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

Functional characterization of the maltose ATP-binding-cassette transporter of Salmonella typhimurium by means of monoclonal antibodies directed against the MalK subunit Anke Stein 1 , Martina Seifert 2 , Rudolf Volkmer-Engert 2 ,Jo¨ rg Siepelmeyer 3 , Knut Jahreis 3 and Erwin Schneider 1 1 Humboldt Universita ¨ t zu Berlin, Institut fu ¨ r Biologie, Berlin, Germany; 2 Humboldt Universita ¨ t zu Berlin, Institut fu ¨ r Medizinische Immunologie, Berlin; 3 Universita ¨ t Osnabru ¨ ck, Fachbereich Biologie/Chemie, Germany The maltose ATP-binding cassette transporter of Salmonella typhimurium is composed of a membrane-associated com- plex (MalFGK 2 ) 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 MalFGK 2 complex with K d values ranging from 10 )6 to 10 )8 M . 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 MalFGK 2 complex in the presence of MalT and enzyme IIA Glc , 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; MalFGK 2 ; enzyme IIA Glc ; 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 F 1 -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, MalFGK 2 ,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 ¨ tI, 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) Eur. J. Biochem. 269, 4074–4085 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03099.x 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 MalFGK 2 complex, when incorporated into liposomes, also exhibits a low intrinsic ATPase activity that, however, is stimulated severalfold in the presence of substrate-loaded MalE and 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 MalE via 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 MalFGK 2 complex 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 MalFGK 2 complex 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. EXPERIMENTAL PROCEDURES Preparative procedures MalK [15], MalFGK 2 [18], and MalE [31] were purified as described. MalE/maltose-loaded proteoliposomes con- taining the MalFGK 2 complex were prepared by a detergent dilution procedure as published elsewhere [18,32]. Enzyme IIA Glc was purified from the cytosolic fraction of E. coli strain BL21 D (pts43crr::kan R ) 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 (malT E.c. on pSE380, p trc ,amp R ) (this study). For competitive inhibition ELISA, N-terminally his-tagged MalT was partially purified from strain JM109, harbouring plasmid pAS9 (malT E.c. on pQE9, p T5 ,amp R )byNi-NTA chromatography. Preparation of mAbs Ten-week-old-femaleBalb/cmicewereimmunizedintra- peritoneally either with native MalK or with an N-terminal fragment (encompassing residues 1–179 [30]) (100 lg each), dissolved in NaCl/P i [35]. On day 12, 25 and 62 the animals were boosted with 50 lgofprotein 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 using 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 m M sodium phosphate buffer, pH 7. After washing off unbound material, mAbs were eluted with 0.1 M glycine/HCl, pH 2.7, and immediately dialyzed against NaCl/P i overnight at 4 °C. Isolation of Fab fragments One millilitre of mAbs (1–3 mg) were mixed with 0.5 mL of papain-agarose beads in 20 m M phosphate buffer, pH 7.5, supplemented with 20 m ML -cysteine and 1 m M EDTA, and incubated overnight at 37 °C. Subsequently, Fab fragments were separated from uncleaved mAbs by incubating the mixture with protein A–Sepharose for 1 h at 4 °C. Unbound material (Fab fragments) was collected, dialyzed overnight against 3 L of 50 m M Tris/HCl, pH 7.5, and stored at 4 °C until use. Ó FEBS 2002 Protein–protein interactions of MalFGK 2 (Eur. J. Biochem. 269) 4075 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) 2 spacer. Epitope mapping The screening of cellulose-bound peptides followed a protocol published elsewhere [39,40]. Peptide libraries were incubated with mAbs overnight at 4 °C 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/H 2 O(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 A ¼ 0.05% trifluoracetic acid in water, and B ¼ 0.05% trifluoracetic acid in acetonitrile. The HPLC had the UV detector at 214 nm, a Vydac C 18 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 m M sodium carbonate buffer, pH 9.6, and incubated overnight at 4 °C. Remaining binding sites were blocked with 2% BSA in NaCl/P i 150 m M NaCl, 3 m M KCl, 8 m M Na 2 HPO 4 · 2H 2 O, 1 m M KH 2 PO 4 ) for 2 h at room temperature. Subsequently, the wells were incubated overnight at 4 °C with mAb diluted in 2% BSA in NaCl/P i /Tween (NaCl/P i 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/ P i /Tween. Antibody binding was detected by adding 100 lL of 62.5 lgmL )1 3,3¢,5,4¢-tetramethylbenzidine, 0.0026% (v/v) H 2 O 2 in 0.1 M sodium acetate/0.1 M citric acid, pH 6. After 10 min at room temperature, the reaction was stopped by addition of 100 lLH 2 SO 4 and 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, MalFGK 2 -containing proteoliposomes, synthetic peptides) the mAbs and an equal volume of free antigen in different concentrations were incubated overnight at 4 °C in the wells coated with MalK (2.5 pmol). The mAbs were used in the following concentrations: 2F9 and 4B3, 2 · 10 )9 M ;4H12, 6.5 · 10 )9 M ;5B5,8· 10 )10 M . 