Functional importance of a conserved sequence motif in FhaC, a prototypic member of the TpsB/Omp85 superfamily Anne-Sophie Delattre 1–4 , Bernard Clantin 5 , Nathalie Saint 6,7 , Camille Locht 1–4 , Vincent Villeret 5 and Franc¸oise Jacob-Dubuisson 1–4 1 Inserm U1019, Center for Infection and Immunity of Lille, France 2 Institut Pasteur de Lille, France 3 Universite ´ Lille Nord de France, Lille, France 4 CNRS UMR8204, Lille, France 5 Institut de Recherche Interdisciplinaire, USR3078 CNRS – Universite ´ de Lille 1 et 2, Villeneuve d’Ascq, France 6 INSERM U554, Universite ´ de Montpellier 1 et 2, France 7 UMR5048 CNRS, Universite ´ de Montpellier 1 et 2, France Keywords Bordetella; outer membrane protein; protein structure; protein transport; two-partner secretion Correspondence F. Jacob-Dubuisson, 1, rue Calmette, 59019 Lille Cedex, France Fax: +33 320 87 11 58 Tel: +33 320 87 11 55 E-mail: francoise.jacob@ibl.fr Database Structural data are available at the Protein Data Bank under the accession number 2QDZ (FhaC WT ) (Received 23 July 2010, revised 8 September 2010, accepted 13 September 2010) doi:10.1111/j.1742-4658.2010.07881.x In Gram-negative bacteria, the two-partner secretion pathway mediates the secretion of TpsA proteins with various functions. TpsB transporters specifi- cally recognize their TpsA partners in the periplasm and mediate their trans- port through a hydrophilic channel. The filamentous haemagglutinin adhesin (FHA) ⁄ FhaC pair represents a model two-partner secretion system, with the structure of the TpsB transporter FhaC providing the bases to deci- pher the mechanism of action of these proteins. FhaC is composed of a b-barrel preceded by two periplasmic polypeptide-transport-associated (POTRA) domains in tandem. The barrel is occluded by an N-terminal helix and an extracellular loop, L6, folded back into the FhaC channel. In this article, we describe a functionally important motif of FhaC. The VRGY tetrad is highly conserved in the TpsB family and, in FhaC, it is located at the tip of L6 reaching the periplasm. Replacement by Ala of the invariant Arg dramatically affects the secretion efficiency, although the structure of FhaC and its channel properties remain unaffected. This substitution affects the secretion mechanism at a step beyond the initial TpsA–TpsB interaction. Replacement of the conserved Tyr affects the channel properties, but not the secretion activity, suggesting that this residue stabilizes the loop in the resting conformation of FhaC. Thus, the conserved motif at the tip of L6 represents an important piece of two-partner secretion machinery. This motif is conserved in a predicted loop between two b-barrel strands in more distant relatives of FhaC involved in protein transport across or assembly into the outer membranes of bacteria and organelles, suggesting a conserved function in the molecular mechanism of transport. Structured digital abstract l MINT-7996294: Fha30 (uniprotkb:P12255) binds (MI:0407)toFhaC (uniprotkb:P35077)by filter binding ( MI:0049) Abbreviations ECL, enhanced chemiluminescence; FHA, filamentous haemagglutinin adhesin; POTRA domain, polypeptide-transport-associated domain; TPS, two-partner secretion; WT, wild-type. FEBS Journal 277 (2010) 4755–4765 ª 2010 The Authors Journal compilation ª 2010 FEBS 4755 Introduction Gram-negative bacteria possess a complex cell enve- lope composed of two membranes. The outer mem- brane, which provides the bacterium with significant protection against toxic agents [1], represents a barrier for the secretion of proteins destined for the cell sur- face or the extracellular milieu. Thus, Gram-negative bacteria have developed a number of pathways specifi- cally devoted to protein secretion. Among these, the two-partner secretion (TPS) pathway is widely repre- sented. The ‘TpsB’ transporters mediate the secretion across the outer membrane of their ‘TpsA’ exoprotein partners, which serve as adhesins, cytolysins, invasins, proteases, etc., to the bacterial cell surface or in the extracellular milieu [2]. The TpsB proteins belong to the TpsB ⁄ Omp85 superfamily of protein transporters, also called polypeptide-transporting b-barrel proteins [3–5]. This superfamily includes transporters located in the outer membranes of Gram-negative bacteria and of organelles of endosymbiotic origin [3,6–12]. These proteins, such as BamA (formerly YaeT) in Escherichia coli, Toc75 and Oep80 in chloroplasts, and Sam50 in mitochondria, are essential parts of complexes involved in protein transport across, or assembly into, the outer membranes of their respective organisms or organelles. The mechanistic principles of transport in the TPS pathway remain to be deciphered. The current model of secretion is as follows. Export of the TpsA precur- sor across the cytoplasmic membrane is mediated by the Sec machinery. TpsA proteins harbour, at their N-terminus, a conserved ‘TPS’ domain, approximately 250 residues long, required for