Isolated CyaA-RTX subdomain from Bordetella pertussis: Structural and functional implications for its interaction with target erythrocyte membranes

Biochemical and Biophysical Research Communications 466 (2015) 76e81

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Isolated CyaA-RTX subdomain from Bordetella pertussis: Structural and functional implications for its interaction with target erythrocyte membranes Riyaz Ahmad Pandit a, Kanungsuk Meetum b, Kittipong Suvarnapunya a, Gerd Katzenmeier b, Wanpen Chaicumpa c, Chanan Angsuthanasombat b, d, * a

Graduate Program in Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand Bacterial Protein Toxin Research Cluster, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand Department of Parasitology and Center of Excellence on Therapeutic Proteins and Antibody Engineering, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand d Laboratory of Molecular Biophysics and Structural Biochemistry, Biophysics Institute for Research and Development (BIRD), Bangkok 10160, Thailand b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 13 August 2015 Accepted 24 August 2015 Available online 29 August 2015

The 126-kDa Bordetella pertussis CyaA-hemolysin (CyaA-Hly) was previously expressed in Escherichia coli as a soluble precursor that can be acylated to retain hemolytic activity. Here, we investigated structural and functional characteristics of a ~100-kDa isolated RTX (Repeat-in-ToXin) subdomain (CyaA-RTX) of CyaA-Hly. Initially, we succeeded in producing a large amount with high purity of the His-tagged CyaARTX fragment and in establishing the interaction of acylated CyaA-Hly with sheep red blood cell (sRBC) membranes by immuno-localization. Following pre-incubation of sRBCs with non-acylated CyaA-Hly or with the CyaA-RTX fragment that itself produces no hemolytic activity, there was a dramatic decrease in CyaA-Hly-induced hemolysis. When CyaA-RTX was pre-incubated with anti-CyaA-RTX antisera, the capability of CyaA-RTX to neutralize the hemolytic activity of CyaA-Hly was greatly decreased. A homology-based model of the 100-kDa CyaA-RTX subdomain revealed a loop structure in Linker II sharing sequence similarity to human WW domains. Sequence alignment of Linker II with the human WW-domain family revealed highly conserved aromatic residues important for proteineprotein interactions. Altogether, our present study demonstrates that the recombinant CyaA-RTX subdomain retains its functionality with respect to binding to target erythrocyte membranes and the WW-homologous region in Linker II conceivably serves as a functional segment required for receptor-binding activity. © 2015 Elsevier Inc. All rights reserved.

Keywords: Bordetella pertussis CyaA-hemolysin CyaA-RTX Erythrocyte-membrane interaction WW domain family

1. Introduction Adenylate cyclase toxin (CyaA) is one of the major virulence factors of Bordetella pertussis, a Gram-negative pathogen that causes whooping cough in humans [1]. The toxin is synthesized as a single polypeptide of 1706 residues and consists of an N-terminal adenylate cyclase (AC) domain of 400 residues and a pore-forming/ hemolysin (Hly) domain of 1306 residues [2]. The CyaA-Hly domain contains of a hydrophobic pore-forming segment (residues 500e800) [3,4] and an acylation region (residues 800e1000) [5].

* Corresponding author. Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand. E-mail address: [email protected] (C. Angsuthanasombat). http://dx.doi.org/10.1016/j.bbrc.2015.08.110 0006-291X/© 2015 Elsevier Inc. All rights reserved.

There is also an RTX (Repeat-in-ToXin) segment, harboring ~40 repeats of Gly-Asp-rich nonapeptides that serve as Ca2þ-binding sites (residues 1006e1600) [6], along with an unprocessed C-terminal secretion signal (residue 1600e1706) [7]. The CyaA toxin is stabilized by extracellular Ca2þ ions which act as a structural stabilizing bridge in a b-roll motif within the RTX region [8,9]. For biological activity, this toxin requires a palmitoyl group be added at Lys983 by CyaC acyltransferase [10]. The RTX subdomain of CyaA is reported to harbor a target receptor-binding region that is required for cell targeting and for AC-domain translocation across the host cell membrane [11]. The AC-catalytic domain would generate cAMP in an uncontrolled manner, which in turn would disrupt transcription of many inflammatory-associated genes in apoptotic pathways leading to the target cell death [12]. CyaA preferentially binds to target cells