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/PAGE and immunoblot analyses were performed as described in [44]. RESULTS 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 MalFGK 2 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 4076 A. Stein et al.(Eur. J. Biochem. 269) Ó FEBS 2002 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 3D structure 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 ¨ tKonstanz). Ó FEBS 2002 Protein–protein interactions of MalFGK 2 (Eur. J. Biochem. 269) 4077 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 MalFGK 2 -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 MalFGK 2 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 MalFGK 2 complex with K d values ranging from 0.1 l M (4H12) to 10 l M (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. 4078 A. Stein et al.(Eur. J. Biochem. 269) Ó FEBS 2002 in lower K d 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 K d values 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 K d values 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 AandB. 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) Ó FEBS 2002 Protein–protein interactions of MalFGK 2 (Eur. J. Biochem. 269) 4079 activity, the coupling between transport and ATP hydrolysis is conveniently assayed by monitoring MalE-maltose- stimulated ATPase activity of MalFGK 2 -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 MalFGK 2 . 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 K d values 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 a12-foldmolarexcessofmAbsoverMalKproteinscontributingto 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 )1 Æmg )1 ;MalFGK 2 ,0.75lmol P i Æmin )1 Æmg )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 2 -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. Antigen 5B5 5B5-Fab 4H12 4H12-Fab 2F9 4B3 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 TSGDLFIG NQVAELQLAH VVEQLGHETQ HLFREDGSACR 4080 A. Stein et al.(Eur. J. Biochem. 269) Ó FEBS 2002 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 MalFGK 2 was reloaded on a Ni-NTA matrix, incubated with a cytosolic fraction of strain JM109 (pAS8) containing MalT, and, after extensive washing, eluted with 250 m M imidazole. Subsequent analysis by SDS/PAGE and immunoblotting then clearly revealed that MalT coeluted with MalFGK 2 (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 MalFGK 2 complex were incubated with partially purified MalT for 4 h at 4 °C prior to the addition of mAbs. After overnight incubation at 4 °C, 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 IIA Glc eliminates binding of mAbs 2F9 and 4B3 to the MalFGK 2 complex The nonphosphorylated form of enzyme IIA Glc 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 IIA Glc 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 IIA Glc by competitive inhibition ELISA. The results are shown in Fig. 6. Clearly, preincubation with enzyme IIA Glc 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). DISCUSSION 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 1m 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 °Cand 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. Ó FEBS 2002 Protein–protein interactions of MalFGK 2 (Eur. J. Biochem. 269) 4081 domain (Fig. 1A). All mAbs bind their epitopes in soluble MalKandintheMalFGK 2 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 MalFGK 2 complex. 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 MalFGK 2 complex 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. EffectofenzymeIIA Glc on competitive inhibition ELISA with proteoliposomes as free antigen. Proteoliposomes (containing 25 lgof 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 )for5hat 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 IIA Glc ;triangles,–con- trol. Representative data are shown. 4082 A. Stein et al.(Eur. J. Biochem. 269) Ó FEBS 2002 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 IIA Glc in 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 MalFGK 2 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 IIA Glc . 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 IIA Glc for binding appears likely. Inhibition of binding of mAb 2F9 by enzyme IIA Glc is 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 IIA Glc , when associated with MalFGK 2 , is expanding into this region, thereby interfering with antibody binding. Interestingly enough, two mutations that restore maltose transport in the presence of enzyme IIA Glc in vivo affect 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 IIA Glc binding 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 IIA Glc results in the inhibition of ATP hydrolysis. Our finding that binding of mAb 4H12 to the MalFGK 2 complex was unaffected by enzyme IIA Glc , Fig. 7. Location of epitopes and of residues putatively involved in MalT and enzyme IIA Glc . 