For Peer Review

Parasitology Research

FhCaBP4: a Fasciola hepatica calcium binding protein with EF-hand and dynein light chain domains

Journal:

Draft

r Fo

Manuscript ID:


Manuscript Type: Date Submitted by the Author:

Complete List of Authors:

Original Paper n/a

er

Pe

Orr, Rebecca; Queen's University, Belfast, School of Biological Sciences Kinkead, Ruth; Queen's University, Belfast, School of Biological Sciences Newman, Richard; Queen's University, Belfast, School of Biological Sciences Anderson, Lindsay; Queen's University, Belfast, School of Biological Sciences Hoey, Elizabeth; Queen's University, Belfast, School of Biological Sciences Trudgett, Alan; Queen's University, Belfast, School of Biological Sciences Timson, David; Queen's University, Belfast, School of Biological Sciences EF-hand, calcium binding protein, liver fluke, DLC, conformational change, calmodulin antagonist, calcium dependent dimerisation, ANS binding

vi

Re

Keywords:

Note: The following files were submitted by the author for peer review, but cannot be converted to PDF. You must view these files (e.g. movies) online.

ew

FhCABP4 with Ca mini.pdb

Editorial Office @ Heinrich-Heine University, Zoomorphology, Cellbiology and Parasitology, 40225 Düsseldorf, Germany

Page 1 of 28

FhCaBP4: a Fasciola hepatica calcium binding protein with EFhand and dynein light chain domains

Rebecca Orr, Ruth Kinkead, Richard Newman, Lindsay Anderson, Elizabeth M Hoey, Alan Trudgett and David J Timson*

School of Biological Sciences, Queen's University Belfast, Medical Biology Centre,

r Fo

97 Lisburn Road, Belfast, BT9 7BL. UK.

* Corresponding author

Pe

Telephone:

+44(0)28 9097 5875

Fax:

+44(0)28 9097 5877

Email:

[email protected]

er ew

vi

Re

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60




Abstract In trematodes, there is a family of proteins which combine EF-hand containing domains with dynein light chain-like domains. A member of this family from the liver fluke, Fasciola hepatica – FhCaBP4 – has been identified and characterised biochemically. FhCaBP4 has an N-terminal domain containing two imperfect EFhand sequences and a C-terminal dynein light-like domain. Molecular modelling predicted that the two domains are joined by a flexible linker. Native gel electrophoresis demonstrated that FhCaBP4 binds to calcium, manganese, barium and

r Fo

strontium ions, but not to magnesium or zinc ions. The hydrophobic, fluorescent probe 8-anilinonaphthalene-1-sulphonate bound more tightly to FhCaBP4 in the

Pe

presence of calcium ions. This suggests that the protein undergoes a conformational change on ion binding which increases the number of non-polar residues on the

er

surface. FhCaBP4 was protected from limited proteolysis by the calmodulin antagonist W7, but not by trifluoperazine or praziquantel. Protein-protein

Re

crosslinking experiments showed that FhCaBP4 underwent calcium ion dependent dimerisation. Since dynein light chains are commonly dimeric, it is likely that

vi

FhCaBP4 dimerises through this domain. The molecular model reveals that the

ew

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 28

calcium ion binding site is located close to a key sequence in the dynein light chainlike domain, suggesting a plausible mechanism for calcium-dependent dimerisation.

Keywords: EF-hand; calcium binding protein; liver fluke; DLC; conformational change; calmodulin antagonist; calcium dependent dimerisation; ANS binding


Page 3 of 28

Introduction Calcium ions are important second messengers in the signalling processes of almost all organisms (Berridge et al. 2000). In order to transduce calcium signals, cells have specialised proteins which sense calcium ion concentrations. Often these proteins respond to calcium ion binding by conformational changes which alter their ability to interact with other proteins, thus allowing them to transduce the signal. A typical eukaryotic cell has a number of these calcium ion sensors, of which the best characterised is calmodulin. This protein consists of two globular domains, joined by

r Fo

an α-helical linker. Each of the globular domains contains two EF-hand structures, each of which can bind one calcium ion (Babu et al. 1985; Babu et al. 1988;

Pe

Kuboniwa et al. 1995; Zhang et al. 1995). On binding calcium ions, the globular heads change conformation revealing a more hydrophobic surface (Gopalakrishna and

er

Anderson 1985; Zhang and Yuan 1998). In some cases these hydrophobic surfaces mediate binding to other proteins. Hydrophobic interactions are also responsible for

Re

interactions with some calmodulin antagonists; for example, trifluoperazine (TFP) binds to calmodulin through hydrophobic clefts on the surface of the protein (Cook et

vi

al. 1994; Matsushima et al. 2000; Vandonselaar et al. 1994).

ew

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


It is becoming clear that flatworm parasites belonging to the class trematoda express a variety of calcium binding proteins in addition to calmodulin. Some of these calcium binding proteins appear to be unique to this group of organisms. All trematodes are parasites, and many are of major medical and economic importance such as the blood flukes Schistosoma mansoni and S. japonicum and the liver flukes Fasciola hepatica, F. gigantica and Clonorchis sinensis. Thus the investigation of key signalling molecules, particularly those which are unique to the parasite, has value in the



identification and characterisation of potential drug targets. In the case of F. hepatica this is especially important given the emergence of resistance to the current drug of choice, triclabendazole (Alvarez-Sanchez et al. 2006; Brennan et al. 2007; Fairweather 2005; Fairweather 2009; Mitchell et al. 1998; Overend and Bowen 1995; Thomas et al. 2000).

