FH8 – a small EF-hand protein from Fasciola hepatica Hugo Fraga 1 , Tiago Q. Faria 2 , Filipe Pinto 1 , Agostinho Almeida 3 , Rui M. M. Brito 2,4 and Ana M. Damas 1,5 1 IBMC, Institute for Molecular and Cell Biology, University of Porto, Portugal 2 Center for Neuroscience and Cell Biology, University of Coimbra, Portugal 3 REQUIMTE, Faculdade de Farma ´ cia, Departamento de Quı ´ mica-Fı ´ sica, University of Porto, Portugal 4 Chemistry Department, Faculty of Science and Technology, University of Coimbra, Portugal 5 ICBAS, Instituto de Cie ˆ ncias Biome ´ dicas de Abel Salazar, University of Porto, Portugal Introduction Fasciola hepatica is a trematode parasite that is respon- sible for fascioliasis. Although traditionally regarded as a parasite of livestock, resulting in a large economic loss to the agricultural community, it remains, in sev- eral countries, an important human parasite, and it is estimated that 2.4 million people are infected with liver fluke worldwide [1]. Infection occurs when the larvae adhering to vegetation are ingested and become infec- tive juveniles in the duodenum. Then, infection pro- ceeds with the rapid penetration of the parasite into the intestinal wall and their entry into the peritoneal cavity, where they break through the liver capsule. Keywords calcium binding protein; fasciolasis; FH8; Fasciola hepatica; sensor protein Correspondence A. M. Damas, IBMC, Institute for Molecular and Cell Biology, University of Porto, R. Campo Alegre 823, 4150-180 Porto, Portugal Fax: +351 226099157 Tel: +351 226074900 E-mail: amdamas@ibmc.up.pt (Received 15 July 2010, revised 24 September 2010, accepted 11 October 2010) doi:10.1111/j.1742-4658.2010.07912.x Vaccine and drug development for fasciolasis rely on a thorough under- standing of the mechanisms involved in parasite–host interactions. FH8 is an 8 kDa protein secreted by the parasite Fasciola hepatica in the early stages of infection. Sequence analysis revealed that FH8 has two EF-hand Ca 2+ -binding motifs, and our experimental data show that the protein binds Ca 2+ and that this induces conformational alterations, thus causing it to behave like a sensor protein. Moreover, FH8 displays low affinity for Ca 2+ (K obs =10 4 M )1 ) and is highly stable in its apo and Ca 2+ -loaded states. Homology models were built for FH8 in both states. It has only one globular domain, with two binding sites and appropriate groups in the positions for coordination of the metal ions. However, an unusually high content of positively charged amino acids in one of the binding sites, when compared with the prototypical sensor proteins, potentially affects the protein’s affinity for Ca 2+ . The only Cys present in FH8, conserved in the homologous proteins of other helminth parasites, is located on the surface, allowing the formation of dimers, detected on SDS gels. These findings reflect specificities of FH8, which are most probably related to its roles both in the parasite and in the host. Structured digital abstract l MINT-8041757: F8 (uniprotkb:Q9NIG5) and F8 (uniprotkb:Q9NIG5) bind (MI:0407)by affinity chromatography technology ( MI:0004) l MINT-8041770: FH8 (uniprotkb:Q9NIG5) and FH8 (uniprotkb:Q9NIG5) bind (MI:0408)by cross-linking study ( MI:0030) Abbreviations ANS, 8-anilonaphthalene-1-sulfonate; BS3, suberic acid bis(3-sulfo-N-hydroxysuccinimide) ester; CaBP, calcium-binding protein; CaM, calmodulin; DLS, dynamic light scattering; R H, hydrodynamic radius; RLU, relative luminescence units; TECP, triphenyl phosphine; T m , melting point; TnC, troponin C. 5072 FEBS Journal 277 (2010) 5072–5085 ª 2010 The Authors Journal compilation ª 2010 FEBS After 8–12 weeks within the liver, they move to the bile ducts, where they mature and produce eggs [1]. Recently, there has been an increase in the number of liver fluke infections of livestock in countries with temperate climates, owing to weather changes that support one of the intermediate hosts of the parasite (Galba truncatula), and the emergence of strains that are resistant to benzimidazole compounds, which are widely used for the treatment of fascioliasis [2]. Further contributing to the spread of this disease is its difficult diagnosis, which is often based on the detec- tion of the worm or lesions in liver sections at the