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through the aMb2-integrin (CD11b/CD18) receptor expressed on the surface of cells in the myeloid lineage, e.g. macrophages [13]. It is noteworthy that the CyaA toxin also exerts its hemolytic activity against sheep erythrocytes, even though they lack the aMb2-intergrin receptor, suggesting an alternative mechanism of target cell binding [14]. Moreover, the 126-kDa truncated CyaA-Hly fragment retains hemolytic activity independent of the N-terminal AC domain [15]. The requirement for Ca2þ binding to the RTX subdomain for its structural stabilization has been clearly established [8,9]. Furthermore, other studies have provided some insights into a plausible receptor-binding segment in the C-terminal domain of CyaA [16]. However, the identity of CyaA-receptor binding residues crucial for target cell recognition remains ambiguous. In the present study, structural and functional characteristics of a ~100-kDa RTX subdomain (CyaA-RTX) were investigated and the results suggested that the CyaA-RTX subdomain can be highly produced as an isolated soluble form in Escherichia coli and that the purified product retains its intrinsic capability to functionally interact with erythrocyte membranes. Moreover, a 3D-modeled structure of the CyaARTX subdomain revealed conserved aromatic residues in the Linker II loop homologous to the human WW domain family that is important for proteineprotein interactions, suggestive of its significance for CyaA-RTX functional activity.

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HP, GE Healthcare Bio-sciences) and run at a flow rate of 1 mL/min. The washing step was performed via elution with 75 mM imidazole (IMZ) in 50 mM HEPES buffer (pH 7.4) containing 2 mM CaCl2. The target His-tagged protein was then eluted with 250 mM IMZ. Elution fractions containing the His-tagged toxin were pooled, analyzed for homogeneity by SDSePAGE and desalted through a desalting column prior to additional analysis. Concentrations of the purified His-tagged protein were determined using the Bradfordbased microassay.

2.4. Western blot analysis Toxin samples separated on SDS-PAGE were transferred to a nitrocellulose membrane blocked with 5% skim milk-PBS (120 mM NaCl, 16 mM Na2HPO4, 4 mM NaH2PO4, pH 7.4) and probed with rabbit anti-RTX polyclonal antisera (1:40,000 dilution) which was raised against the 100-kDa purified CyaA-RTX fragment as described previously [10]. Toxin-antibody complexes were detected with alkaline phosphatase (AP)-conjugated goat anti-rabbit IgG antibodies (Pierce; 1:7000 dilution) and visualized by incubation with BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium). The presence of 6  His tag was verified by probing with AP-conjugated anti-His C-term antibodies (Invitrogen; 1:2000 dilution) and BCIP/NBT detection.

2. Material and methods 2.1. Construction of recombinant plasmid with His-tagged fusion

2.5. Immuno-localization assay

Incorporation of a 6  His tag in the previously constructed pCyaA-RTX plasmid [9] was performed by using the pCyaAC-PF/H6 plasmid [17] as a template. pCyaA-RTX were digested with NdeI and SacI, yielding the CyaA-RTX-coding fragment (1211 bp) which was gel purified and ligated with pCyaAC-PF/H6 (6358 bp) pre-cut with NdeI and SacI. CyaC-coding gene from pCyaAC-PF/H6 was deactivated by using Not1 which deleted a 103-bp 30 -end segment (encoding 34 amino acids) of the cyaC gene. The resulting pCyaARTX/H6 plasmid with a 6  His-tag was transformed into E. coli strain JM109 and verified by restriction digestion. The plasmid was re-transformed into the expression host, a protease-deficient E. coli strain BL21(DE3)pLysS and the target gene segment was verified by DNA sequencing.

