature sequence (LSGGQ motif) of still unknown

function [3] The crystal structures of several prokaryotic

ABC domains have been solved in recent years that agree

largely on the overall folds Accordingly, the structures can

be subdivided in an F1-type ATP-binding domain, encom-passing both Walker sites, a specific a-helical subdomain, containing the LSGGQ motif and a specific antiparallel-b-subdomain [4–7]

The binding protein-dependent maltose/maltodextrin transporter of enterobacteria, such as Escherichia coli and Salmonella typhimurium, is a well-characterized model system for studying the mechanism of action of the ABC transport family [8] Based on computational analysis, it belongs to a subclass of ABC importers designated CUT1 (carbohydrate uptake transporter) [9] or OSP (oligosaccha-rides and polyols) [10], respectively Members of this subclass transport a variety of di- and oligosaccharides, glycerol phosphate and polyols and are recognized by their common subunit composition (two individual membrane-spanning subunits and two copies of a single ABC protein) and by an extension of approximately a hundred amino acid residues at the C-terminus of the ABC protein [11] The maltose transporter of E coli/S typhimurium is composed of the periplasmic maltose binding protein, MalE, and of the membrane-associated complex, MalFGK2, con-sisting of one copy each of the hydrophobic subunits MalF and MalG and two copies of the nucleotide-binding subunit MalK [12] Crystals of Salmonella MalK are available [13] but their structure could not be solved yet However, the tertiary structure of a MalK homolog, isolated from the

Correspondence to E Schneider, Humboldt Universita¨t zu Berlin,

Mathematisch-Naturwissenschaftliche Fakulta¨t I,

Institut fu¨r Biologie, Bakterienphysiologie, Chausseestr 117,

D-10115 Berlin, Germany.

Tel.: + 49 (0)30 2093 8121, Fax: + 49 (0)30 2093 8126,

E-mail: erwin.schneider@rz.hu-berlin.de

Abbreviations: IF-medium, Iscove’s DMEM/NUT MIX F12.

(Received 27 March 2002, revised 6 June 2002, accepted 8 July 2002)

hyperthermophilic archaeon Thermococcus litoralis, was

recently determined [5] Two molecules are present per

asymmetric unit that contact each other through the ATPase

domains with the C-terminal domains attached at opposite

poles Based on these data, a 3D model of the E coli MalK

protein was recently presented [14]

Enterobacterial MalK can be purified in fairly large

amounts [15] and displays a spontaneous ATPase activity

that is insensitive to inhibition by vanadate, a typical

inhibitor of ABC transporters [16] The purified MalFGK2

complex, when incorporated into liposomes, also exhibits a

low intrinsic ATPase activity that, however, is stimulated

severalfold in the presence of substrate-loaded MalEand is

vanadate-sensitive [12,17–19]

According to a current transport model, the presence of

substrate in the medium is thought to be signalled by

liganded MalEvia interaction with externally exposed

peptide loops of MalF and MalG [20] As a consequence,

conformational changes of the latter are transmitted to the

MalK subunits which, in turn, become activated

Hydro-lysis of ATP would then trigger subsequent conformational

changes that eventually lead to the translocation of the

substrate molecule Recent findings suggested that these

steps occur rather simultaneously [21]

Interaction of MalK with the hydrophobic subunits

involves contact of residues in the helical subdomain

with conserved cytoplasmic loops (EAA motifs) in MalF

and MalG [22–24] This view, based on suppressor

mutational analyses and cross-linking studies, is largely

consistent with the recently solved crystal structure of

MsbA, an ABC transporter mediating the export of

the lipid A component of the E coli outer membrane

[25]

Besides acting as an import system for

maltose/malto-dextrins, the MalFGK2complex is involved in the

regula-tion of genes belonging to the maltose regulon [8] In the

absence of substrate, the idle transporter is thought to

interact with the positive transcriptional regulator, MalT,

via the MalK subunits, thereby preventing MalT from

binding to its target sequences upstream of

maltose-regulated promotors When the transporter becomes

engaged in translocating maltose across the membrane,

MalT is released and transcription of maltose-regulated

genes can occur [26]

In addition, the maltose transporter is subject to

inhibi-tion by binding of dephosphorylated enzyme IIA of the

glucose transporter (phosphoenolpyruvate

phosphotrans-ferase system) to the MalK subunits in a process called

inducer exclusion in the context of global carbon regulation

in enteric bacteria [27]

Both regulatory activities of MalK are largely mediated

by the C-terminal domain of the protein [5,28–30]

Obviously, specific protein–protein interactions within

the MalFGK2complex as well as between the transporter

and regulatory proteins are crucial for its role in intact cells

However, in the absence of tertiary structural information

on the complete transporter, these interactions are still

poorly understood at the molecular level Here, we describe

the use of monoclonal antibodies raised against the MalK

subunit as tools to gain further insights in the structural

basis of transporter functions

E X P E R I M E N T A L P R O C E D U R E S

Preparative procedures MalK [15], MalFGK2 [18], and MalE[31] were purified

as described MalE/maltose-loaded proteoliposomes con-taining the MalFGK2 complex were prepared by a detergent dilution procedure as published elsewhere [18,32]