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 IIA Glc (B) binding are shown in yellow and orange, respectively. Numbers correspond to the position of the indicated residue in the E. coli MalK 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. Ó FEBS 2002 Protein–protein interactions of MalFGK 2 (Eur. J. Biochem. 269) 4083 [...]... changes of MalK, a bacterial ATP binding cassette transporter protein J Biol Chem 269, 20456–20461 Reyes, M & Shuman, H.A (1988) Overproduction of the MalK protein prevents expression of the Escherichia coli mal regulon J Bacteriol 170, 4598–4602 Panagiotidis, C.H., Boos, W & Shuman, H.A (1998) The ATPbinding cassette subunit of the maltose transporter MalK antagonizes MalT, the activator of the Escherichia... Functional purification of a bacterial ATP-binding cassette transporter protein (MalK) from the cytoplasmic fraction of an overproducing strain Protein Expression Purif 6, 10–14 Morbach, S., Tebbe, S & Schneider, E (1993) The ATP-binding cassette (ABC) transporter for maltose/ maltodextrins of Salmonella typhimurium Characterization of the ATPase activity associated with the purified MalK subunit J Biol Chem... P.-Q., Ames, G.F.-L & Kim, S.-H (1998) Crystal structure of the ATP-binding subunit of an ABC transporter Nature 396, 703–707 5 Diederichs, K., Diez, J., Greller, G., Muller, C., Breed, J., Schnell, ¨ C., Vonrhein, C., Boos, W & Welte, W (2000) Crystal structure of MalK, the ATPase subunit of the trehalose /maltose ABC transporter of the archaeon Thermococcus litoralis EMBO J 19, 5951–5961 6 Yuan, Y.-R.,... are potent inhibitors of the ATPase activity of the reconstituted bacterial ATP-binding cassette transporter for maltose (MalFGK2) Biochem Biophys Res Commun 216, 589–594 Landmesser, H., Stein, A., Bluschke, B., Brinkmann, M., Hunke, ¨ S & Schneider, E (2002) Large-scale purification, dissociation and functional reassembly of the maltose ATP-binding cassette transporter (MalFGK2) of Salmonella typhimurium. .. domain in the MalK subunit of the ATP-binding-cassette transport system for maltose of Salmonella typhimurium (MalFGK2) is crucial for interaction with MalF and MalG A study using the LacK protein of Agrobacterium radiobacter as a tool Mol Microbiol 22, 555–566 ´ Hunke, S Mourez, M Jehanno, Dassa, E & Schneider, E (2000) ATP modulates subunit subunit interactions in an ATP-bindingcassette transporter. .. of the bacterial ATP-binding-cassette (ABC) -protein MalK Acta Crystallogr D-Biol Cryst 55, 285–286 Bohm, A., Diez, J., Diederichs, K., Welte, W & Boos, W (2001) ¨ Structural model of MalK, the ABC subunit of the maltose transporter of Escherichia coli: Implications for mal gene regulation, inducer exclusion and subunit assembly J Biol Chem 277, 3708–3717 Schneider, E., Linde, M & Tebbe, S (1995) Functional. .. are located within or close to the epitope (Figs 1A and 7B), may be interpreted in favour of this notion In summary, the monoclonal antibodies described here have proven to be useful tools in further elucidating protein–protein interactions of the maltose ABC transporter Possible future applications, especially of 4H12 and 5B5, will include studies on the assembly of the MalFGK2 complex in vitro which... (1997) Purification and characterization of HisP, the ATP-binding subunit of a traffic ATPase (ABC transporter) , the histidine permease of Salmonella typhimurium J Biol Chem 272, 27745–27752 Schneider, E & Walter, C (1991) A chimeric nucleotide-binding protein, encoded by a hisP -malK hybrid gene, is functional in maltose transport in Salmonella typhimurium Mol Microbiol 5, 1375–1383 Schneider, E., Wilken,... van Dam, K & Postma, P.W (1994) Quantification of the regulation of glycerol and maltose metabolism by IIAGlc of the phosphoenolpyruvate-dependent glucose phosphotransferase system in Salmonella typhimurium J Bacteriol 176, 3518–3526 Mannering, D.E., Sharma, S & Davidson, A.L (2001) Demonstration of conformational changes associated with activation of the maltose transport complex J Biol Chem 276, 12362–12368... (1998) Domain structure of the ATPbinding-cassette (ABC) protein MalK of Salmonella typhimurium as assessed by coexpressed half molecules and LacK¢- MalK chimeras J Bacteriol 180, 5299–5305 31 Honer zu Bentrup, K., Schmid, R & Schneider, E (1994) Maltose ¨ transport in Aeromonas hydrophila: purification, biochemical characterization and partial protein sequence analysis of a periplasmic maltose- binding protein . Functional characterization of the maltose ATP-binding-cassette transporter of Salmonella typhimurium by means of monoclonal antibodies directed against the MalK subunit Anke Stein 1 ,. [44]. RESULTS 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. 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