These unusual trematode calcium binding proteins include molecules which are predicted to have similar overall structures to calmodulin, for example the F. hepatica

r Fo

calmodulin-like proteins FhCaM2 and FhCaM3 (Russell et al. 2007; Russell et al. 2012). There are also proteins which, although they contain one or more EF-hands,

Pe

do not conform to the overall structure of calmodulin. For example, there is a family of small (8 kDa) proteins in trematodes which contain only one EF-hand which

er

includes Fh8 from F. heptatica, Sm8 from S. mansoni and SjCa8 from S. japonicum (Abath et al. 2002; Fraga et al. 2010; Hu et al. 2008). There are also proteins which

Re

incorporate EF-hands in combination with other domains, for example those which combine EF-hands with a dynein light chain (DLC) domain. Such proteins have been

vi

identified in S. mansoni (Sm22.6), S. japonicum (Sj22.6), S. haematobium (Sh22.6), F.

ew

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 28

gigantica (FgCaBP1, FgCaBP2 and FgCaBP4), F. hepatica (Fh22), Opisthorchis viverrini (OvCaBP) and C. sinensis (CsTP31.8 and CsTegu21.6) (Fitzsimmons et al. 2004; Huang et al. 2007; Kim et al. 2011; Ruiz de Eguino et al. 1999; Senawong et al. 2012; Stein and David 1986; Subpipattana et al. 2012; Vichasri-Grams et al. 2006; Waine et al. 1994). Several of these proteins have been localised to the tegument (Subpipattana et al. 2012; Vichasri-Grams et al. 2006), the outer surface of trematodes which provides the interface between the worm and its host and which is the route of entry for many anthelmintic drugs (Alvarez et al. 2007). Consistent with this


Page 5 of 28

tegumental localisation, Sj22.6 and Sh22.6 have been shown to initiate an IgEmediated immune response in infected humans (Fitzsimmons et al. 2004; Santiago et al. 1998). In this study, we characterise the biochemical properties of the F. hepatica equivalent of FgCaBP4, which we name FhCaBP4.

r Fo er

Pe ew

vi

Re

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60




Materials and Methods Cloning, expression and purification of FhCaBP4 The coding sequence for FhCaB4 was amplified from a F. hepatica EST library (clone Fh49c10) by PCR using primers which incorporated sequences which enabled cloning into the expression vector pET46 Ek/LIC (Merck, Nottingham, UK). This vector inserts bases coding for the amino acid sequence MAHHHHHHVDDDDK at the 5′ end of the coding sequence. Following cloning into this vector using the manufacturer’s recommended protocol, insertion was verified by restriction digestion,

r Fo

PCR and DNA sequencing of the insert (MWG Biotech, Ebersburg, Germany). The coding sequence has been submitted to the GenBank database with accession number JQ792170.

er

Pe

The plasmid was transformed into competent E. coli HMS174(DE3) and a colony resulting from this transformation was grown, shaking overnight at 37 °C in Luria-

Re

Bertani medium supplemented with 100 µg.ml-1 ampicillin. This culture was then diluted into 1 l of LB (supplemented with 100 µg.ml-1 ampicillin) and grown shaking

vi

at 37 °C until A600nm reached 0.6 to 1.0 (typically 3-4 h). At this time the cultures

ew

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 28

were induced with 1 mM IPTG and grown for a further 2-3 h. Cultures were harvested by centrifugation (4200 g for 15 min at 4 °C), resuspended in approximately 20 ml buffer R (50 mM Hepes-OH, PH 7.5, 150 mM NaCl, 10%(v/v) glycerol) and stored, frozen at -80 °C.

Cell suspensions were thawed and the cells disrupted by sonication on ice (three 100 W, 30 s pulses with 30-60 s gaps in between for cooling). Solid matter was removed by centrifugation (20,000 g for 20 min at 4 °C). The supernatant was applied to a 1


Page 7 of 28

ml nickel-agarose column (His-Select, Sigma, Poole, UK) which had been previously equilibrated in buffer W (buffer R, except 500 mM NaCl) and allowed to pass through under gravity. The column was then washed with buffer W (20 ml) and the protein eluted by the application of three 2 ml aliquots of buffer E (buffer W supplemented with 250 mM imidazole). Fractions containing FhCaBP4 were identified by 10% SDS-PAGE, dialysed overnight against buffer D (buffer R supplemented with 2 mM DTT), divided into 50-100 µl aliquots and stored, frozen at -80 °C. Protein concentrations were determined by the method of Bradford (Bradford 1976), using

r Fo

BSA as a standard.