Immuno-localization was performed to determine the binding of CyaA-Hly to erythrocyte membranes. A 30-ml suspension of sRBCs (5  108 cells/mL) were diluted in 80 mL of PBS buffer (pH 7.4) containing 50 mM glycine, 5 mM glucose and 75 mM sucrose, followed by addition of CyaA-Hly toxin (~10 mg). The treated cells were pelleted, washed (3 times), re-suspended in 200 mL of PBSGly/sugar buffer (pH 7.4) and incubated with anti-RTX polyclonal antisera (1:200 dilution) at 37  C for 30 min. Cell complexes were collected by centrifugation, washed 3 times and followed by another 30-min incubation with FITC-conjugated anti-rabbit IgG antibodies (SigmaeAldrich; 1:50 dilution). After washing, the stained cells were collected by centrifugation, deposited on the glass slide, mounted with a cover slip, and then examined on a confocal laser scanning microscope (Olympus FV1000) under an oil-immersion lens (60).

2.2. Expression of His-tagged proteins All His-tagged recombinant toxins were expressed in E. coli strain BL21(DE3)pLysS cells at 30  C in Luria-Bertani medium supplemented with ampicillin (100 mg/mL) and chloramphenicol (34 mg/mL). Protein expression was induced with isopropyl-b-Dthiogalactopyranoside (IPTG) at final concentration of 0.1 mM, and E. coli cells were harvested by centrifugation, re-suspended in 20 mM HEPES buffer (pH 7.4) containing 2 mM CaCl2 and 1 mM protease inhibitors (phenylmethylsulfonylfluoride, PMSF, and 1,10phenanthroline, PNT). After addition of lysozyme (0.1 mg/mL final concentration), cell suspension was incubated on ice overnight and subsequently disrupted by sonication. The cell lysate was centrifuged at 10,000  g, 4  C for 45 min. Concentrations of soluble proteins in the supernatant were determined by Bradford-based protein microassay. 2.3. Purification of His-tagged proteins 6  His-tagged toxins were purified via immobilized metal affinity chromatography (IMAC). 15-mL lysate supernatant (5 mg/mL) was loaded onto an affinity-based Ni2þ-NTA column (5 mL-HisTrap

2.6. Hemolytic activity assay In vitro hemolytic activity of the protein toxin against sRBCs was assayed in 1.5-mL microcentrifuge tube as previously described [15], with some modifications. 200 mL of purified toxins (~10 mg) were gently mixed with sRBCs (5  108 cells) in 800 mL of assay buffer (120 mM TriseHCl, pH 7.4, 50 mM NaCl2 and 2 mM CaCl2) and incubated at 37  C for 6 h. At the end of the incubation, the mixture was centrifuged at 12,000  g for 2 min to remove nonlysed sRBCs and cell debris, and the supernatant was transferred to a flat-bottom 96-well microtiter plate for measuring the released hemoglobin by spectrophotometer at OD540. The same amount of total proteins in soluble cell lysate containing pET-17b was used as a negative control while an equal amount of erythrocytes lysed with 0.1% Triton-X 100 was defined as 100% hemolysis. Percent hemolysis for each toxin sample was calculated by {[OD540 sample  OD540 negative control]/[OD540 of 100% hemolysis  OD540 negative control]}  100. All samples were tested in triplicate for three independent experiments.