slaughterhouse, hampering systematic diagnosis of the disease in farm animals [1]. Excreted–secreted antigens have been shown to be useful in the diagnosis of human fascioliasis. Besides their importance in screening, the secreted proteins are essential for pathogenesis, as they are involved in several physiological processes of the parasite [3–6]. Indeed, F. hepatica secretes a large array of proteins into the host, and transcriptomic and proteomic approaches, during the different life stages of the para- site, have been applied to investigate the importance of these proteins for the parasite–host interaction [7]. In one of those studies, an ORF corresponding to a 69 amino acid protein was isolated from a screen of an F. hepatica cDNA bank [8]. This protein was called FH8, because of its molecular mass of 8 kDa, and was detected in the early stages of infection (1–3 weeks postinfection) [8]. Immunofluorescence studies demon- strated that it was present on the surface of the para- site and in secreted fluids, probably resulting from the shedding of the worm glycocalyx. As FH8 is expressed on the surface of the parasite, during its cercarial stage, it is a good candidate for vaccine and drug development. Three proteins homologous to FH8 were also described in the fluke parasites Schistosoma man- soni (SM8) [9], Clonorchis sinensis (CH8) and Schisto- soma japonicum (SJ8) [10,11]. Besides FH8, two calmodulin (CaM)-like proteins from F. hepatica have been identified [2]. One of them (FhCaM1) is highly similar to mammalian CaM (98% identity), whereas the other (FhCaM2) has only 41% identity. Both of them bind Ca 2+ , and homology models have been obtained [2]. An analysis of FH8 amino acid sequence revealed the presence of two EF-hands, which are helix–loop– helix structural motifs involved in Ca 2+ coordination. The most common EF-hand motif, also called the canonical EF-hand, is present in CaM and troponin C (TnC), and contains a 12 amino acid binding loop that provides most of the oxygens that coordinate Ca 2+ . However, the composition and length of the Ca 2+ -binding loops can vary among EF-hand proteins [12,13]. EF-hand proteins are organized into structural domains, containing two or more EF-hands, which form highly stable helical bundles. The minimum functional unit present in EF-hand calcium-binding proteins (CaBPs) is a domain with two EF-hands, whose stability is maintained by a small antiparallel b-sheet (EF-hand b-scaffold), formed by two stretches of the Ca 2+ -bind- ing loops. The two EF-hands are covalently bonded via the B ⁄ C linker, which connects the exiting helix (B) of the first EF-hand to the first helix (C) of the second EF-hand. Despite the similarities in sequence and three- dimensional structure of EF-hands, it is known that CaBPs perform a diverse range of functions [13,14], and they are normally classified into two groups: Ca 2+ sen- sors, represented by CaM and TnC, and Ca 2+ buffers, such as calbindin D 9k and parvalbumin. The sensor proteins display Ca 2+ -dependent conformational changes, whereas Ca 2+ buffers, which are involved in Ca 2+ signal modulation, undergo minimal structural changes upon Ca 2+ binding. It was reported that when Ca 2+ binds to sensor proteins, it triggers a switch from a closed to an open conformation, owing to the reorien- tation of the four helices of each functional domain, exposing a hydrophobic region that acts as a target- binding surface. The molecular and structural features responsible for the differences between the two classes of CaBP, although studied by several groups, are not completely understood [13–16]. Some researchers refer to the importance of the B ⁄ C linker in distinguishing between the sensor and buffer proteins [13,15]. More- over, it has been reported that CaM and TnC have the shortest B ⁄ C linkers, and also that the N-terminal domain of CaM is less hydrophobic than that of calbin- din D 9k [13,15]. FH8 is one of the smallest CaBPs described until now, and this makes it a very particular case study protein for the as yet unclear structure–function relationships in the EF-hand family of proteins. Here, we report the cloning, expression and initial biochemical and structural characterization of FH8 from F. hepatica. Results FH8 cloning and purification Because of