Bioinformatics

Pe

Sequence alignments were carried out using ClustalW (Thompson et al. 2002) as

er

implemented at the Kyoto University Bioinformatics Centre (http://www.genome.jp/tools/clustalw/) in the “slow, accurate” mode using the gap

Re

opening penalty of 10, a gap extension penalty of 0.05 and the BLOSUM weighting matrix.

ew

Molecular modelling

vi

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The protein sequence of FhCaBP4 was submitted to the Phyre2 server (http://www.sbg.bio.ic.ac.uk/phyre2) for homology modelling in the intensive mode (Kelley and Sternberg 2009). The resulting model was solvated and energy minimised using YASARA (http://www.yasara.org/minimizationserver.htm) (Krieger et al. 2009). A calcium ion was introduced into the structure by reference to a template structure (mouse Reps1 EH domain; PDB ID: 1FI6 (Kim et al. 2001)) and



the model reminimised using YASARA. This final model is provided as supplementary data to this paper.

Native gel electrophoresis Divalent metal ion binding was assessed using native gel electrophoresis under conditions based on previous studies of calmodulin-like proteins (Russell et al. 2007). FhCaBP4 (80 µM) was incubated at room temperature in the presence of EGTA (2 mM) and cations (4 mM) for 25 min. Then an equal volume of native gel loading

r Fo

buffer (120 mM Tris pH 6.8, 20% (v/v) glycerol, 5% (w/v) bromophenol blue, 1% (w/v) DTT) was added. Samples were analysed on 10% (w/v) native polyacrylamide

Pe

gels (pH 8.8) at 20 mA (constant current). Gels were stained with Coomassie blue (dissolved in 45 % (v/v) ethanol, 10 % (v/v) glacial acetic acid) and destained in

er

0.75 %(v/v) glacial acetic acid, 0.5 %(v/v) ethanol.

ANS fluorescence assays

Re

FhCaBP4 (5 µM) was incubated for 1 h at 37 °C with either 1 mM CaCl2 or 1 mM

vi

EGTA in the presence of 20 µM 8-anilinonaphthalene-1-sulphonate (ANS) in a total

ew

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 28

volume of 150 µl in a FluoroNunc™ black 96-well plate. The negative control contained 10 mM HEPES pH 8.8 and 20 µM ANS. The fluorescence spectrum (440 nm to 510 nm; excitation wavelength 350 nm) was recorded using a Spectra Max Gemini XS fluorescence plate-reader and SOFTmax PRO software.

Crosslinking FhCaBP4 (30 µM) was incubated for 1 h at 37 °C with 1 mM EGTA or 1 mM CaCl2. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) was then


Page 9 of 28

added to a final concentration of either 7 µM or 70 µM. The reactions were allowed to proceed for 30 min at 37 °C and were halted by the addition of an equal volume of SDS-PAGE loading buffer (120 mM TrisHCl pH 6.8, 4% (w/v) SDS, 20% (v/v) glycerol, 5% (w/v) bromophenol blue, 1% (w/v) DTT) and heating to 95 °C for 5 min. Products were analysed by 15% SDS-PAGE.

Limited proteolysis FhCaBP4 (40 µM) was incubated for 5 min at 37 °C in the presence of TFP (TFP), N-

r Fo

(6 aminohexyl)-5-chloro-1-naphthalenesulphonamide (W7) or praziquantel (each 250 µM; stocks initially dissolved in DMSO and further diluted in buffer R such that the

Pe

final concentration of DMSO in the reactions was 1% (v/v)). Chymotrypsin was added to a final concentration of 36 nM. Digestion proceeded for 30 min at 37 °C.

er

Products were analysed by 15% SDS-PAGE.

Results

Re

FhCaBP4 is a member of a family of trematode calcium binding proteins which

vi

contain EF-hand and dynein light chain-like domains

ew

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The deduced protein sequence of FhCaBP4 is identical to that of FgCaBP4 (figure 1). This is not surprising since the two species are very closely related. Other than FgCaBP4, the most similar, currently known protein to FhCaBP4 is a tegumental antigen from C. sinensis (accession number: GAA56892). FhCaBP4 has a predicted molecular mass of 22.3 kDa and an estimated pI of 5.0. Sequence analysis predicted that the protein has a domain containing two EF-hand-like sequences at the Nterminus (residues 1-86) and a DLC-like domain at the C-terminus (residues 103-190).