Enzyme IIAGlcwas purified from the cytosolic fraction of

E colistrain BL21 D (pts43crr::kanR) harbouring plasmid pCRL13 (crr on pET23A) [33] by Ni-NTA affinity chro-matography

Crude extract containing MalT was prepared according

to [34] from E coli strain JM109 (Stratagene), carrying plasmid pAS8 (malTE.c.on pSE380, ptrc, ampR) (this study) For competitive inhibition ELISA, N-terminally his-tagged MalT was partially purified from strain JM109, harbouring plasmid pAS9 (malTE.c.on pQE9, pT5, ampR) by Ni-NTA chromatography

Preparation of mAbs Ten-week-old-female Balb/c mice were immunized intra-peritoneally either with native MalK or with an N-terminal fragment (encompassing residues 1–179 [30]) (100 lg each), dissolved in NaCl/Pi [35] On day 12, 25 and 62 the animals were boosted with 50 lg of protein each The final boost was given 4 days prior to the fusion For hybridoma production spleen cells were isolated and fused with myeloma cells SP2/0 as described [36] using poly(ethylene glycol) 1500 as fusion agent Selection of hybridoma cells was performed in hypoxanthine, aminop-terin and thymidine selection medium supplement Grow-ing hybridomas were screened by ELISA usGrow-ing MalK as bound antigen Selected hybrid cell lines were cloned at least three times by limiting dilution Cloned hybridoma cells were maintained in 20% IF-medium, supplemented with 70% fetal bovine serum and 10% dimethylsulfoxide for several days at )80 C and subsequently stored in liquid nitrogen For the production of mAbs, cells were grown in IF or RPMI 1690 medium (Bichrom KG, Berlin) in 2 L culture flasks

mAbs were purified by loading concentrated culture supernatant on a Protein G-Sepharose 4 fast flow matrix equilibrated with 20 mM sodium phosphate buffer, pH 7 After washing off unbound material, mAbs were eluted with 0.1M glycine/HCl, pH 2.7, and immediately dialyzed against NaCl/Piovernight at 4C

Isolation of Fab fragments One millilitre of mAbs (1–3 mg) were mixed with 0.5 mL of papain-agarose beads in 20 mMphosphate buffer, pH 7.5, supplemented with 20 mM L-cysteine and 1 mMEDTA, and incubated overnight at 37C Subsequently, Fab fragments were separated from uncleaved mAbs by incubating the mixture with protein A–Sepharose for 1 h at 4C Unbound material (Fab fragments) was collected, dialyzed overnight against 3 L of 50 mM Tris/HCl, pH 7.5, and stored at 4C until use

Determination of isotypes

Isotopes of the mAbs were determined by using Roche’s

ISO STRIP-mouse isotyping kit according to the

manufac-turer’s instructions

Peptide synthesis on cellulose membranes – SPOT

synthesis

Cellulose-bound peptide libraries were automatically

pre-pared on Whatman 50 paper (Whatman, Maidstone, UK)

according to standard SPOT synthesis protocols [37] using a

SPOT synthesizer (Abimed GmbH, Langenfeld, Germany)

as described elsewhere [38–41] The sequence files were

generated with the software DIGEN (Jerini AG, Berlin,

Germany) The peptides were derived from S typhimurium

MalK Libraries consisting of 10meric peptides (overlapping

by 9 amino acids) and peptide-substitutional analyses were

synthesized All peptides are C-terminally attached to

cellulose via a (a-Ala)2spacer

Epitope mapping

The screening of cellulose-bound peptides followed a

protocol published elsewhere [39,40] Peptide libraries

were incubated with mAbs overnight at 4C in blocking

buffer (10% blocking reagent, Roche, in TNT, 10%

sucrose) and binding was detected with

peroxidase-conjugated goat anti(mouse IgG) antibody on hyperfilm

(Amersham/Pharmacia, Braunschweig, Germany) using the

Western Blot Chemiluminescence Reagent Plus System of

NEN (Boston, MA, USA)

Peptide synthesis

Peptides, structurally derived from the epitopes identified

after screening of the peptide libraries as described above

were synthesized on solid phase (50 lmol scale) on Tentagel

SRam resin (Rapp Polymere, Tu¨bingen, Germany) by using

PyBOP activation and a standard Fmoc-chemistry-based

protocol of an AMS 422 Peptide Synthesizer (Abimed,

Langenfeld, Germany) Side-chain protections of amino

acids are as follows: Glu, Asp (OtBu); Ser, Thr, Tyr,

Trp (tBu); His, Lys (Boc); Asn, Gln (Trt); Arg (Pbf)

Trifluoracetic acid /phenole/triisopropylsilane/H2O (9.4 :