secretion. In the peri- plasm, the TPS domain in an extended conformation is recognized by the periplasmic domain of its TpsB partner [13]. This molecular interaction is then fol- lowed by the initiation of TpsA translocation through a hydrophilic channel formed by the transporter [14]. As secretion proceeds, the exoprotein folds progres- sively at the cell surface into a long b-helix. The TPS domain itself adopts a right-handed b-helical structure with short extrahelical segments [15–17]. Two subtypes of TPS system have been identified, which differ by the sequences of the TPS domains of the TpsA proteins and by those of their TpsB transporters [16,18]. Never- theless, the structure of the TPS domain is highly con- served between the two subtypes [15–17], indicating that the TPS pathway is dedicated to the secretion of b-solenoid proteins [19]. Our model TPS system is the filamentous haemag- glutinin adhesin (FHA) ⁄ FhaC pair of the whooping cough agent Bordetella pertussis. FHA is a major adhesin of this respiratory pathogen, and FhaC is its specific TpsB transporter [20]. The structure of FhaC has been solved by X-ray crystallography [21]. FhaC is monomeric and comprises a 16-stranded b-barrel (height, 35 A ˚ ) joined by short periplasmic turns and longer surface loops, called L1–L8. The N-terminus of the protein is located in the extracellular milieu and folds into a 20-residue-long a-helix, H1, that passes right through the transmembrane barrel. The C-termi- nus of H1 emerges into the periplasm and is connected to two tandem polypeptide transport-associated (PO- TRA) domains [22] via a 30-residue-long linker unde- fined in the crystal structure. The extracellular loop L6 that joins b-strands 11 and 12 of the barrel is folded as a hairpin in the barrel interior, with its tip reaching the periplasm. The barrel of FhaC forms an ion-per- meable channel in lipid bilayers, and we have proposed that this pore represents the FHA-conducting channel [14,23]. However, because it is almost totally occluded in the structure, significant conformational changes must take place in the transport process. Other pro- teins of the superfamily have also been shown to form ion-permeable channels in lipid bilayers [9,24–29]. However, the role of the pore for the mechanism of integration of membrane proteins remains unknown. Contrary to H1, which can be removed without sig- nificant loss of function, deletions of POTRA1, POTRA2 or L6 abolish FhaC activity [14,21]. Interest- ingly, the L6 loop of FhaC harbours a conserved motif found in most members of the superfamily [3,4]. In FhaC, the highly conserved V449RGY452 tetrad is located at the tip of L6 close to the periplasmic side of the barrel. In this study, we demonstrate that the replacement of Arg by Ala in this conserved motif dra- matically affects the secretion activity of FhaC, but not the properties of the FhaC channel or its structure. In contrast, replacement of the conserved Tyr affects the pore properties of FhaC, but not its secretion activity, indicating a more subtle role for this residue. This work thus provides the first identification of a functionally important motif for the molecular mecha- nism of TpsB transporters. Because the VRGY ⁄ F motif highlighted here is conserved in the TpsB ⁄ Omp85 superfamily, it is also likely to be func- tionally relevant for other members of the superfamily. Results Conservation of L6 sequence in the superfamily The VRGY motif is located at the tip of L6 of FhaC, which reaches the periplasm. Alignments of the Importance of conserved motif in transporter FhaC A S. Delattre et al. 4756 FEBS Journal 277 (2010) 4755–4765 ª 2010 The Authors Journal compilation ª 2010 FEBS predicted L6 sequences of a number of TpsB trans- porters show that the VRGY ⁄ F tetrad is conserved in both TpsB subtypes, with Arg totally invariant and only slight variations at the first, third and fourth posi- tions of the motif ([18] and Fig. 1). In the rest of L6, a few other residues are conserved between the two TpsB subtypes, which otherwise appear to have distinct sig- natures. To enlarge our analysis, the sequence of L6 was also aligned with the corresponding regions of rep- resentative members of the superfamily, including E. coli YaeT (BamA), Neisseria meningitidis Omp85, Arabidopsis thaliana Toc75-III and Oep80, Saccharo- myces cerevisiae Sam50, and TeOmp85 of the cyano- bacterium Thermosynechococcus elongatus [30] (Fig. 1). In all proteins, a VRGY-related motif is found in a segment predicted to form a loop between two strands of the b-barrel and located close to, and at a conserved distance from, the C-terminus. Of note, the length of the predicted loops varies between proteins [18]. In all proteins, the Arg residue of the VRGY-related motif is invariant (Fig. 1). The other three residues of the motif are not strictly invariant, but their physicochemical features are well conserved. Additional similarities are conspicuous