Molecular modelling predicts that these two domains fold essentially independently of each other and are joined by a flexible, partly unstructured linker (figure 2a). Neither of the two EF-hands contains a perfect, consensus sequence for calcium binding (figure 1, 2b,c). Indeed in the highest ranked template to include a bound ion (mouse Reps1 EH domain; PDB ID: 1FI6), contains only one calcium in the structure, corresponding to the second EF-hand in FhCaBP4 (Kim et al. 2001). This calcium ion was used to model the position of the equivalent ion into FhCaBP4 (figure 2a,c). In a typical EF-hand six residues (known as X, Y, Z, -X, -Y and –Z) provide

r Fo

pentagonal bipyramidal coordination of the divalent cation (Gifford et al. 2007). In both EF-hands the seventh amino acid residue (or –Y position) fails to match the

Pe

consensus. In the first EF-hand this residue is isoleucine and in the second it is lysine (figure 2b,c). However, calcium binding appears to be possible at the second EF-hand

er

because the positively charged side chain of the lysine is orientated away from the ion (figure 2c). The DLC-like domain showed some differences to typical DLC structures.

Re

In the top-ranked template (human PIN/LC8; PDB ID: 1CMI) dimerisation is mediated by an inter subunit β-sheet (Liang et al. 1999). In the model of FhCaBP4,

vi

the equivalent region does not adopt any recognisable secondary structure (figure 2a).

FhCaBP4 binds calcium and other divalent cations

ew

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 28

Recombinant FhCaBP4 can be expressed in, and purified from, E. coli (figure 3). Typical yields were 14 mg of FhCaBP4 per litre of bacterial culture. The ability of this recombinant protein to interact with calcium and other divalent ions was assessed by native gel electrophoresis. The addition of the calcium chelating agent EGTA increased the mobility of the protein in native gel electrophoresis (figure 4a). Addition of EGTA and calcium chloride in a 1:2 molar ratio restored the mobility of


Page 11 of 28

FhCaBP4 to that of the untreated protein. This suggests that the protein, as purified from E. coli, was largely in a calcium bound form. Manganese, barium and strontium, but not magnesium or zinc chlorides, also induced the slower migrating form when added in two-fold excess over EGTA (figure 4a).

Calmodulin, and some other EF-hand containing proteins, undergo a conformational change in the presence of calcium ions which exposes hydrophobic residues on the surface of the protein (Gopalakrishna and Anderson 1985; Zhang and Yuan 1998).

r Fo

This increased surface hydrophobicity can be detected through the binding of ANS, which undergoes a marked increase in fluorescence intensity when bound in a

Pe

hydrophobic environment (Bunick et al. 2004; Fraga et al. 2010; LaPorte et al. 1980; Stryer 1965). In the case of FhCaBP4, the addition of calcium chloride causes an

er

increase in the ANS fluorescence emission intensity compared to spectra collected in the presence of EGTA (figure 4b).

Re

FhCaBP4 forms dimers in the presence of calcium ions

vi

Many EF-hand proteins, including calmodulin and the S100 family, can form dimers

ew

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and higher order oligomers (Lafitte et al. 1999; Streicher et al. 2010). Crosslinking of FhCaBP4 with EDC revealed the presence of dimers in the presence of calcium chloride, but not in the presence of EGTA (figure 5a).

FhCaBP4 interacts with the calmodulin antagonist W7 The interaction of FhCaBP4 with two calmodulin antagonists, TFP and W7 was probed by limited proteolysis. The interaction with praziquantel, which has been shown to interact with the structurally related protein, myosin regulatory light chain



(Pelikan-Conchaudron et al. 2011), was also investigated. FhCaBP4 can be degraded by sub-stoichiometric amounts of chymotrypsin (figure 5b). The solvent used to dissolve the drugs (DMSO) does not affect the pattern of proteolysis; neither does TFP or praziquantel. However, the presence of W7 resulted in protection from limited proteolysis (figure 5b).

Discussion FhCaBP4 is a member of an extensive family of trematode proteins which contain EF-

r Fo

hand and DLC domains. Despite imperfection in the consensus sequences of the EFhands, the protein is able to bind at least one calcium ion. The binding site also

Pe

tolerates other divalent cations. However, it is likely that calcium represents the normal, physiological ligand for FhCaBP4 since the other ions are unlikely to be

er

present at significant concentrations in the organism’s cells. Binding to calcium ions has two effects on the protein: it causes increased surface hydrophobicity,

Re

presumably in a similar manner to other EF-hand proteins and it induces homodimerisation of the protein. The structural nature of this dimerisation is

vi

unknown. Both EF-hand proteins and DLCs are known to dimerise (Barbar et al.

ew

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 28

2001; Lafitte et al. 1999). Indeed, the majority of DLC structures are dimers. For FhCaBP4 to undergo calcium-dependent dimerisation, it would either need to dimerise through the EF-hand containing domain or calcium binding to the EF-hands would need to influence the DLC-like domain. The first possibility would require an unusual DLC-like domain which is unable to dimerise; the second would require allosteric communication within the molecule. The molecular model of the protein generated a monomer, in which a key β-strand normally seen in DLCs is in an unstructured conformation. In dimeric DLCs, this sequence forms an inter-subunit β-


Page 13 of 28

sheet across the dimer interface (Liang et al. 1999; Mohan and Hosur 2009). Interestingly, the putative calcium ion binding site is located adjacent to this region (figure 2a). Therefore, it is entirely possible that calcium binding can influence its structure and, thus, dimerisation.