0.1 : 0.3 : 0.2) was used for resin cleavage and side-chain

deblocking The crude peptides were purified to

homogen-eity by RP-HPLC using the linear solvent gradient 5–60% B

in A for 30 min, with A¼ 0.05% trifluoracetic acid in

water, and B¼ 0.05% trifluoracetic acid in acetonitrile

The HPLC had the UV detector at 214 nm, a Vydac C18

column of 20· 250 mm, and a flow rate 10 mLÆmin)1 The

MS were performed on a matrix-assisted laser desorption

ionization-time of flight mass spectrometer (Laser

Bench-TopII, Applied Biosystems) The purity of the product was

characterized by analytical HPLC

ELISA

For ELISA, microtiter plates were coated with purified

MalK (2.5 pmol) diluted in 100 mM sodium carbonate

buffer, pH 9.6, and incubated overnight at 4C Remaining

binding sites were blocked with 2% BSA in NaCl/P

150 mMNaCl, 3 mMKCl, 8 mMNa2HPO4· 2 H2O, 1 mM

KH2PO4) for 2 h at room temperature Subsequently, the wells were incubated overnight at 4C with mAb diluted in 2% BSA in NaCl/Pi/Tween (NaCl/Pi containing 0.5% Tween 80) Incubation with the second antibody (HRP-conjugated goat anti(mouse IgG); 5· 10)2-fold dilution] occurred for 2 h at room temperature After each step the excess protein was removed by fourfold washing with NaCl/

Pi/Tween Antibody binding was detected by adding 100 lL

of 62.5 lg mL)1 3,3¢,5,4¢-tetramethylbenzidine, 0.0026% (v/v) H2O2in 0.1Msodium acetate/0.1Mcitric acid, pH 6 After 10 min at room temperature, the reaction was stopped

by addition of 100 lL H2SO4and the color development was measured at 450 nm

Binding constants of mAbs were measured by com-petitive inhibition ELISA according to [42] Suitable concentrations of mAbs were first determined by adding various amounts of antibody to microtiter wells, coated with various amounts of MalK, under experimental conditions identical to those used in binding experiments (ELISA)

The competitive inhibition ELISA was essentially carried out as described above In order to allow competition between bound antigen (MalK) and free antigen (MalK, MalFGK2-containing proteoliposomes, synthetic peptides) the mAbs and an equal volume of free antigen in different concentrations were incubated overnight at 4C in the wells coated with MalK (2.5 pmol) The mAbs were used in the following concentrations: 2F9 and 4B3, 2· 10)9M; 4H12, 6.5· 10)9M; 5B5, 8· 10)10M

Analytical methods Hydrolysis of ATP was assayed in microtiter plates essentially as described in [43] Protein was assayed using the BCA kit from Bio-Rad SDS/PAGEand immunoblot analyses were performed as described in [44]

R E S U L T S

Monoclonal antibodies recognize epitopes

in the N-terminal (ATPase) domain and in the C-terminal domain of MalK, respectively

Monoclonal antibodies were prepared against the MalK subunit of the S typhimurium maltose ABC transporter using purified, nondenatured MalK or an N-terminal MalK fragment (MalKN1, encompassing residues 1–179 [30]), as antigen for immunization Nine individual hybridoma cell lines producing antibodies of the Ig subclass IgG1 were obtained and immunoblot analyses revealed a specific reaction with the corresponding antigen in each case when purified MalK or MalFGK2 complex were separated by SDS/PAGE

Immunoblots using truncated MalK proteins [30] suggested that five mAbs, obtained with MalKN1, recognize epitopes in the N-terminal (ATPase) domain while the remaining four mAbs, obtained with intact MalK, bind to the C-terminal (regulatory) domain For precise determination of the epitopes overlapping deca-peptides corresponding to the entire MalK sequence were synthesized on cellulose membranes by SPOT-synthesis [37–41] The results for the binding analyses of the

different mAbs are shown in Fig 2 Four peptide epitopes

were identified One mAb (5B5) recognizes the peptide

53-ETITSGDLTRM-67, located close to the Walker A

motif, four (4H12, 6E6, 3A12, 4D8) bind to 111-NQRVNQVAEVLQL-123, located within the helical subdomain (helices 2–4, Fig 1A), three (2F9, 1D8, 2G4)

Fig 1 Location of epitopes in the amino acid sequence of S typhimurium MalK (A) and in the modelled 3Dstructure of E coli MalK (B) (A) The Walker A and B motifs and the ABC signature are highlighted in yellow The epitopes recognized by the mAbs are highlighted in red Residues that when mutated render E coli MalK insensitive to inducer exclusion are underlined while residues that cause a regulatory phenotype when mutated are doubly underlined [14] a-Helices and b-strands that have been identified in the structure of T litoralis MalK [5] are indicated above the sequences as broken and dotted lines, respectively Please note that the primary structures of S typhimurium (acc no X54292) and E coli MalK (acc no J01648) differ only by 16 amino acid changes and by the lack of the dipeptide PM in S typhimurium MalK after residue L258 Furthermore, A320 (underlined) corresponds to S322 in E coli MalK (B) Stereo representation of the model of monomeric E coli MalK [14] The epitopes recognized by the mAbs are indicated in red The figure was drawn with RasMol 2.6 (http://www.umass.edu/microbio/rasmol) using the coordinates provided by W Welte (Universita¨t Konstanz).