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2.7. Hemolysis-inhibition assay The assay was performed in 1.5-mL microcentrifuge tube by preincubating 30 ml of sRBCs (5  108 cells/mL) with either CyaA-RTX or NA/CyaA-Hly (10, 20 and 30 mg) suspended in 80 mL of assay buffer (120 mM TriseHCl, pH 7.4, 50 mM NaCl2 and 2 mM CaCl2) at 37  C for 1 h. CyaA-Hly toxin (~10 mg) was added to each mixture suspension which was then adjusted to 1-mL final volume prior to additional incubation at 37  C for 5 h. The extent of hemolysis inhibition exerted by CyaA-RTX or NA/CyaA-Hly was calculated as described above for the hemolysis assay. 2.8. In vitro toxin neutralization assay The ability of anti-RTX antisera to interfere with binding of CyaA-RTX to erythrocyte membranes was assessed by preincubating CyaA-RTX (~10 mg) with different concentrations of anti-RTX polyclonal antisera at 37  C for 2 h. Following addition of 30 mL of sRBCs (5  108 cells/mL) suspended in assay buffer (120 mM TriseHCl, pH 7.4, 50 mM NaCl2 and 2 mM CaCl2), the mixture was further incubated at 37  C for 2 h. The treated cells were collected and washed 3 times with the assay buffer before incubating with CyaA-Hly (~10 mg) for another 5 h. A parallel assay was carried out for neutralization of CyaA-Hly toxin activity. The toxin (~10 mg) was pre-incubated with varied concentrations of anti-RTX polyclonal antisera at 37  C for 2 h prior to further incubation with 30 mL of sRBC (5  108 cells/mL) for 5 h. For both neutralization assays, the non-lysed cells were centrifuged (12,000  g for 2 min) and the supernatant containing the released hemoglobin was measured by spectrophotometer at OD540. 2.9. Homology-based modeling For homology-based modeling, the CyaA-RTX sequence (residues 1001e1660) was submitted to the Raptor web (http://raptorx. uchicago.edu), a template-based protein structure modeling server. To each model, Ca2þ ions were incorporated by 3D fitting the template molecule of Pseudomonas sp. MIS38 lipase (PDB ID: 2Z8X). To give the best inter-residue contact as well as Ca2þ ion contact, the rotamers of some residues were manually adjusted. Finally, an overall structure of CyaA-RTX with Ca2þ ions was applied to energy minimization using REFMAC5 (CCP4: Supported Program; http:// www.ccp4.ac.uk/html/refmac5.html). Geometries of all-atom contact and structure validation of the final model were performed using the MolProbity program (http://molprobity.biochem.duke. edu). Stereo-chemical quality and Ramachandran plot analyses of the CyaA-RTX modeled structure were performed using MolProbity.

Fig. 1. Expression, purification and Western blotting of CyaA-RTX/H6. Left panel: SDSPAGE analysis (Coomassie brilliant blue-stained 10% gel) of crude extracts from E. coli cells expressing the 100-kDa CyaA-RTX/H6 subdomain protein (lane 1), IPTG-induced E. coli insoluble fraction (lane 2) and its soluble fraction (lane 3). Lane 4 is the IMAC-purified CyaA-RTX/H6 protein. Lane M denotes the molecular mass standards. Right panel: Western blot analysis of the CyaA-RTX/H6 soluble fraction probing with anti-RTX (lane 1) or anti-His antibodies (lane 2). An arrow indicates the band corresponding to the 100-kDa CyaA-RTX/H6 protein.

purification of the His-tagged target protein from crude lysates via IMAC (see data below). Following IPTG-induced expression driven via the T7 promoter, the CyaA-RTX/H6 fragment was vastly produced as its 100-kDa soluble form (Fig. 1, left panel). Analysis by Western blotting confirmed that CyaA-RTX/H6 was recognized by both anti-His and anti-RTX antisera (Fig. 1, right panel), verifying the presence of a His-affinity tag as well as its RTX identity. This result also indicated that addition of a 6  His tag to CyaA-RTX did not affect recognition by its specific antibodies. Nonetheless, it should be noted that the

3. Results and discussion 3.1. Optimization of His-tagged CyaA-RTX expression Previously, although we were able to express a high-yield soluble form of the CyaA-RTX subdomain in E. coli from pCyaARTX under T7 RNA polymerase-driven expression and that its 100-kDa stable product was obtained with high purity via a single-step anion-exchange chromatography, a large quantity of the purified CyaA-RTX fragment could not be achieved [9]. In the present study, we have employed the same strategy that has recently proven effective for producing the CyaA-Hly toxin at large quantity and high purity [17]. We thus re-constructed the recombinant plasmid, pCya-RTX/H6, encoding the CyaA-RTX subdomain fused at the C-terminus with a 6  His tag (Cya-RTX/H6) which was subsequently exploited to enable an efficient one-step