FgCaBP4 has been localised to the tegument, as have a number of other members from this protein family although the absence of an immune response to it in infected animals would suggested that it is neither secreted or exposed on the external surface

r Fo

of the apical plasma membrane (Kim et al. 2011; Subpipattana et al. 2012). We assume that FhCaBP4 has a similar localisation. However, the physiological role of

Pe

this, and other similar proteins, remains unclear. The existence of several family members within a single species suggests that each protein might have specialised

er

roles in calcium sensing and signalling. Thus, identification of their protein binding partners would assist in predicting functions, as would selective knock-down using

Re

RNAi. The latter approach would also determine whether, or not, any of these proteins are essential for the organism’s life cycle and are thus potential drug targets.

vi

That W7 interacts with FhCaBP4, whereas TFP does not, suggests that there are

ew

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


differences in the structure of the binding site, compared to calmodulin and other, related proteins. These differences could be exploited in the design of specific antagonists. In addition, experimental determination of their structures in the calcium-bound and calcium-free forms, would enhance our understanding of calciuminduced conformation changes. Structural work would also form the foundation for structure-based drug design in the event that these proteins are shown to be viable drug targets for the treatment of liver fluke infections.



References Abath FG, Xavier EM, Montenegro SM, Werkhauser RP (2002) Partial molecular characterization of Sm8, a tegumental antigen of Schistosoma mansoni. Mem Inst Oswaldo Cruz 97 Suppl 1:91-93 Alvarez LI, Mottier ML, Lanusse CE (2007) Drug transfer into target helminth parasites. Trends Parasitol 23:97-104 Alvarez-Sanchez MA, Mainar-Jaime RC, Perez-Garcia J, Rojo-Vazquez FA (2006) Resistance of Fasciola hepatica to triclabendazole and albendazole in sheep in Spain.

r Fo

Vet Rec 159:424-425

Babu YS, Bugg CE, Cook WJ (1988) Structure of calmodulin refined at 2.2 Å

Pe

resolution. J Mol Biol 204:191-204

Babu YS, Sack JS, Greenhough TJ, Bugg CE, Means AR, Cook WJ (1985) Three-

er

dimensional structure of calmodulin. Nature 315:37-40 Barbar E, Kleinman B, Imhoff D, Li M, Hays TS, Hare M (2001) Dimerization and

Re

folding of LC8, a highly conserved light chain of cytoplasmic dynein. Biochemistry 40:1596-1605

vi

Berridge MJ, Lipp P, Bootman MD (2000) The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol 1:11-21

ew

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 28

Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254 Brennan GP, Fairweather I, Trudgett A, Hoey E, McCoy, McConville M, Meaney M, Robinson M, McFerran N, Ryan L, Lanusse C, Mottier L, Alvarez L, Solana H, Virkel G, Brophy PM (2007) Understanding triclabendazole resistance. Exp Mol Pathol 82:104-109


Page 15 of 28

Bunick CG, Nelson MR, Mangahas S, Hunter MJ, Sheehan JH, Mizoue LS, Bunick GJ, Chazin WJ (2004) Designing sequence to control protein function in an EF-hand protein. J Am Chem Soc 126:5990-5998 Cook WJ, Walter LJ, Walter MR (1994) Drug binding by calmodulin: crystal structure of a calmodulin-trifluoperazine complex. Biochemistry 33:15259-15265 Fairweather I (2009) Triclabendazole progress report, 2005-2009: an advancement of learning? J Helminthol 83:139-150 Fairweather I (2005) Triclabendazole: new skills to unravel an old(ish) enigma. J

r Fo

Helminthol 79:227-234

Fitzsimmons CM, Stewart TJ, Hoffmann KF, Grogan JL, Yazdanbakhsh M, Dunne

Pe

DW (2004) Human IgE response to the Schistosoma haematobium 22.6 kDa antigen. Parasite Immunol 26:371-376

er

Fraga H, Faria TQ, Pinto F, Almeida A, Brito RM, Damas AM (2010) FH8--a small EF-hand protein from Fasciola hepatica. FEBS J 277:5072-5085