recognize 304-VVEQLGHETQIHIQIP-319 and one (4B3)

binds to 352-LFREDGSACR-361, both located in the

C-terminal domain (Fig 1A and B) The fact that in each

case strong signals with successive overlapping peptides

were obtained argues in favour of linear rather than

discontinuous epitopes Only mAbs 5B5, 4H12, 2F9, and

4B3 were further characterized

In order to identify those amino acid residues that are

indispensable for binding within each epitope

substitu-tional analyses of the peptides were performed In these

experiments every position was substituted one-at-a-time

by all other genetically encoded amino acids Thus, all

possible single site substitution analogs were synthesized

and screened Discrete substitution patterns were

identified (Fig 3) and the results are summarized in

Table 1

In the case of 4H12, four residues at the N-terminus

(N111–V114) are not essential for binding However, the

third and fourth position of the peptide are nonetheless

required as revealed by an additional analyses using

peptides that varied in length at the N- or C-terminal end

or both (not shown) Thus, the minimum epitope

encom-passes residues R113 to L123 Furthermore, E119 and Q122

can be replaced by various amino acids without loss of

binding

Binding of mAb 5B5 is strongly dependent on the

residues L60–G63 which are either indispensable or can be

substituted only by chemically related amino acids

(Fig 3B) This result was confirmed by length analysis

(not shown)

Similarly, the data clearly revealed that the peptide G309-I314 is absolutely essential for binding of mAb 2F9 (Fig 3C) The observation that 4B3 bound only to one spot (out of 150) in the peptide scan (Fig 2D) already suggested that the peptide L352–R361 would be the minimum epitope This notion was basically confirmed by mutational analyses (Fig 3D) and by the failure of the mAb

to recognize peptides lacking residues at either the N- or C-terminus (not shown) Interestingly, substitution of several residues, in particular L352C, R354N, E355M, A359I/V and C360F/L, resulted in significantly increased binding of 4B3

ATP affects binding of mAbs to soluble MalK but not to MalFGK2-containing proteoliposomes The affinities of the mAbs for their respective antigens were determined by competitive inhibition ELISA according to Friguet et al (1987) [42], using soluble MalK, proteolipo-somes containing the MalFGK2 complex or synthetic soluble peptides as free antigen The resulting dissociation constants are summarized in Table 2

All mAbs have largely similar affinities for their respective epitopes in both MalK and the MalFGK2complex with Kd values ranging from 0.1 lM (4H12) to 10 lM (4B3) This finding is not only consistent with the surface-exposed localization of the epitopes in the tertiary structure of MalK [5] (Fig 1B) but also suggests that complex assembly is not accompanied by a significant change in accessibility None-theless, the use of synthetic peptides as free antigen resulted

Fig 2 Binding of mAbs to MalK-derived peptide scans (10-mers) The MalK fragments given below were scanned with cellulose-bound peptides shifted by one amino acid The numbers of spots in each row and the total number of rows are indicated above and at the right-hand side of each blot, respectively Blots were incubated with mAbs and developed as described in Experimental procedures (A) mAbs 4H12, 3A12, 4D8, 6E6: fragment G104–L134, elongated at the N-terminal end by the tripeptide QAA (42 spots in total); peptide sequences read as follows: row 1/spot 1, empty; 1/2, QAAG104AKKEVM-110; 1/3, AAG104AKKEVMN-111; 1/4, AG104AKKEVMNQ-112 and so forth (B) 5B5: fragment G51–F98, elongated at the C-terminal end by the dipeptide RP (41 spots in total); peptide sequences read as follows: row 1/spot 1, 51-GLETITSGDL60; 1/2, 52-LETITSGDLF-61; 1/3, 53-ETITSGDLFI-62 and so forth (C) 2F9, 1D8, 2G4: fragment R211-V369 (150 spots in total); peptide sequences read

as follows: row 1/spot 1, 211-RVAQVGKPLE220; 1/2, 212-VAQVGKPLEL221; 1/3, 213-AQVGKPLELY222 and so forth D 4B3: fragment R211-V369 (150 spots in total); peptide sequences read as in C See Fig 1 A for sequence information.