Fig. 2. Hemolytic activities of purified CyaA-Hly/H6, NA-CyaA-Hly/H6 and CyaA-RTX/ H6 proteins tested against sRBCs. The crude cell lysate containing the pET-17b vector alone was used as a negative control. Error bars indicate standard errors of the mean from three independent experiment each performed in triplicate. The toxin showing significant hemolysis is denoted by shading. Inset, SDS-PAGE analysis (Coomassie brilliant blue-stained 10% gel) of the IMAC-purified proteins used in the assay; CyaARTX/H6 (lane 1, ~5 mg), CyaA-Hly/H6 (lane 2, ~2 mg) NA-CyaA-Hly/H6 (lane 3, ~2 mg). Arrows indicate the band corresponding to the 100-kDa CyaA-RTX/H6 protein (red) and 126-kDa of CyaA-Hly/H6 or NA-CyaA-Hly/H6 (blue). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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100-kDa CyaA-RTX/H6 protein appeared to migrate on SDSePAGE with a relatively larger size than the 116-kDa b-galactosidase standard marker (Fig. 1, left panel), and the explanation for its anomalous migration behavior in SDS-gels remains ambiguous [9]. Previously, we demonstrated that 5 mM CaCl2 was necessary for the structural stability of the CyaA-RTX molecule [9]. Herein, we initially expressed CyaA-RTX/H6 in 5 mM CaCl2, a concentration that yielded low amount of the soluble protein. When the concentration of CaCl2 was incrementally reduced from 5 mM to 2 mM, however, there was a steady increase in solubility, with maximal recovery of the soluble protein product at 2 mM CaCl2 (see Fig. 1, lane 3). In contrast, when CyaA-RTX/H6 was expressed at 7 mM CaCl2, there was a substantial reduction in protein solubility (data not shown). These data indicate that optimal Ca2þ concentration may not only be involved in structural stability but also plays a significant role in toxin solubility. At CaCl2 concentrations higher than 2 mM there may also be an increase in CyaA-RTX/H6 aggregation and inclusion body formation resulting in lower yields. Protein purification of the CyaA-RTX/H6 subdomain fragment from whole lysate supernatant was herein accomplished via a one-

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step affinity-based Ni2þ-NTA column chromatography. As analyzed by SDS-PAGE and Western blotting, a high-yield protein band with >95% purity of the 100-kDa CyaA-RTX/H6 fragment bound to NiNTA was successfully recovered by a stepwise elution with 250 mM IMZ (Fig. 1, lane 4). It is worth mentioning that 2 mM CaCl2 as well as protease inhibitors (i.e., PMSF and PNT) are necessary for preparation of CyaA-RTX/H6. These optimized conditions facilitated us to obtain sufficient amounts of the purified CyaA-RTX/H6 protein fragment (~20e60 mg/L of culture) for further functional characterization. 3.2. Isolated CyaA-RTX fragment retains the capacity to bind erythrocyte membranes To determine if, and to what extent, CyaA-RTX alone has hemolytic activity, the purified CyaA-RTX/H6 protein (10 mg) was incubated with sRBCs and its hemolytic activity was compared with that of 10 mg CyaA-Hly/H6 and 10 mg NA/CyaA-Hly/H6, respectively (Fig. 2, inset). Since it is well established that acetylation of CyaA and/or its hemolysin domain (CyaA-Hly) at Lys983 is essential for