Re

Gifford JL, Walsh MP, Vogel HJ (2007) Structures and metal-ion-binding properties of the Ca2+-binding helix-loop-helix EF-hand motifs. Biochem J 405:199-221

vi

Gopalakrishna R, Anderson WB (1985) The effects of chemical modification of

ew

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


calmodulin on Ca2+-induced exposure of a hydrophobic region. Separation of active and inactive forms of calmodulin. Biochim Biophys Acta 844:265-269 Hu S, Law P, Lv Z, Wu Z, Fung MC (2008) Molecular characterization of a calciumbinding protein SjCa8 from Schistosoma japonicum. Parasitol Res 103:1047-1053 Huang Y, Zhou Z, Hu X, Wei Q, Xu J, Wu Z, Yu X (2007) A novel tegumental protein 31.8 kDa of Clonorchis sinensis: sequence analysis, expression, and immunolocalization. Parasitol Res 102:77-81



Kelley LA, Sternberg MJ (2009) Protein structure prediction on the Web: a case study using the Phyre server. Nat Protoc 4:363-371 Kim S, Cullis DN, Feig LA, Baleja JD (2001) Solution structure of the Reps1 EH domain and characterization of its binding to NPF target sequences. Biochemistry 40:6776-6785 Kim YJ, Yoo WG, Lee MR, Kim DW, Lee WJ, Kang JM, Na BK, Ju JW (2011) Identification and characterization of a novel 21.6-kDa tegumental protein from Clonorchis sinensis. Parasitol Res

r Fo

Krieger E, Joo K, Lee J, Lee J, Raman S, Thompson J, Tyka M, Baker D, Karplus K (2009) Improving physical realism, stereochemistry, and side-chain accuracy in

Pe

homology modeling: Four approaches that performed well in CASP8. Proteins 77 Suppl 9:114-122

er

Kuboniwa H, Tjandra N, Grzesiek S, Ren H, Klee CB, Bax A (1995) Solution structure of calcium-free calmodulin. Nat Struct Biol 2:768-776

Re

Lafitte D, Heck AJ, Hill TJ, Jumel K, Harding SE, Derrick PJ (1999) Evidence of noncovalent dimerization of calmodulin. Eur J Biochem 261:337-344

vi

LaPorte DC, Wierman BM, Storm DR (1980) Calcium-induced exposure of a

ew

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 28

hydrophobic surface on calmodulin. Biochemistry 19:3814-3819

Liang J, Jaffrey SR, Guo W, Snyder SH, Clardy J (1999) Structure of the PIN/LC8 dimer with a bound peptide. Nat Struct Biol 6:735-740 Matsushima N, Hayashi N, Jinbo Y, Izumi Y (2000) Ca2+-bound calmodulin forms a compact globular structure on binding four trifluoperazine molecules in solution. Biochem J 347 Pt 1:211-215 Mitchell GB, Maris L, Bonniwell MA (1998) Triclabendazole-resistant liver fluke in Scottish sheep. Vet Rec 143:399


Page 17 of 28

Mohan PM, Hosur RV (2009) Structure-function-folding relationships and native energy landscape of dynein light chain protein: nuclear magnetic resonance insights. J Biosci 34:465-479 Overend DJ, Bowen FL (1995) Resistance of Fasciola hepatica to triclabendazole. Aust Vet J 72:275-276 Pelikan-Conchaudron A, Le Clainche C, Didry D, Carlier MF (2011) IQGAP1 is a calmodulin-regulated barbed end capper of actin filaments: possible implications in its function in cell migration. J Biol Chem 286:35119-35128

r Fo

Ruiz de Eguino AD, Machin A, Casais R, Castro AM, Boga JA, Martin-Alonso JM, Parra F (1999) Cloning and expression in Escherichia coli of a Fasciola hepatica

Pe

gene encoding a calcium-binding protein. Mol Biochem Parasitol 101:13-21 Russell SL, McFerran NV, Moore CM, Tsang Y, Glass P, Hoey EM, Trudgett A,

er

Timson DJ (2012) A novel calmodulin-like protein from the liver fluke, Fasciola hepatica. Biochmie Under review.

Re

Russell SL, McFerran NV, Hoey EM, Trudgett A, Timson DJ (2007) Characterisation of two calmodulin-like proteins from the liver fluke, Fasciola hepatica. Biol Chem 388:593-599

ew

vi

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Santiago ML, Hafalla JC, Kurtis JD, Aligui GL, Wiest PM, Olveda RM, Olds GR, Dunne DW, Ramirez BL (1998) Identification of the Schistosoma japonicum 22.6kDa antigen as a major target of the human IgE response: similarity of IgE-binding epitopes to allergen peptides. Int Arch Allergy Immunol 117:94-104 Senawong G, Laha T, Loukas A, Brindley PJ, Sripa B (2012) Cloning, expression, and characterization of a novel Opisthorchis viverrini calcium-binding EF-hand protein. Parasitol Int 61:94-100