in lower Kd values, except for 4H12, indicating that the

epitopes are not fully exposed when part of the folded

polypeptide chain

Remarkably however, when the binding assays were

performed in the presence of ATP, the Kdvalues determined

with soluble MalK increased for all mAbs between

two-(2F9) and sevenfold (4H12) (Table 2) These data suggest

that the epitopes become less accessible in the ATP-bound

form of the subunit, thereby probably reflecting

ATP-induced structural alterations previously observed by limited proteolysis [45] In this study, ATP was found to render the peptide fragment between residues R66 and R146 more resistant to protease [45] Our finding that both mAbs for which the strongest reduction in affinity was observed (5B5, 4H12) recognize epitopes located within this fragment

is consistent with this result In addition, the ATP-induced global conformational change apparently also affects the C-terminal domain as the Kdvalues of 4B3 and, to a lesser extent, 2F9, were increased too In contrast, ATP did not change the affinity of the mAbs for their epitopes in complex-associated MalK, although ATP-induced confor-mational changes were also observed with the transport complex [24,49] Thus, this finding gives rise to the speculation that these changes must differ from those of soluble MalK

Fab fragments of 5B5 and 4H12 only slightly inhibit ATPase activity of MalK and of proteoliposomes Having established the binding properties of the mAbs, we analyzed their possible effects on transporter functions While the spontaneous ATPase activity exhibited by purified MalK can be taken as a measure for the catalytic

Fig 3 Substitutional analyses of the peptide epitopes recognized by the mAbs Each amino acid of the four peptide epitopes (indicated at the left-hand side of each blot) which are recognized by the mAbs (identified by the analysis shown in Fig 2) is substituted by all other 20 L -amino acids (rows) in alphabetical order (shown on top of each blot) and tested for binding to the respective mAb All spots in the left column comprise the wild type (wt) sequence of the epitopes (A) mAb 4H12, peptide analyzed: 111-NQRVNQVAEVLQL123; (B) mAb 5B5, peptide analyzed 53-ETITSGDLTRM67; (C) mAb 2F9, peptide analyzed: 304-VVEQLGHETQIHIQIP319; (D) mAb 4B3, peptide analyzed: 352-LFREDG SACR361 See Fig 1A for sequence information.

Table 1 Location of recognition sites of mAbs in MalK See also Fig 1

A and B.

mAbs Recognition sequence Location

5B5 60-LFIG-63 C-terminus of Walker A

(within b4) 4H12 113-RVNQVAEVLQL-123 Helical subdomain

(within a3) 2F9 309-GHETQI-314 C-terminal domain

(between b17 and b18) 4B3 352-LFREDGSACR-361 C-terminal domain

(end of b21 and beyond)

activity, the coupling between transport and ATP hydrolysis

is conveniently assayed by monitoring

MalE-maltose-stimulated ATPase activity of MalFGK2-containing

proteoliposomes Thus, MalK and proteoliposomes were

incubated with excess mAbs and subsequently assayed for ATP hydrolysis The results are shown in Fig 4 While 4H12 and 5B5 that bind to epitopes in the N-terminal ATPase domain strongly reduced the enzymatic activity of MalK, 4B3 and 2F9, both recognizing epitopes in the C-terminal domain, did not (Fig 4A) Similar results were obtained with proteoliposomes, except that the inhibitory effect of 5B5 was only moderate (Fig 4B) These findings are consistent with the assumed roles of the N- and C-terminal domains of MalK in catalytic and regulatory functions of the transporter, respectively

However, when using intact (divalent) mAbs the possi-bility that inhibition, where observed, resulted from steric effects caused by the Fc regions of these mAbs cannot be excluded To address this possibility, Fab fragments of mAbs 5B5 and 4H12 were prepared and tested to determine whether these monovalent fragments are also capable of inhibiting the ATPase activity of MalK and MalFGK2 Indeed, the hydrolytic activity of MalK was only moder-ately affected by the Fabs whereas in proteoliposomes, the observed effect was negligible (Fig 4C) In control experi-ments, we assured that this result was not due to a loss in binding affinity of the Fab fragments for their epitopes As shown in Table 2, both Fab fragments have similar or even better affinities for the protein antigen than the correspond-ing mAbs Moreover, the Kdvalues also changed in the

Fig 4 Effects of mAbs and Fab fragments on ATPase activity of MalK and MalFGK 2 containing proteoliposomes ATPase activities were monitored after incubation of mAbs with MalK (A) or MalE-maltose-loaded proteoliposomes (B) in 50 m M Tris/HCl, pH 7.5, for 1 h at room temperature at molar ratios of 1.5 : 1 and 3 : 1, respectively (molecular mass of MalK, 40 kDa; molecular mass of complex,

171 kDa) In the case of proteoliposomes, this actually corresponds to

a 12-fold molar excess of mAbs over MalK proteins contributing to enzymatic activity This calculation is based on the finding that only 25% of the added complex protein becomes incorporated into the liposomes with the MalK subunits facing the medium [18] (C) ATP hydrolysis was assayed after incubation of Fab fragments of 4H12 and 5B5 with MalK or proteoliposomes as above at molar ratios of 1.5 : 1 and 10 : 1, respectively (corresponding to a 40-fold excess over medium-exposed MalK in proteoliposomes) The data represent the average of at least three independent experiments Control activities: MalK, 0.12 lmol P i Æmin)1Æmg)1; MalFGK 2 , 0.75 lmol P i Æmin)1Æmg)1 (these values correspond to approximately half of the routinely meas-ured activities due to the removal of dithiothreitol from the buffer by dialysis in order to avoid dissociation of the antibodies) PLS, MalFGK -containing proteoliposomes.