Fig. 3. (A) Dose-dependent inhibition of CyaA-Hly/H6 mediated hemolysis by NA-CyaA-Hly/H6 or CyaA-RTX. sRBCs were pre-incubated with NA-CyaA-Hly or CyaA-RTX at the indicated ratios for 2 h at 37  C. CyaA-Hly/H6 (~10 mg) was then added in the reaction and incubated for 5 h at 37  C. The extent of inhibition was calculated by percent of hemolysis induced by 0.1% Triton-X. Error bars indicate standard errors of the mean from three independent experiments each performed in triplicate. The level of significance is denoted by shading (p values  0.05), where black shading represents the most significant effect. (B) Focal immuno-localization of CyaA-Hly/H6 on sRBC. Interaction of CyaA-Hly/H6 with membrane-bound sRBC molecules was directly visualized by using secondary antibodies conjugated with FITC against primary anti-RTX antisera. Nomarski interference contrast view of CyaA-Hly/H6 treated-sRBC (Top). Overlay of the contrast view superimposed with fluorescence signal (Middle). Fluorescence image (Bottom). Scale bar is 10 mm. (C) In vitro neutralization assay of CyaA-Hly/H6 with anti-RTX antisera. CyaA-Hly/H6 (-) or CyaA-RTX/H6 (:) was pre-incubated with the indicated concentrations of anti-RTX antisera for 2 h at 37  C. Pre-incubated CyaA-Hly was incubated with sRBCs for 5 h at 37  C. Anti-RTX antiserum-treated CyaA-RTX was re-incubated with sRBCs for 2 h at 37  C. sRBCs were washed and incubated with CyaA-Hly for 5 h at 37  C. Results are expressed in percent hemolysis of three independent experiments. Inset, SDS-PAGE analysis (Coomassie brilliant bluestained 10% gel) of anti-RTX antiserum proteins (~20 mg). An arrow denotes the denatured and reduced rabbit IgG heavy chain of ~50 kDa.

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toxin binding to the integrin aMb2 receptor [18,19], binding and hemolytic activity of the acylated CyaA-Hly/H6 toxin were incorporated as controls. As expected, neither CyaA-RTX/H6 nor NA/ CyaA-Hly/H6 showed hemolytic activity (5 ± 2% and 6 ± 3% of complete hemolysis, respectively) compared with CyaA-Hly (~70 ± 5% of complete hemolysis) (see Fig. 2). Further findings via hemolysis-inhibition assays revealed that when sRBCs were pre-incubated with either CyaA-RTX/H6 or NA/ CyaA-Hly/H6, there was a slight reduction in subsequent CyaA-Hly/ H6-induced hemolysis (62 ± 5% and 60 ± 4%, respectively) at 1:1 concentration with CyaA-Hly/H6 (Fig. 3A). Moreover, when sRBCs were pre-incubated with CyaA-RTX/H6 or NA/CyaA-Hly/H6 at 2and 3-fold increases in concentrations, interestingly, CyaA-Hlyhemolysis was markedly reduced (Fig. 3A). Taken together, these data suggest that the CyaA-RTX subdomain maintains its nativefolded conformation and its ability to block CyaA-Hly binding to its target molecule on the erythrocyte membrane, thereby inhibiting CyaA-Hly-mediated hemolytic activity. Despite its inhibitory capacity, however, no plausible binding site for CyaA-Hly on the erythrocyte membrane has yet been identified. We have now performed immuno-colocalization assays that validate the CyaA-Hly binding on sRBCs and show that binding appears as focal associations (Fig. 3B). The observation that pre-incubation of sRBCs with CyaA-RTX/H6 can inhibit the hemolytic activity of CyaA-Hly led us to ask whether

anti-RTX antisera can neutralize and thereby interfere with CyaARTX binding and CyaA-Hly hemolytic activity. When tested, the anti-RTX antisera effectively inhibited CyaA-Hly hemolytic activity and also inhibited the interaction between CyaA-RTX and sRBCs in a dose dependent manner (Fig. 3C), suggesting that anti-RTX antisera blocks the capability CyaA-RTX to bind sRBC membranes and interferes with CyaA-Hly hemolytic activity. Although epitope data for the anti-RTX antisera used in this study are not yet available, recent studies have demonstrated that the CyaA-RTX subdomain retains at least three epitopes capable of eliciting neutralizing antibodies [20]. 3.3. CyaA-RTX homology-based model supportive of receptor binding To date, the lack of crystal structure for the CyaA-Hly toxin or the CyaA-RTX fragment has hampered the experimental characterization of the CyaA-RTX-binding region. To gain critical insights into the architecture of the CyaA-RTX subdomain fragment, attempts were made to build a plausible 3D-modeled structure of the 100kDa CyaA-RTX fragment. The Ramachandran plot of the CyaA-RTX model revealed that over 93.3% of the residues stay in their original most favored sites with additional allowed positional flexible. This observation indicates that the modeled structure remains in sterically favorable main-chain conformations.