Stein LD, David JR (1986) Cloning of a developmentally regulated tegument antigen of Schistosoma mansoni. Mol Biochem Parasitol 20:253-264 Streicher WW, Lopez MM, Makhatadze GI (2010) Modulation of quaternary structure of S100 proteins by calcium ions. Biophys Chem 151:181-186 Stryer L (1965) The interaction of a naphthalene dye with apomyoglobin and apohemoglobin. A fluorescent probe of non-polar binding sites. J Mol Biol 13:482495 Subpipattana P, Grams R, Vichasri-Grams S (2012) Analysis of a calcium-binding

r Fo

EF-hand protein family in Fasciola gigantica. Exp Parasitol 130:364-373 Thomas I, Coles GC, Duffus K (2000) Triclabendazole-resistant Fasciola hepatica in

Pe

southwest Wales. Vet Rec 146:200

Thompson JD, Gibson TJ, Higgins DG (2002) Multiple sequence alignment using

er

ClustalW and ClustalX. Curr Protoc Bioinformatics Chapter 2:Unit 2.3 Vandonselaar M, Hickie RA, Quail JW, Delbaere LT (1994) Trifluoperazine-induced

Re

conformational change in Ca2+-calmodulin. Nat Struct Biol 1:795-801 Vichasri-Grams S, Subpipattana P, Sobhon P, Viyanant V, Grams R (2006) An

vi

analysis of the calcium-binding protein 1 of Fasciola gigantica with a comparison to

ew

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 28

its homologs in the phylum Platyhelminthes. Mol Biochem Parasitol 146:10-23 Waine GJ, Becker MM, Scott JC, Kalinna BH, Yang W, McManus DP (1994) Purification of a recombinant Schistosoma japonicum antigen homologous to the 22kDa membrane-associated antigen of S. mansoni, a putative vaccine candidate against schistosomiasis. Gene 142:259-263 Zhang M, Yuan T (1998) Molecular mechanisms of calmodulin's functional versatility. Biochem Cell Biol 76:313-323


Page 19 of 28

Zhang M, Tanaka T, Ikura M (1995) Calcium-induced conformational transition revealed by the solution structure of apo calmodulin. Nat Struct Biol 2:758-767

r Fo er

Pe ew

vi

Re

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60




Figure legends Figure 1: The sequence of FhCaBP4 (a) The protein sequence of FhCaBP4 is shown with the EF-hands marked (. indicates potential ion coordinating residues) and the interdomain linker marked with ~. (b) Bioinformatic analysis (alignment by ClustalW followed by construction of a rooted, unweighted pair group method with arithmetic mean tree with branch length) shows the sequence relationship between FhCaBP4 and similar proteins. Sm22.6, S. mansoni 22.6 kDa calcium binding protein (AAA29922); Sh22.6, S. haematobium 22.6 kDa calcium binding protein (AAW49250); Sj22.6, S.

r Fo

japonicum 22.6 kDa calcium binding protein (AAB52407); Fh22, F. hepatica 22 kDa calcium binding protein (CAA06036); FgCaBP1, F. gigantica calcium binding

Pe

protein 1 (AAZ20312); FgCaBP3, F. gigantica calcium binding protein 3 (AEX92828); FgCaBP4, F. gigantica calcium binding protein 4 (AEX92829);

er

CsTegAnt, C. sinensis tegumental antigen (GAA56892); OvCaBP, O. viverrini calcium binding protein (no accession number currently available); CsTegu21.6, C.

Re

sinensis 21.6 kDa tegumental antigen (AEI69651).

vi

Figure 2: The predicted structure of FhCaBP4. (a) The overall predicted fold is

ew

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 28

shown with the N-terminal, EF-hand containing domain in the upper left and the Cterminal DLC-like domain in the lower right. The calcium ion bound at the second EF-hand is shown as a sphere and the unstructured region which forms an inter subunit β-sheet in dimeric DLCs is indicated with an arrow. (b) The structure of the first EF-hand-like region is shown with the potential ion coordinating residues in stick form. (c) The structure of the second EF-hand-like region is shown with the ion coordinating residues in stick form and the calcium ion as a sphere.


Page 21 of 28

Figure 3: Expression and purification of FhCaBP4. Samples from various stages from the expression and purification process (see Materials and Methods) were taken and analysed by 10% SDS-PAGE. U, extract from uninduced cells; I, extract from cells 2.5 h after induction; S, soluble material following sonication and centrifugation; F, material which flowed through the nickel-agarose column; W, material removed from the column in the wash step; E, material eluted from the column. The molecular masses of the markers (in kDa) are shown to the left of the gel.

r Fo

Figure 4: FhCaBP4 binds divalent cations and undergoes conformational changes in response to calcium (a) FhCaBP4 (80 µM) was resolved on native gels

Pe

(10 %, pH 8.8) in the presence of 2 mM EGTA and, where indicated, 4 mM divalent cations; -, protein not treated with EGTA or ions; E, protein treated with EGTA only.

er

Decreased mobility, compared to the EGTA treated sample, provides evidence for a conformational change induced by ion binding. (b) The fluorescence spectrum of

Re

ANS (20 µM; excitation wavelength 350 nm) was essentially unchanged in the presence of EGTA (1 mM) treated FhCaBP4 (5 µM). However, in the presence of

vi

calcium ions (1 mM), the fluorescence intensity was enhanced, suggesting increased

ew

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


binding of ANS to hydrophobic regions on the surface of the protein.