Table 2 Binding constants of mAbs and Fab fragments K d values ( M ) were determined as described in [42] Values given are means ± SEM from at least three different experiments ND, not determined.

MalK 4.9 ± 3.7 · 10)7 5.4 ± 1.3 · 10)8 1.1 ± 0.5 · 10)7 3.8 ± 1.2 · 10)8 2.6 ± 0.2 · 10)6 1.1 ± 0.1 · 10)5 MalK + ATP

(2 m M )

2.7 ± 1.1 · 10)6 2.6 ± 0.5 · 10)7 7.6 ± 1.6 · 10)7 3.4 ± 1.3 · 10)7 5.5 ± 0.5 · 10)6 5.5 ± 2.6 · 10)5 MalFGK 2 4.9 ± 1.2 · 10)7 3.8 ± 0.5 · 10)7 1.7 ± 0.2 · 10)7 4.2 ± 0.8 · 10)7 1.0 ± 0.8 · 10)6 4.8 ± 1.4 · 10)6 MalFGK 2 + ATP

(2 m M )

4.8 ± 0.2 · 10)7 2.3 ± 0.3 · 10)6 1.7 ± 0.7 · 10)7 4.2 ± 0.8 · 10)7 1.2 ± 0.2 · 10)6 7.1 ± 0.5 · 10)6 Peptide 5.7 ± 0.2 · 10)8 ND 4.7 ± 4.4 · 10)7 ND 3.4 ± 1.2 · 10)7 8.0 ± 1.2 · 10)7

presence of ATP when soluble MalK was used as the free

antigen Thus, neither of the epitopes is likely to be directly

involved in the enzymatic reaction

MalT interferes with binding of mAbs 4B3 and 2F9

to their epitopes

The maltose transporter of E coli and S typhimurium is

involved in the regulation of transcription of genes

belong-ing to the maltose regulon [26] This notion is based on

mutational analyses [28,30,44,46] and supported by

dem-onstrating physical interaction of MalK and MalT in

coelution experiments [47] However, evidence that MalT

also binds to the intact transporter is lacking Thus, before

studying the possible effects of mAbs on MalT binding, we

set out to directly demonstrate complex–MalT interaction

by using a coelution approach similar to that in [47] To this

end, purified MalFGK2was reloaded on a Ni-NTA matrix,

incubated with a cytosolic fraction of strain JM109 (pAS8)

containing MalT, and, after extensive washing, eluted with

250 mMimidazole Subsequent analysis by SDS/PAGEand

immunoblotting then clearly revealed that MalT coeluted

with MalFGK2 (data not shown), thereby indicating a

specific interaction of MalT with the complex

Then, we studied whether binding of MalT to the

reconstituted transport complex would prevent any of the

mAbs from getting access to their epitopes by competitive

inhibition ELISA To this end, proteoliposomes containing

the MalFGK2 complex were incubated with partially

purified MalT for 4 h at 4C prior to the addition of

mAbs After overnight incubation at 4C, binding assays

were performed in microtiter plates coated with purified

MalK as described in Experimental Procedures The results

are shown in Fig 5 Preincubation with MalT reduced the

interaction of 4B3 (Fig 5B), and to a lesser extent, 2F9

(Fig 5A), with the transport complex, while no such

reaction was observed with 4H12 (Fig 5C) and 5B5 (Fig 5D), recognizing epitopes in the N-terminal domain Thus, these findings clearly suggest that MalT binds to the transport complex by interaction with the C-terminal domains of the MalK subunits

Enzyme IIAGlceliminates binding of mAbs 2F9 and 4B3

to the MalFGK2complex The nonphosphorylated form of enzyme IIAGlc of the phosphoenolpyruvate phosphotransferase system blocks maltose transport by inhibition of ATPase activity in the process of inducer exclusion [18,29,48] The majority of missense mutations that render maltose uptake insensitive

to inducer exclusion is clustered in the C-terminal domain of MalK [14,28,29], indicating an interaction of enzyme IIAGlc with the MalK subunits Again, to obtain direct evidence in favour of this notion, we investigated a possible overlap of binding sites for mAbs and enzyme IIAGlcby competitive inhibition ELISA The results are shown in Fig 6 Clearly, preincubation with enzyme IIAGlc resulted in similarly reduced binding of mAbs 2F9 and 4B3 that both recognize epitopes in the C-terminal domain (Fig 6A,B), while binding of 4H12 and 5B5 remained unaffected (Fig 6C,D)

D I S C U S S I O N

In this communication we describe the isolation and characterization of nine monoclonal antibodies raised against the MalK subunit of the maltose ABC transporter

of S typhimurium that bind to four nonoverlapping linear epitopes Two epitopes, recognized representatively by mAbs 5B5 and 4H12 are located in the N-terminal ATPase domain of MalK in between the Walker A and B motifs while those recognized representatively by mAbs 2F9 and 4B3 are located in the C-terminal regulatory