in the same region (Fig. 1). Furthermore, the analysis of the sequences of a large number of pre- dicted Toc75, Sam50 and Omp85 homologues indi- cated the presence of a closely related tetrad in the vast majority of these proteins (not shown). In most proteins, the first position of this motif harbours a hydrophobic residue (Val, Ile or Leu), although other residues occur occasionally. Arg and Gly residues are found overwhelmingly at the second and third posi- tions, exceptionally replaced by Lys or Ser and by Ala or Ser, respectively. The fourth position of the tetrad is occupied by Phe or Tyr in the vast majority of pro- teins or, occasionally, by other hydrophobic residues or His. This region is the best conserved in the super- family, which strongly suggests that it is important for the structure or function of these transporters. Importance of conserved motif for FhaC function The complete deletion of L6 in FhaC has been shown to abolish the secretion of FHA, indicating the impor- tance of this loop for transport activity [21]. However, the channel properties of FhaC are also strongly Fig. 1. Sequence alignments of representative proteins of the TpsB ⁄ Omp85 superfamily. The sequence around the conserved VRGY ⁄ F tet- rad is shown. The first line shows the FhaC sequence, with the first six and last three residues belonging to b-strands 11 and 12 of the b-barrel, respectively. Note that the loop corresponding to L6 is predicted to be longer in some other proteins of the superfamily [18]. Only proteins that have been characterized to some degree were selected for the alignment, excluding predicted proteins. The first 12 proteins belong to the TpsB family, with the first seven belonging to subtype I TpsB and the last five to subtype II TpsB transporters. The other six proteins belong to other groups of the superfamily. The more conserved motifs are highlighted. At, Arabidopsis thaliana; Bp, Bordetella per- tussis; Ech, Erwinia chrysanthemi; Ec, Escherichia coli; Et, Edwardsiella tarda; Hd, Haemophilus ducreyi; Hi, Haemophilus influenzae; Nm, Neisseria meningitidis; Pf, Pseudomonas fluorescens; Pm, Proteus mirabilis; Sc, Saccharomyces cerevisiae; Sm, Serratia marcescens; Te, Thermosynechococcus elongatus; Ye, Yersinia enterocolitica. A S. Delattre et al. Importance of conserved motif in transporter FhaC FEBS Journal 277 (2010) 4755–4765 ª 2010 The Authors Journal compilation ª 2010 FEBS 4757 affected by the 34-residue-long deletion, which suggests that it might perturb significantly the structure of the protein. In order to probe more finely the function of the conserved motif of L6, the VRGY tetrad was tar- geted by site-directed mutagenesis to alter its physico- chemical properties. Because of the poor resolution of L6 in the FhaC structure, no information is available regarding putative interactions between the side chains of these residues and the rest of the protein. We thus chose to replace Arg and Tyr based on their strong conservation in the superfamily, whereas Gly was not targeted because it might be involved in the structure of the loop. Arg450 and Tyr452 were replaced by Ala separately or together, thus creating FhaC R450A , FhaC Y452A and FhaC R450A+Y452A . The three FhaC variants were co-expressed with a gene encoding a secretion-competent, N-terminal FHA derivative called Fha44 in E. coli [31], and the secretion of Fha44 in culture supernatants was determined by semiquantita- tive immunoblotting using anti-FHA IgG1’s (Fig. 2). In parallel, the localization and abundance of FhaC in the outer membrane were analysed by immunoblotting of membrane extracts with an anti-FhaC serum (Fig. 2). The R450A substitution and the double sub- stitution both reduced FHA secretion by approxi- mately 90% relative to the wild-type (WT) control (FhaC WT ), whereas FhaC was present in similar amounts in all strains. In contrast, the Y452A replace- ment appeared to have no significant effect on FHA secretion. Thus, the invariant Arg residue, but not the conserved aromatic residue, is essential for FhaC activity. Effect of the substitutions on FHA recognition In the TPS pathway, the first step of secretion is a spe- cific recognition between the two partners in the peri- plasm. This is followed by the translocation of the TpsA partner through the channel formed by its TpsB transporter. We have shown that the POTRA-contain- ing periplasmic portion of FhaC binds FHA in vitro [13,21]. Because the tip of the L6 loop reaches the peri- plasm, it is conceivable that it also participates in the initial interaction with the substrate. To determine whether the interaction between FHA and FhaC is affected by the introduced substitutions, the FhaC variants were tested for their ability to rec- ognize an immobilized FHA fragment harbouring the TPS domain [13,21]. Using this overlay assay, FhaC R450A and FhaC Y452A bound to the FHA frag- ment quite efficiently (Fig. 3). To obtain a semiquanti- tative assessment