the small size of the protein and its hypo- thetical Ca 2+ -binding properties, the expression and purification of recombinant FH8, with the use of conventional affinity tags, was not appropriate. As an alternative, a construct was prepared with the H. Fraga et al. FH8 from Fasciola hepatica FEBS Journal 277 (2010) 5072–5085 ª 2010 The Authors Journal compilation ª 2010 FEBS 5073 IMPACT system [17]. This system uses the inducible self-cleavage activity of inteins to purify recombinant native proteins by a single affinity step. Although the protein eluted from the chitin column contained high molecular mass impurities, FH8 was purified to homo- geneity with an Amicon 30 kDa molecular filter (Fig. 1A). In addition to the FH8 band, a band around the 15 kDa marker was observed. This band was stronger when the SDS sample buffer contained no reducing agents. As the FH8 sequence has one Cys (Cys36), this was an indication that the 15 kDa band corresponded to a dimer formed with the oxidation of the FH8 single Cys, and a reducing agent was there- fore included in the assays [2 mm triphenyl phosphine (TECP), unless otherwise indicated]. Results from MS analysis led to the identification of a major peak corresponding to the product of the N-terminal hydrolysis of FH8 Met (7532.7 gÆmol )1 ). The cleavage of the N-terminal Met is a common post- translational modification catalyzed by Escherichia coli aminopeptidades when the side chain in the penulti- mate residue is Ala, Cys, Pro, Ser, Thr, and Val. FH8 contains a Pro at position 2, and this process was therefore very likely to occur during the overnight cleavage with dithiothreitol. Because of its position, the removal of this Met will not influence any of the biochemical data. MALDI-TOF MS also allowed the identification of a small peak of the FH8 dimer (15 076 gÆmol )1 ). All of the purification steps were per- formed in the presence of 1 mm EDTA, and the puri- fied FH8 was free of Ca 2+ as confirmed by atomic absorption spectroscopy. Sequence analysis The FH8 sequence was initially compared with those of the two other CaM-like proteins from F. hepatica, FhCaM1 and FhCaM2. Identities were only 19% and 26%, and 22% and 33%, for the N-termini and C-ter- mini of FhCaM1 and FhCaM2, respectively (Fig. 2A). Moreover, sequence similarity revealed that other helminths have CaBPs similar to FH8 (Fig. 2B), namely C. sinensis (CH8, 55% identity), S. mansoni [9] (SM8, 40% identity) and S. japonicum [11] (SJ8, 37% identity). Interestingly, just like FH8, the S. japonicum protein is localized in the parasite surface, and is expressed at the initial stages of infection [9,11]. In order to obtain an indication of the type of CaBP that FH8 might be, several sequence alignments were performed, using CaM and TnC as representatives of the sensor proteins, and calbindin D 9k , as a model for the buffer CaBPs. They contain one (calbindin D 9k )or two (CaM and TnC) globular domains, each of them with two EF-hand Ca 2+ -binding motifs linked by an EF-hand b-scaffold. Figure 2C shows the alignment of FH8 with the N-terminal and C-terminal fragments of CaMs and also with calbindin D 9k . The C-terminal domain of TnC is also shown, because of its close similarity to CaMs and because some amino acids in the FH8 sequence are different from those in CaM but are homologous to TnC residues. The Ca 2+ -binding sites are presented in red. Whereas the CaM and TnC families of proteins are characterized by a binding loop with 12 amino acids, calbindin D 9k has 14 amino acids in the corresponding loop. In most EF-hand proteins, Ca 2+ is coordinated to seven oxygen atoms, arranged in a pentagonal bipyra- mid; six are provided by the protein, and one by a water molecule. Positions X, Y and Z indicate the first three Ca 2+ ligands of the loop, each of them contrib- uting one oxygen; the Glu in the last position of the loop ()Z) contributes two oxygens of its c-carboxyl group, and the central residue of the loop ()Y) binds Ca 2+ with the main chain carbonyl oxygen. In most structures, the seventh ligand is a water molecule, in position )X, provided indirectly by the protein. Next to residue )Y, there is a hydrophobic amino acid, Ile in most CaMs and Val and Leu in FH8, whose main chain forms two hydrogen bonds with the equivalent