Fig 5 Effect of MalT on competitive inhibition ELISA with proteoliposomes as free antigen Proteoliposomes (containing 15 lg of complex protein) were incubated with partially purified MalT (12 lg) in 50 m M Tris/HCl, pH 7.5, containing 100 m M KCl, 10% (v/v) glycerol, 5 m M MgCl 2 and

1 m M ATP for 4 h at 4 C in a total volume of 150 lL Subsequently, aliquots were removed, further incubated with mAbs overnight at 4 C and assayed for binding by competitive inhibition ELISA as described in Experimental procedures In control experiments, the mAbs were replaced with

an equal volume of buffer (A) 2F9; (B) 4B3; (C) 4H12; (D) 5B5 Symbols: squares, + MalT; triangles, control Representative data are shown.

domain (Fig 1A) All mAbs bind their epitopes in soluble

MalK and in the MalFGK2 complex both in the

denatured and native states, suggesting a surface exposure

of the respective peptide fragments This notion is

consistent with the three-dimensional structure of the

close homolog MalK of T litoralis [5], which is used as a

model for the E coli/S typhimurium MalK protein [14]

(Fig 1B)

The peptide 60-LFig-63 (mAb 5B5) is located

carboxy-terminal of the Walker A motif in a region that consists of

antiparallel b sheets (Fig 1A,B) Binding of the intact mAb

strongly inhibited the ATPase activity of the MalK subunit

but had only a moderate effect on ATP hydrolysis catalyzed

by the reconstituted MalFGK2complex In contrast, Fab

fragments displayed the same minor effect on the catalytic

activity of both systems, indicating that the epitope is not

essential for transport function per se This conclusion is in

agreement with the lack of reports on mutations in this

region that cause a defect in maltose uptake

The peptide recognized by mAb 4H12 is located in the

helical subdomain of MalK (encompassing helices 2–4),

covering most of the C-terminal part of a3 (Fig 1A) The

helical subdomain is supposedly next to the membrane

components as suggested from suppressor analyses [22,23]

and crosslinking experiments [24] In particular, residues

V114, V117, and L123, all part of the epitope to which

4H12 binds, were proposed to participate in interaction

with MalFG Interestingly, V114 was found to be located

in close proximity to MalF only in the presence of ATP,

suggesting an ATP-induced conformational change that

affects the relative positions of helix 3 and the EAA loop

These data gave rise to the speculation that during

transport residues in helix 3 might participate in

trans-mitting signals to the membrane-integral subunits via the

conserved EAA loops or vice versa [24] The results

presented here provide further insight into the putative

role of helix 3

At first glance, the finding that MalE-maltose stimulated

ATPase activity of the reconstituted transport complex was

only slightly inhibited by Fab fragments of 4H12 might be

taken as evidence against a direct role of the peptide

fragment and thus, of helix 3, in transport function However, this does not exclude that the epitope is nonethe-less affected by (ATP-induced) conformational changes during substrate translocation The observation that bind-ing of the intact mAb was not tolerated with respect to ATPase activity in both MalK and the MalFGK2complex

is consistent with this notion Moreover, residues V114, V117 and L123 are largely buried within the MalK dimer [14] despite the fact that helix 3 as such is located at the surface of the protein Thus, in the assembled complex, interactions with MalFG may take place within a hydro-phobic pocket inaccessible to the antibody This view would

be in line with the observation that V114 is dispensable for antibody binding but is in contrast to the result from substitutional analysis that V117 and L123 are almost fully essential for antibody recognition (Fig 3A) However, the accessibility of these residues in the folded polypeptide and

in the context of the assembled and reconstituted complex is likely to differ from that in synthetic peptides Thus, binding

of complete 4H12 or of its Fab fragments to the epitope in the native environment might preferentially occur via those residues that, according to the MalK structure [14], are clearly surface-exposed These include N115, Q116, E119, V120 and Q122 (Fig 7A or B) Then, binding of Fab fragments would not interfere with subunit–subunit inter-actions

None of the mAbs recognizing epitopes in the C-terminal domain inhibited the catalytic activity of MalK or the transport cycle This finding is consistent with the C-terminal extension being a unique structural feature of the MalK-subfamily of ABC proteins [11] and with its presumed role in regulatory functions However, two highly conserved residues have been identified in the C-terminal domain that, when mutated, abolish transport Substituting lysine for glutamate at position 306 in S typhimurium MalK (E308 in E coli MalK), caused a substantially reduced ATPase activity of the protein [50], while the F355Y mutation in E coli MalK resulted in a defect in maltose utilization [14] E306 is located close to the epitope

of mAb 2F9 while F355 (F353 in S typhimurium MalK) constitutes part of the recognition site of 4B3 (Fig 1A)

Fig 6 Effect of enzyme IIAGlcon competitive inhibition ELISA with proteoliposomes as free antigen Proteoliposomes (containing 25 lg of complex protein) were incubated with purified enzyme IIA Glc (230 lg) in 50 m M Tris/HCl,

pH 7.5, containing 100 m M NaCl, 12.5% (v/v) glycerol, for 5 min at 37 C in a total volume of 150 lL Subsequently, aliquots were removed, further incubated with mAbs (final concentration: 0.16 lgÆmL)1) for 5 h at

4 C and analyzed by competitive inhibition ELISA as described in Experimental proce-dures In control experiments, the mAbs were replaced with an equal volume of buffer (A) 2F9; (B) 4B3; (C) 4H12; (D) 5B5 Symbols: diamonds, + enzyme IIAGlc; triangles, – con-trol Representative data are shown.