of their binding, we performed densitometry scanning of the FhaC bands from several overlays. Using the WT band as a reference (100%), relative binding values of 84 ± 6% and 105 ± 25% were observed for FhaC R450A and FhaC Y452A , respec- tively. This indicates that the tip of the L6 loop does not appear to play a significant role for the initial recruitment of FHA in the periplasm. Structure of FhaC R450A In order to test whether R450 is essential for the struc- tural integrity of FhaC, e.g. for the position of func- tionally important elements such as the POTRA domains or L6, the structure of FhaC R450A was solved by X-ray crystallography. FhaC R450A crystallized in the same conditions as its FhaC WT counterpart [21]. Data collection and refinement statistics are given in A B Fig. 2. Role of R450 of the VRGY tetrad for FhaC activity. (A) Secretion activity of the FhaC variants. Escherichia coli UT5600 har- bouring two plasmids, pFJD12 that encodes an efficiently secreted FHA derivative called Fha44 and pFcc3 (encoding FhaC), pFcc3- R450A , pFcc3- Y452A or pFcc3- R450A+Y452A , was grown to mid- exponential phase, and expression of the recombinant fha44 gene was induced for 3 h. Equal amounts of total membranes and non- concentrated culture supernatants from all recombinant strains were collected and analysed by immunoblotting using anti-FHA IgG1’s and an anti-FhaC serum (top and bottom panels, respec- tively). A representative experiment is shown. (B) Quantification of the secretion efficiency. The amounts of protein were quantified by densitometry scanning, and the Fha44 ⁄ FhaC ratio was calculated for each recombinant strain, with the secretion activity of the strain producing WT FhaC set to 100%. The experiments were per- formed several times (> 3) for quantification. Importance of conserved motif in transporter FhaC A S. Delattre et al. 4758 FEBS Journal 277 (2010) 4755–4765 ª 2010 The Authors Journal compilation ª 2010 FEBS Table 1. Although determined at a limited resolution of 3.5 A ˚ , this structure allows us to compare the R450A variant with WT FhaC. Overall, the structure of the FhaC R450A variant is very similar to the FhaC WT structure. This is exemplified by the rmsds for the Ca superimposition of the barrel (0.44 A ˚ ) and the two POTRAs (0.61 A ˚ ). The overall rmsd is 0.53 A ˚ (Fig. 4A). The relative orientation of the two POTRA domains is the same in both structures, as well as the presence and orientation of helix H1 and loop L6 inside the b-barrel. The moderate resolution of both the WT and R450 structures does not allow the fine analysis of differences between them. However, H1 clearly occupies a similar position in both structures (Fig. 4B). With regard to L6, the electron density of the R450A variant allows the unambiguous positioning of residues Q433 to I441 and G463 to T469 inside the b-barrel at positions similar to those observed in the WT structure. Other residues of the L6 loop are not seen in the FhaC R450A structure, as a result of a high mobility of the loop and ⁄ or the limited resolution. Nevertheless, the conformational constraints imposed by residues Q433 to I441 and G463 to T469 on L6 demonstrate that this loop occupies similar positions inside the b-barrel in both FhaC WT and the FhaC R450A variant. Thus, our data strongly argue that the R450A substitution has no significant effect on the structure of the FhaC barrel and the POTRA domains, or on the position of L6 in the channel, and therefore the Arg450 residue is probably conserved for a functional rather than a structural purpose. Channel properties of the FhaC variants Because L6 is not clearly defined in the FhaC R450A structure, we tested whether the low secretion activity of FhaC R450A could be explained by pore alteration using electrophysiological techniques. Indeed, we have observed previously that FhaC variants with low secre- tion activities generally have altered pore properties [14]. The electrophysiological properties of FhaC R450A and FhaC Y452A inserted in lipid bilayers were analysed in comparison with those of FhaC WT . Initial character- ization was performed by inserting a large number (about 100) of FhaC molecules in a membrane submit- ted to slow ramps of voltage. Asymmetric I ⁄ V record- ings, similar to those observed previously with FhaC WT (Fig. 5A, part a), were obtained for both variants (Fig. 5B, part a; Fig. 5C, part a). This asym- metric I ⁄ V profile indicated that both proteins have a preferred sense of insertion into the lipid bilayers, simi- lar to FhaC WT . The two mutants showed a linear I ⁄ V relationship from +120 mV to around )60 mV, indi- cating a voltage-independent ion conductance in this voltage range (Fig. 5B, part a; Fig. 5C, part a). From )60 to )120 mV, the I ⁄ V curve of both variants lost its linearity and the current recorded at the two Table 1. Data collection and refinement statistics. FhaC R450A Data collection Cell parameters (A ˚ ) 107.55, 