residue of the paired EF-hand, forming the EF-hand b-scaffold. The structural integrity of the two-EF-hand domain is maintained by this short b-sheet and by hydrophobic contacts between the protein helices. The last three residues of each Ca 2+ -binding loop are helical, and form the first turn of the exiting helix. AB EDTA Ca 2+ Fig. 1. (A) Purification of recombinant FH8 with the IMPACT expression system. Ext, E. coli extract after overnight induction of the 63 kDa FH8–intein tag construct (*); FT, flowthrough of the chi- tin column; Elu, eluted protein after overnight incubation in reducing buffer (dithiothreitol, 50 m M); 30 K, purified FH8 after 30 kDa molecular exclusion. (B) Native gel retardation assay. FH8 displays the characteristic mobility shift observed for EF-hand CaBPs in the presence of Ca 2+ . Runs were performed in the presence of 1 mM EDTA or 5 mM Ca 2+ . FH8 from Fasciola hepatica H. Fraga et al. 5074 FEBS Journal 277 (2010) 5072–5085 ª 2010 The Authors Journal compilation ª 2010 FEBS The results show that FH8 contains two EF-hand motifs, which most probably form a globular domain. The size and amino acid content of the Ca 2+ -binding sites are very similar to those in CaM and TnC, but not to those in calbindin D 9k . The conserved amino acids that coordinate Ca 2+ (positions X, Y, Z and )Z) are preserved between CaM and FH8, with the exception of amino acids at position Y in both loops, Asn17 ⁄ Asp and Asn53 ⁄ Asp (amino acids refer to FH8 ⁄ CaM). Interestingly, these substitutions also occur in TnC and on the first EF- hand of FhCaM2. It is known that the replacement of Glu at position )Z with other amino acids causes a dramatic decrease in Ca 2+ affinity, whereas mutations at other Ca 2+ -coordinating positions do not have such drastic consequences [18,19]. As reports on structural and biochemical data indicate that the mechanisms of Ca 2+ -induced conformational changes in CaM and TnC are similar [20], we foresee that the Asn ⁄ Asp modifications will not impair Ca 2+ binding. In positions )Y are Lys21 and Lys57, which are different from the amino acids in CaM and TnC. However, the coordination from this position is per- formed through the oxygen of the main chain carbonyl group. Although the side chain is not directly involved in Ca 2+ coordination, it is possible that the positively charged side chain will influence Ca 2+ binding through charge repulsion. Finally, we decided to check the hydrophobicity of the FH8 sequence, and compare it with the CaM N-terminal and TnC C-terminal sequences, CaM and TnC being the proteins used for the homology model- ing studies. The N-terminal and C-terminal fragments were the domains of each protein that were most simi- lar to FH8. The hydropathy plots are presented in Fig. 3, and they show small, but noticeable, differences between FH8 and the other two domains. The N-ter- minal region up to amino acid 31 is similar for the three proteins; then there is a region in FH8, involving Asp32 and Asp33, which is clearly negatively charged, whereas CaM and TnC have identical and positive charges. Cys36-Pro37-Leu38 from FH8 is less hydro- philic than the corresponding aligned regions in CaM and TnC; it includes the only Cys present in FH8, A B C Fig. 2. Sequence alignment of FH8 with: (A) the CaM-like proteins FhCaM1 and FhCaM2; (B) the helminth proteins CH8, SH8 and SM8; and (C) the prototypical Ca 2+ sensor protein CaM together with the C-terminal fragments of TnC and Ca 2+ buffer calbindin D 9K . As CaM and TnC have two domains, only the N-terminal or C-terminal fragments that had higher homology with FH8 are presented in the alignment. The sequence numbering is shown at the beginning and at the end of each fragment. The Protein Data Bank codes of the protein structures used as templates for the molecular modeling of FH8 are presented in parentheses. The Ca 2+ -binding sites are colored red, and positions within sites I and II that are involved in chelating Ca 2+ are labeled X, Y, Z, )Y, )X and )Z. The consensus symbols denoting the degree of conservation in each column between FH8 and CaMs and FH8 and calbindin D 9K proteins are colored blue and have the following meaning: ‘*’, identical in all sequences in the alignment; ‘:’, conserved substitutions; ‘.’, semiconserved