Because a direct role for both residues in the enzymatic

reaction is highly unlikely, structural disorders caused by the

different chemical nature of the replacing residue may

account for the observed phenotypes Our finding that both

mAbs failed to inhibit the ATPase activity of MalK is at

least not contradictory to this notion

Based on extensive mutational analyses, the activities of

the maltose transporter in transcriptional regulation and as

target for enzyme IIAGlcin the process of inducer exclusion

have been largely attributed to the C-terminal domain of

MalK [14,28–30] The observation that MalK

phenotypi-cally acts as a repressor of maltose-regulated genes was

interpreted in favour of a direct interaction with the positive

regulator, MalT, of the mal regulon, for which biochemical

evidence was presented [47] Our results confirm and extend

the current knowledge on MalT-transporter interplay by

demonstrating binding of MalT to the purified MalFGK2

complex in a coelution experiment Furthermore, the

finding that MalT reduced binding of mAb 4B3, and to a

lesser extent, 2F9 to their respective epitopes in

proteo-liposomes provides the first biochemical evidence for the

C-terminal domain of MalK being the site of interaction

with MalT

Bo¨hm et al [14] recently showed that in E coli MalK,

residues that when mutated diminish or abolish the

repressing effect on the mal regulon (Fig 1A) are exposed

on one surface of the C-terminal domain only They mark

the contours of a putative MalT binding site that may cover

the top and central part (Fig 7A, residues highlighted in

yellow) None of these residues are included in the epitopes

recognized by mAbs 2F9 and 4B3, respectively (Fig 7A,

residues highlighted in red) Most of the residues

constitu-ting the epitope recognized by 2F9 are located on the same

site but at the bottom part of the C-terminal domain The

only moderate effect of MalT on binding of 2F9 argues

against these residues being part of the MalT–MalK interaction face Rather, the epitope may be located at its periphery The peptide fragment to which 4B3 binds is largely exposed on the opposite surface but significantly protrudes into the cavity between the N- and C-terminal domain Thus, it is very well possible that MalT when bound to the MalK subunits sterically hinders 4B3 from gaining access to its epitope

Our results also indicate that both C-terminal epitopes are likely to overlap with a putative binding site of enzyme IIAGlc Mutations known to render maltose transport insensitive to inducer exclusion are all but two located in the C-terminal domain [14,28,29] (Fig 1A) However, compared to the residues constituting a putative MalT binding site they are exposed on the opposite surface of the protein (Fig 7B) Clearly, the epitope recognized by mAb 4B3 is in such close contact to R228 and F241 that a competition with enzyme IIAGlcfor binding appears likely Inhibition of binding of mAb 2F9 by enzyme IIAGlcis less obvious This epitope is mostly exposed on the opposite surface with only one residue, H310 (N312 in E.coli.) protruding at the bottom of the C-terminal domain (Fig 7B) Thus, one may speculate that enzyme IIAGlc, when associated with MalFGK2, is expanding into this region, thereby interfering with antibody binding

Interestingly enough, two mutations that restore maltose transport in the presence of enzyme IIAGlc in vivoaffect residues in the N-terminal domain of MalK (E119, A124) (Figs 1 and 7B) This location makes it highly unlikely that both residues are taking part in an enzyme IIAGlcbinding site Rather, as already discussed by Bo¨hm et al [14], the residues may be involved in the signalling pathway that upon binding of enzyme IIAGlcresults in the inhibition of ATP hydrolysis Our finding that binding of mAb 4H12 to the MalFGK2complex was unaffected by enzyme IIAGlc,

Fig 7 Location of epitopes and of residues putatively involved in MalT and enzyme IIAGlc Space-fill representation of the model of monomeric

E coli MalK Residues that correspond to epitopes recognized by the mAbs and to amino acid residues identified by mutational analyses in E coli MalK [14,31,32] (see also Fig 1A) are highlighted in different colors: Epitopes of mAbs are colored red and residues putatively involved in MalT (A) and enzyme IIAGlc(B) binding are shown in yellow and orange, respectively Numbers correspond to the position of the indicated residue in the

E coliMalK protein (see Fig 1A) Please note that in Fig 7B, the epitope of 5B5 is located on the back side of the protein and therefore not visible This is indicated by a broken line.