Figure 5: Biochemical properties of FhCaBP4 (a) Crosslinking of FhCaBP4 (30 µM) with EDC revealed calcium-dependent dimerisation. Products of the crosslinking reactions were resolved on 15% SDS-PAGE. The masses of the markers are indicated (in kDa) to the left of the gel. A band corresponding to a FhCaBP4 dimer was seen in the presence of 1 mM calcium chloride, but not in the presence of 1 mM EGTA. (b) W7, but not TFP or praziquantel, protects FhCaBP4 from limited



proteolysis. FhCaBP4 (40 µM) was subjected to digestion with 36 nM chymotrypsin in the presence of 1 mM calcium chloride and the products of the reaction resolved on 15% SDS-PAGE. C, untreated FhCaBP4; +, FhCaBP4 digested with chymotrypsin; D, FhCaBP4 digested in the presence of 1% (v/v) DMSO; T, FhCaBP4 digested in the presence of 250 µM TFP; W, FhCaBP4 digested in the presence of 250 µM W7; P, FhCaBP4 digested in the presence of 250 µM praziquantel. Of these compounds, only W7 protects the protein from proteolysis.

r Fo er

Pe ew

vi

Re

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 28


Page 23 of 28


1 2 3 4 . . . . . . 5 MGEVALEGSN LEKMIQLFLQ LDRNRDDIVD ENELRQACAE HKLPEEEVSR 50 6 7 . . . . . . ~~~~ ~~~~~~~~~~ 8 WLDMFDADEN GKITLEEFCR ALGLRTAEMR VEKMEREEVR AGRGRPMPED 100 9 ~~ 10 VEVIASTMSQ EKKVEVTEKF KEFLAKTGGK PEDMNLVVKQ LKDYLDERHG 150 11 12 RVWQTLVLTG SYWMKFSHEP FMSLQFKVGP NIVLVWRTPSN 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 Office @ Heinrich-Heine University, Zoomorphology, Cellbiology and Parasitology, 40225 Düsseldorf, Germa itorial 56 57 58

(a)

w ie

ev

rR

ee

rP

Fo

(b)


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Page 24 of 28

(b)

D26

N24

(a)

D22

I28

Fo

E33

rP

D30

ee

rR

C N

(c)

ev

D58

iew N60

D56 E67

K62 T64 Editorial Office @ Heinrich-Heine University, Zoomorphology, Cellbiology and Parasitology, 40225 Düsseldorf, Germany

Page 25 of 28


w ie

ev

rR

ee

rP

Fo

1 2 3 U I S F W E 4 5 116 6 7 66 8 9 10 45 11 35 12 13 14 25 FhCaBP4 15 16 18 17 18 14 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 Office @ Heinrich-Heine University, Zoomorphology, Cellbiology and Parasitology, 40225 Düsseldorf, Germa itorial 56 57 58


Page 26 of 28

1 2 2+ 2+ 2+ 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 2+ 2+ 2+ 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 800 38 39 40 600 41 +Ca2+ 42 43 +EGTA 400 44 Control 45 46 200 47 48 49 0 50 430 440 450 460 470 480 490 500 510 520 51 52 Wavelenghth/nm 53 54 55 Office @ Heinrich-Heine University, Zoomorphology, Cellbiology and Parasitology, 40225 Düsseldorf, Germa itorial 56 57 58

(a)

-

E

E

Mg

Mn

Ba

Zn

Sr

Fluorescence Intensity

(b)

w ie

ev

rR

ee

rP

Fo -

Ca

Page 27 of 28


w ie

ev

rR

ee

rP

Fo

1 2 (a) 3 +Ca2+ EGTA 4 5 0 0 7 70 7 70 [EDC]/μM 6 7 116 8 66 dimer 9 10 45 11 35 12 13 monomer 25 14 15 18 16 14 17 18 19 20 21 22 (b) 23 C + D T W P 24 25 26 27 28 29 FhCaBP4 30 31 Digestion 32 products 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 Office @ Heinrich-Heine University, Zoomorphology, Cellbiology and Parasitology, 40225 Düsseldorf, Germa itorial 56 57 58


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Page 28 of 28

(b)

D26

N24

(a)

D22

I28

Fo

E33

rP

D30

ee

rR

C N

(c)

ev

D58

iew N60

D56 E67

K62 T64 Editorial Office @ Heinrich-Heine University, Zoomorphology, Cellbiology and Parasitology, 40225 Düsseldorf, Germany