139.39, 113.08 Space group C222 1 Wavelength (A ˚ ) 0.93340 Resolution (A ˚ ) a 48.6–3.5 (3.6–3.5) Completeness (%) 99.7 (100) Redundancy 14.5 (14.9) I ⁄ rI 30.0 (6.1) Rmrgd-F (%) 6.4 (28.2) Beamline ESRF ID14-1 Refinement R work (%) b 33.0 R free (%) 37.3 Rmsd Bond lengths (A ˚ ) 0.013 Bond angles (deg) 2.3 a Number in parentheses is the statistic for the highest resolution shell. b R factor = R||F o | ) |F c || ⁄ R|F o |, where |F o | and |F c | are the observed and calculated structure factor amplitudes, respectively. A B Fig. 3. Overlay assay showing FHA–FhaC interactions. The FHA derivative Fha30, immobilized on separate nitrocellulose strips, was used as bait for the indicated FhaC myc variants. Following incuba- tion of the strips with each of the FhaC variants, the Fha30–FhaC complexes were detected with an anti-c-myc IgG1 followed by ECL reaction. The strips were aligned and the ECL reaction was carried out simultaneously for all strips, using a single autoradiographic film. A representative experiment is shown. (A) Amounts of Fha30 used as bait as analysed in a duplicate electrophoresis gel. The gel was stained with Coomassie blue. (B) FhaC myc detected after the overlay. A S. Delattre et al. Importance of conserved motif in transporter FhaC FEBS Journal 277 (2010) 4755–4765 ª 2010 The Authors Journal compilation ª 2010 FEBS 4759 voltage sweep directions revealed an hysteresis. Thus, the curves of the current measured (I) as a function of the voltage applied (V) do not superpose when V increases or decreases, indicating a delay in the con- ductance in response to voltage changes. This hystere- sis was similarly observed with FhaC WT (Fig. 5A, part a). However, the two variants displayed different behaviour in response to voltage, as revealed by a vari- ation of the hysteresis shape, suggesting that they may have different channel characteristics (Fig. 5D, part a). The electrical properties of the two FhaC variants were further examined in single-channel experiments by measuring their conductance values. FhaC R450A showed discrete current transitions at positive and neg- ative applied potentials (Fig. 5B, part b). The calcu- lated conductance value from the two major peaks of the current amplitude histogram was 1240 ± 130 pS at positive voltage (Fig. 5D, part b), very similar to that of FhaC WT [14]. Previous single-channel analyses of FhaC WT have revealed different behaviours at posi- tive and negative potentials, with noisier current and the appearance of conductance substates at negative polarity (Fig. 5A, part b). Moreover, WT channels at negative potential usually display different opening and closing kinetics than the channels recorded at positive voltage (Fig. 5A, part b). FhaC R450A shares these characteristics as, at negative potential, its cur- rent recordings showed more rapid oscillations between the open and closed states of the channel, and substates giving rise to smaller conductance values (1010 ± 160 pS). Interestingly, at both polarities, Fha- C R450A channels displayed opening and closing kinetics faster than those of WT. In contrast with FhaC R450A , FhaC Y452A exhibited a majority of very noisy channels at both polarities (Fig. 5C, part b). Moreover, the current amplitude his- togram at positive voltages displayed a broader distri- bution of events compared with FhaC R450A , clearly indicating the presence of many conductance substates and impairing the determination of a precise value of conductance (Fig. 5C, part c). At negative potentials, the channels were as noisy as those obtained at posi- tive voltages and had a tendency to also display reduced current amplitudes. Altogether, the substitution of Arg450 by Ala does not have a drastic effect on channel activity and thus External environment AB Outer membrane Periplasm POTRA 2 POTRA 1 L6 H1 VRGY Fig. 4. Superposition of the crystal structure of FhaC R450A determined at 3.5 A ˚ resolution (red) and the structure of FhaC WT (blue). (A) The proteins are oriented with their surface side at the top. POTRA1 and 2, H1 and L6 are labelled. All structural elements, including the barrel, the POTRA domains and H1 superpose neatly. (B) The VRGY motif is positioned on the WT structure. Because of the relatively low resolu- tion for FhaC R450A , the position of L6 inside the barrel cannot be traced until the tip of the loop. However, the surface-proximal portions of L6, which are defined by residues Q433 to I441 and G463 to T469, superpose well between the two proteins, arguing against a major con- formational change of the loop. Structural data are available in the Protein Data Bank under the accession numbers 2QDZ (FhaC WT ) and 3NJT (FhaC R450A ). Importance of conserved motif in transporter FhaC A S. Delattre et al. 4760 FEBS Journal 277 (2010) 4755–4765 ª 2010 The Authors Journal compilation ª 2010 FEBS does not appear to affect the FhaC pore. In contrast, the ion channel properties of FhaC Y452A are markedly