Fig. 4. (A) 3D-modeled structure of the 100-kDa CyaA-RTX subdomain, showing five blocks (Blocks IeV as colored in dark blue, cyan, yellow, orange and purple, respectively) of the nonapeptide repeats using its unique b-roll structure to bind to Ca2þ ions (violet balls). Linker regions are colored in green. Inset, the zoomed region of a unique loop structure of Linker II illustrating the three highly conserved aromatic side-chains (i.e. Trp1239, Tyr1250 and Tyr1251) which are depicted as van der Waals spheres. The structure was generated by using PyMOL program. (B) Multiple amino acid sequence alignments of Linker II-loop sequence of CyaA-RTX with the representative sequences of human WW domains. Acronyms of each WW sequence are shown on the left side of the alignment. Highly conserved amino acid residues across the alignment are bolded with yellow background. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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As can be inferred from Fig. 4A, the overall 3D-modeled structure of CyaA-RTX from Gly1001 to Tyr1660 displays an organization of five structurally similar blocks (Block I 1015e1088, Block II 1138e1211, Block III 1247e1353, Block IV 1377e1485 and Block V 1529e1591) arranged in a roughly linear fashion in which Block III is centrally located. Moreover, there are at least five potential Ca2þ-binding sites in each of these modeled blocks joined by linker sequences (Linkers IeIV) which are relatively diverse. Of particular interest, Linker II joining Blocks II and III contains a loop sequence with ~40% similarity to the sequence of an artificial WW-domain loop structure (PDB 1YZM) (see Fig. 4B, blue-lined box). In an effort to understand the functional importance of the Linker II-loop structure, its amino acid sequence was also aligned with those of representative family members of the human WW domains, a small protein module that mediates specific proteineprotein interactions and that harbors highly conserved aromatic residues [21,22] (Fig. 4B). The resulting alignment revealed that Trp1239, Gly1247, Tyr1250 and Tyr1251 are the most conserved residues in Linker II (Fig. 4B, yellow shaded). It is worth noting that Ile1261 at CyaA-RTX Linker II is replaced by Trp, a more versatile residue, at the same aligned position in all of the human WW domains [23]. Recently, a Y65C mutation in the WW domain of PQBP1 (polyGln tract-binding protein 1) renders it incapable of binding with its ligand WBP11 (WW domain-binding protein 11) [24], thus signifying the importance of the invariant Tyr65 in proteineprotein interactions. Tyr65 of PQBP1 corresponds to Tyr1251 of the CyaA-RTX Linker II. The invariant conservation of the three aromatic residues within all of the WW domains and the Linker II domain, i.e. Trp1239, Tyr1250 and Tyr1251 (denoted in Fig 4A as van der Waals spheres), together with the loss of binding capacity due to the Y65C mutation, supports the contention that these three conserved residues in Linker II could play an important role in its interaction with a receptor counterpart. This notion is supported by a previous study which suggests that a segment of CyaA encompassing the region between residues 1166e1287 is a major CD11b-binding motif [18]. This proposition is further strengthened by the finding that the CyaA-neutralizing 6E1 monoclonal antibody recognizes a region between residues 1156 and 1319 for its binding and neutralizing activity. In that study, incubation of the 6E1-monoclonal antibody with the 177-kDa CyaA full-length toxin produced partial inhibition of CyaA toxin-induced hemolysis without affecting cytotoxicity, suggesting that this region is important for interaction with sRBC membrane [25]. Acknowledgment We are highly grateful to Prof. Peter J. Stambrook, University of Cincinnati, for his critical comments. This work was supported in part by grants from Mahidol University (MU 49/2557) and the Thailand Research Fund (IRG-57-8-0009). A Royal Golden Jubilee Ph.D. Scholarship (to K.M.) is gratefully acknowledged. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2015.08.110. References [1] J.A. Melvin, E.V. Scheller, J.F. Miller, P.A. Cotter, Bordetella pertussis pathogenesis: current and future challenges, Nat. Rev. Microbiol. 12 (2014) 274e288.

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