altered, indicating that the pore is modified by the sub- stitution, possibly because interactions between L6 and the b-barrel are affected. Discussion The structure of FhaC, the first and thus far the only full-length structure available for the TpsB ⁄ Omp85 superfamily, has provided useful insights into the architecture of TpsB and related transporters [21]. The mechanistic principles that govern TPS remain to be elucidated, however. In this work, we provide the first identification of a motif functionally important for TPS transport. Using a combination of sequence align- ments and structure–function analyses of FhaC vari- ants obtained by site-directed mutagenesis, we show that a conserved motif at the tip of the long extracellu- lar loop L6 is required for the activity of the trans- porter. Its strong conservation in TpsB proteins strongly indicates a conserved role for this motif in TPS systems. Altogether, our data show that Arg450 is essentially important for function. Thus, although the FhaC R450A variant is strongly disabled with respect to secretion activity, its global structure is not affected. The elec- trophysiological properties of its channels are similar to those of FhaC WT . In contrast, the complete deletion of L6 both abolished the activity of FhaC and affected its channel properties, arguing that it significantly perturbed the structure of the protein [21]. The FhaC R450A channels are, nevertheless, slightly less stable than those of FhaC WT , suggesting that the substitution may have caused minor structural changes to the channel. FhaC R450A appears to recognize its substrate as efficiently as FhaC WT , ruling out a role for this conserved Arg in the initial TpsA–TpsB inter- action. We thus propose that Arg450 contributes to a later step in the secretion process related to the trans- location of the substrate. The crucial position of L6 strongly argues that it is probably involved in the conformational changes expected to take place on translocation. Understanding its molecular function in the translocation process will most probably involve the characterization of secretion intermediates. A (a) (b) (c) (a) (b) (c) (a) (b) (a) (b) (c) B C D Fig. 5. Electrophysiological properties of FhaC variants. (A) Characterization of the channels formed by FhaC WT (figure repro- duced from ref. [14]). (B, C) Characterization of the channels formed by FhaC R450A and FhaC Y452A , respectively. In (A–C), the I ⁄ V curves between )100 and +100 mV are shown in (a); the direction of the voltage ramp is marked by the arrow near each curve. The single-channel recordings at positive and negative applied voltages are shown in (b) (BL, baseline). In (c), the associated amplitude histograms at +60 mV are shown. (D) Comparison of the electro- physiological behaviour of FhaC R450A (grey) and FhaC Y452A (black) with superposition of the I ⁄ V curves in (a) and of the amplitude histograms in (b). The perturbed behaviour of FhaC Y452A can be seen most clearly from the absence of defined conductance levels. A S. Delattre et al. Importance of conserved motif in transporter FhaC FEBS Journal 277 (2010) 4755–4765 ª 2010 The Authors Journal compilation ª 2010 FEBS 4761 Interestingly, we showed that the replacement of Tyr452 in the same conserved L6 motif modifies the pore properties of FhaC. The FhaC Y452A channel has no defined conductance levels, indicating that it is less stable. A similar behaviour has been observed previ- ously with other variants harbouring peptide insertions in L6 [14]. Of note, in the latter cases, the secretion activity of FhaC was abolished, whereas the single Tyr452 to Ala substitution did not affect the secretion activity in a significant manner. Thus, Tyr452 most probably participates in the positioning of L6 or the regulation of L6 mobility in the channel in the absence of substrate, but its substitution by Ala does not prevent L6 from adopting its ‘active’ conforma- tion during translocation. Therefore, a possible func- tion for Tyr452 may be to stabilize L6 in the channel when the protein is in the ‘resting’ conformation, i.e. not actively translocating FHA. The aromatic charac- ter of this residue is well conserved in the family, which suggests a conserved role for this residue in the channel. Together with L6, the H1 helix also runs through the channel. Unlike L6, however, H1 is not conserved in the TpsB family, with at least one TpsB transporter being devoid of a helix before the POTRAs [18]. We have also shown that it is not important for the secre- tion activity of FhaC [14]. One possible function of H1 may be to plug the resting channel between two cycles of secretion. We have obtained indications that one FhaC molecule secretes several molecules of FHA (F. Jacob-Dubuisson, unpublished data). If FhaC cycles between several conformations, we have most probably captured its ‘resting’ conformation by crystal- lography, whereas structural rearrangements of the channel are expected when FhaC is in action. If H1 moves out of the pore in vivo, this may trigger a repo- sitioning of L6 for the secretion mechanism. Further work will aim to test this hypothesis. So far, our studies have identified two major players in the TPS pathway: the TPS domain of the TpsA pro- tein and L6 of the TpsB transporter [13,21] (this work). A strong argument that these pieces of the TPS machinery function together comes from sequence alignments. Thus, it is striking that the two subtypes of TPS system can be distinguished by specific signa- tures in both elements [16,18] (this work). Similarly, the sequences of the two POTRA domains differ between the two subtypes of TpsB transporter [18]. The POTRAs are also essential for FhaC activity [13,21], and therefore they also constitute essential parts of the TPS machinery. All members of the super- family share similar structural features, namely a C-terminal b-barrel preceded by 1–7 POTRA domains [30]. In addition, the VRGY ⁄ F sequence motif is par- ticularly well conserved, and its position relative to the C-terminus of the barrel appears to be similar in all proteins. In all cases, it is predicted to be part of an extracellular loop between two b-strands of the barrel. If, as suggested by its conservation, the VGRY⁄ F tetrad is also functionally relevant in other members of the TpsB ⁄ Omp85 superfamily, it implies that the barrel actively participates in the mechanism of these trans- porters. Given the size of the substrates handled by Omp85 transporters, however, these mechanisms remain to be understood. Similar to that proposed for FhaC, it is possible that the loop harbouring the con- served motif is involved in critical conformational changes in these other transporters. Materials and methods Plasmids and constructions PCRII-TOPO12TM was constructed as follows: the fhaC fragment encoding residues Pro275 to Phe454 was amplified by PCR using pFcc3 [32] as template and the oligonucleo- tides Fc12UP (5¢-TTAGATCTCCGCTGGGGCGTACGC G-3¢) and FcA2Lo (5¢-CCAAGCTTCCGGGCTCAGAA ACTGAGG-3¢) as primers. The amplicon was inserted into pCRII-TOPO Ò (Invitrogen, Cergy-Pontoise, France) and sequenced, yielding pCRII-TOPO12TM. The point muta- tions were introduced using the QuikChange II XL Site- Directed Mutagenesis Kit (Agilent Technologies, La Jolla, CA, USA), according to the manufacturer’s instructions. PCRII-TOPO12TM was used as a template with the following primers : R450AUp (5¢-GACGAGTACACGGT GGCCGGATACAACCTCAGGA-3¢) and R450ALo (5¢-TC CTGAGGTTGTATCCG GCCACCGTGT ACTCGTC-3¢); Y452AUp (5¢-ACACGGTGCGCGGAGCCAACCTCAAG ACGTC-3¢) and Y452ALo (5¢-GACGTCCTGAGGTTGG CTCCGGCCACCGTGT-3¢); RA+YAUp (5¢-ACACGGT GGCCGGAGCCAACCTCAGGACGTC-3¢) and RA+YA Lo (5¢-GACGTCCTGAGGTTGGCTCCGGCCACCGTG T-3¢). After mutagenesis and sequence verification, the BsiWI-HindIII fragments of these vectors were exchanged for the WT fragment of pFcc3, yielding pFcc3-R 450 A, pFcc3- Y 452 A and pFcc3-R 450 A+Y 452 A. The BsiWI-HindIII frag- ments were similarly exchanged into pFJD118 [14], yielding pFJD118-R 450 A and pFJD118-Y 452 A, which were used to produce 6-His-tagged FhaC variants for electrophysiology analyses [14]. pFJD118 encodes full-length FhaC with an N- terminal 6-His tag. For crystallography, pT7FcA 450 -noHis was used. It was generated by restricting pFJD118-R 450 A with BamHI and re-ligating to eliminate the 6-His tag coding sequence. pFJD140 [13] was used for the production of c-myc- tagged FhaC WT for the overlay assay. The PstI-HindIII frag- Importance of conserved motif in transporter FhaC A S. Delattre et al. 4762 FEBS Journal 277 (2010) 4755–4765 ª 2010 The Authors Journal compilation ª 2010 FEBS ments of pFcc3-R 450 A and pFcc3-Y 452 A were introduced into the same sites of pFJD140, replacing the WT fragment, generating pFJD140-R 450 A and pFJD140-Y 452 A. Secretion assay E. coli UT5600 harbouring pFJD12 (encoding an 80-kDa N-terminal portion of FHA called Fha44 which can be effi- ciently secreted in E. coli [31]) was transformed with pFcc3, pFcc3-R 450 A, pFcc3-Y 452 A or pFcc3-R 450 A+Y 452 A. The cells were grown at 37 °C in liquid Luria–Bertani medium until the cultures reached the late exponential phase (A 600 = 0.8), and the expression of fha44 was induced with 1mm isopropyl thio-b-d-galactoside for 3 h at 37 °C. Bacte- ria were then harvested by centrifugation at 10 000 g for 15 min at 4 °C. The culture supernatants were separated by SDS ⁄ PAGE and analysed by western blot with a mixture of anti-FHA monoclonal IgG1’s (F1, F4 and F5) [33]. The cell pellets were resuspended in 50 mm Tris ⁄ HCl (pH 8.0), 150 mm NaCl, 10 lgÆmL )1 DNAse I (Sigma, Lyon, France) and a cocktail of protease inhibitors (Complete EDTA Free, Roche Ò , Rosny-sous-Bois, France), and the bacteria were broken using a French pressure cell. After clarification of the lysates by centrifugation at 20 000 g for 20 min, mem- brane proteins were harvested by ultracentrifugation at 100 000 g for 1 h at 13 °C. Each pellet was resuspended in 200 lL of the same buffer as above. Equal amounts of sam- ples were separated by SDS ⁄ PAGE and analysed by western blot with an anti-FhaC serum. This antibody was raised in rats against the periplasmic domain of FhaC and prepared by Eurogentec (Seraing, Belgium). The amounts of Fha44 and FhaC were quantified by densitometry scanning of the immunoblots, followed by data analysis with imagequant tl software (GE HealthCare, Saclay, France). Protein production and purification The FhaC variants were produced and purified as described previously [21]. Overlay assay Fha30 is a 30-kDa, secreted FHA truncate encompassing the TPS domain and first three repeats. It was used as bait and FhaC myc and its variants as prey. Fha30 was produced and purified as described previously [15]. Identical amounts of Fha30 (5 lg) were loaded onto several lanes of SDS ⁄ PAGE gels and, following electrophoresis, the protein was blotted onto nitrocellulose. The strips of nitrocellulose were each incubated with purified FhaC myc (WT or mutant), and bound FhaC myc was detected using an anti- c-myc IgG1 and chemiluminescence as in ref. [13]. The strips were aligned and the development reaction [enhanced chemiluminescence (ECL); GE Healthcare] was carried out simultaneously for all strips using a single autoradiography film, for comparison purposes. The assay was repeated three times. Crystallization, data collection, structure determination and refinement FhaC R450A crystals were obtained at 20 °C using the hang- ing drop vapour diffusion method. The protein and precipi- tant solutions were mixed in a 1 : 1 ratio. Crystals were grown at a protein concentration of 26 mgÆmL )1 in 28% poly(ethylene glycol) 1000, 1% b-octyl-glucoside and 500 mm imidazole (pH 6.5). The FhaC R450A crystals were similar, with regard to space group and asymmetric unit composition, to the native crystals reported previously [21]. The diffraction data were collected at 100 K on beamline ID14-1 at the European Synchrotron Radiation Facility (Grenoble, France). The diffraction data were processed with xds [34]. Data collection and refinement statistics are summarized in Table 1. The three-dimensional structure of FhaC R450A was solved using the FhaC WT structure (PDB code: 2QDZ) as the starting model. Rigid-body refinements were performed with cns [35,36] using H1, L6, POTRA1, POTRA2 and the b-barrel as independent bodies. The final refinement steps were performed using the maximum likelihood algorithm, and grouped B-factor calculation was performed with cns. The refinement led to R work of 33.0% and R free of 37.3% using all data to 3.5 A ˚ . The final model does not comprise the first two residues, the loop between H1 and POTRA1 (residues 31–52) and the extracellular loops L1 (221–228), L3 (295–301), L4 (342–350), L5 (384–397) and L8 (532– 542), which are not visible in the electron density map. In the FhaC WT structure, the tip of L6 (residues 443–458) was not well defined in the electron density map and was there- fore built as a polyalanine chain. In the FhaC R450A struc- ture, the residues 441–463 of L6 were not visible in the electron density map and are not included in the final model. Structural data are available in the Protein Data Bank database under the accession number 3NJT. Channel analysis The planar lipid bilayer recordings were performed as described in ref. [14]. Virtually solvent-free planar lipid bilayers were formed over a 125–200-lm hole in a 10-lm- thick polytetrafluoroethylene film pretreated with a mixture of 1 : 40 (v ⁄ v) hexadecane–hexane and sandwiched between two half glass cells. Phosphatidylcholine from soy beans (azolectin from Sigma type IV S), dissolved in hexane (0.5%), was spread on the top of the electrolyte solution [1 m KCl, 10 mm Hepes (pH 7.4)] on both sides of the bilayer chamber. Bilayer formation was achieved by lower- ing and then raising the levels in one compartment and monitoring capacity responses. The trans chamber was con- nected to ground and the cis chamber to the input of a A S. Delattre et al. Importance of conserved motif in transporter FhaC FEBS Journal 277 (2010) 4755–4765 ª 2010 The Authors Journal compilation ª 2010 FEBS 4763 BLM 120 amplifier (Bio-Logic, Halifax, Canada). The puri- fied FhaC proteins were added to the cis side (5–100 ngÆmL )1 ) of the bilayer chamber. For the macroscopic conductance experiments, doped membranes were subjected to slow voltage ramps (10 mVÆs )1 ), and the transmembrane currents were ampli- fied (BBA-01; Eastern Scientific, Rockville, MD, USA). The current–voltage curves were stored on a computer and analysed using scope software (PowerLab, ADI Instru- ments, Sydney, Australia). For single-channel recordings, currents were amplified by a BLM 120 amplifier. 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Structural data are available in the Protein Data Bank database under the accession