substitutions. H. Fraga et al. FH8 from Fasciola hepatica FEBS Journal 277 (2010) 5072–5085 ª 2010 The Authors Journal compilation ª 2010 FEBS 5075 which is conserved in similar proteins from fluke para- sites (CH8, SH8, SM8; see Fig. 2B). Clearly, Cys36 is a valid target for mutagenesis in future functional studies. Finally region 47–60, which belongs to the sec- ond EF-hand and corresponds to four amino acids of the first helix plus the Ca 2+ -binding site without the two last residues, is substantially different from that in CaM and remarkably similar to that in TnC. These differences may influence protein stability and ⁄ or Ca 2+ -binding affinity. Ca 2+ -induced conformational alterations FH8 sequence homology revealed the presence of two EF-hand motifs, and therefore the first question to be explored was the protein’s ability to bind Ca 2+ . This was initially tested using the PAGE mobility assay, as it has been reported that, in the presence of Ca 2+ , functional CaBPs display significant mobility shifts in electrophoresis [2]. In fact, we observed retardation in FH8 migration in a 10% native gel with 5 mm Ca 2+ (Fig. 1B). This preliminary result, demonstrating the functionality of FH8 EF-hand motifs, was further substantiated with an equilibrium dialysis experiment with Ca 2+ (5 mm), where a ratio of 2.0 ± 0.3 Ca 2+ per FH8 macromolecule was observed, indicating that both Ca 2+ -binding sites are functional. As FH8 was able to bind Ca 2+ , possible structural modifications associated with Ca 2+ coordination were explored. This question was particularly relevant, as it is associated with the functionality of FH8, as a buffer or sensor protein. To probe for possible conformational modifications, intrinsic amino acid fluorescence was used. FH8 does not contain any Trp or Tyr residues, which are commonly used for this approach, but it has two Phe residues (Phe30 and Phe46). Phe has relatively weak fluorescence, which is negligible in the presence of Trp or Tyr, but it was previously used to monitor Ca 2+ binding [21]. Accordingly, Phe fluorescence was mea- sured in the presence of EDTA and Ca 2+ , and we observed an increase of 30% in the Ca 2+ -loaded state (Fig. 4A), indicating a conformational alteration. Figure 5 shows the molecular model obtained for FH8 by homology modeling. The side chains of Phe30 and Phe46 are represented in the model, and they are located close to binding site I (Phe30) and the EF-hand b-scaffold (Phe46), which are regions that undergo large conformational alterations as a result of Ca 2+ binding (Figs 5 and 6). As can be seen in Fig.6, a hydrophobic patch becomes exposed upon Ca 2+ binding in the case of CaM, and for FH8 the solvent- exposed hydrophobic area is also larger in the Ca 2+ - loaded state. Dynamic light scattering (DLS) is another technique that can be used as a screen for major conformational changes in proteins, and has previously been used by other researchers to monitor CaM conformational alterations upon Ca 2+ binding [22]. We measured the hydrodynamic radius (R H ) of FH8 in the presence and absence of Ca 2+ . Despite the large standard deviation of the results, it is clear that FH8 displays a larger R H Fig. 3. Hydropathy profiles of FH8, the CaM N-terminus and the TnC C-terminus. Fig. 4. (A) Phe fluorescence. Emission spectrum of FH8 Phe in the presence of 20 m M Ca 2+ (circles) and 3 mM EDTA (crosses). (B) R H of FH8 in the presence of 20 mM Ca 2+ (circles) and 1 mM EDTA (crosses) determined by DLS. FH8 shows an increase in R H that is consistent with a more elongated shape in its Ca 2+ -loaded state. FH8 from Fasciola hepatica H. Fraga et al. 5076 FEBS Journal 277 (2010) 5072–5085 ª 2010 The Authors Journal compilation ª 2010 FEBS (2.7 ± 1.1 nm) in the Ca 2+ -loaded state than in the apo state (1.8 ± 0.7 nm; Fig. 4B). Interestingly, these results are in agreement with the data reported for CaM [22], supporting the idea that Ca 2+ coordination results in alterations in FH8 structure. After determining that FH8 undergoes conforma- tional changes upon Ca 2+ binding, we decided to test whether these alterations resulted in an increase in the hydrophobicity of the protein surface, which is a char- acteristic of Ca 2+ sensors. The binding of the hydro- phobic probe 8-anilonaphthalene-1-sulfonate (ANS) to FH8 was monitored by fluorescence spectroscopy. ANS binds noncovalently to hydrophobic segments of proteins, and it was reported that Ca 2+ sensors show strong enhancement of ANS fluorescence upon Ca 2+ binding [15]. Consistent with the previous hypothesis, Ca 2+ was added to FH8, and ANS fluorescence detec- tion resulted in a blue shift and enhancement of ANS fluorescence (Fig. 7A). Moreover, no changes in ANS fluorescence emission were observed in the presence of 200 mm NaCl, which excludes the effect of ionic strength, or 20 mm Mg 2+ , demonstrating the specific- ity of the conformational change for Ca 2+ (Fig. 7B). The failure of Mg 2+ to induce hydrophobic residue exposure may result from the noncoordination of this ion or just its inability to promote a conformational change, as previously reported for CaM [23]. In order to determine whether the increase in hydro- phobic residue exposure in FH8 was a consequence of the formation of dimers or aggregates induced by Ca 2+ , the primary amine cross-linker suberic acid bis(3-sulfo-N-hydroxysuccinimide) ester (BS3) was used A B B A C Site I Site II Site II Site I D D C Fig. 5. The overall modeled structure of FH8 in the open (green) and closed (yellow) conformations, represented by cartoon ribbons. The protein has two EF-hand Ca 2+ -binding sites (represented by site I and site II). The Ca 2+ -binding loops are presented in more detail; the side chains of atoms that participate in Ca 2+ coordination are shown as sticks and labeled; the calcium ions are represented by gray spheres. In site II, FH8 Asp59 ()X position) is also shown. The backbone of the residues forming the EF-hand b-scaffold is shown in a ball-and-stick rep- resentation. The positions of Cys36, Phe30 and Phe46 are highlighted. The four helices are labeled A, B, C and D. The molecular models were obtained by homology modeling, using the SWISS-MODEL and SWISS-PDB VIEWER programs [41]. The figures were prepared with PYMOL (http://www.pymol.org). A a b d c a b d c a b d c a b d c B Fig. 6. Molecular surface representation of CaM and FH8 in the closed and open conformations. The hydrophobic accessible sur- faces, as defined by the side chains of Val, Ile, Leu and Phe, are in green. (A) The N-terminal domain of R. norvegicus CaM in the apo (Protein Data Bank code: 1QX5) and Ca 2+ -loaded (Protein Data Bank code: 3B32) conformations. (B) FH8 modeled structures for both conformations. The location of Cys36 is shown as yellow sticks. The figures were prepared with PYMOL (http://www.pymol.org). H. Fraga et al. FH8 from Fasciola hepatica FEBS Journal 277 (2010) 5072–5085 ª 2010 The Authors Journal compilation ª 2010 FEBS 5077 in the presence of different concentrations of Ca 2+ .As shown in Fig. 7C, it is clear that the FH8 apo state is a monomer, although some residual dimerization was observed for concentrations of Ca 2+ above 1 mm. The conformational alterations induced by Ca 2+ and, in particular, the sensitivity of the ANS assay were used to assess FH8 affinity for this ion, as previ- ously described for other proteins [24,25]. Figure 7D shows the fluorescence of ANS titrated with Ca 2+ in the presence of FH8. The Ca 2+ titration curve is sig- moidal, an indication of cooperative binding, and fit- ting of the experimental data to a simple allosteric model gave a Hill coefficient of 1.6 ± 0.09 and a K obs of 590 ± 20 lm. This result is in agreement with the findings of experiments on several EF-hand CaBPs, where cooperativity in Ca 2+ binding was observed. However, the K obs was unusually high in comparison with the canonical proteins of the sensor family. In order to corroborate this result, equilibrium dialysis with 250 lm Ca 2+ was performed. Consistent with the low affinity suggested by the Ca 2+ titration curve, a ratio of 0.2 Ca 2+ per FH8 was determined, confirm- ing that FH8 has a low affinity for Ca 2+ (data not shown). In fact, EF-hand CaBPs have binding con- stants for Ca 2+ that span a wide range (10 3 –10 9 m )1 ), with no obvious correlation with the type or arrange- ment of the Ca 2+ ligands [14], although it is known that exposure of hydrophobic residues results in a lower affinity for Ca 2+ , and sensor proteins invariably show lower affinities than buffer proteins. Sequence alignments show that the EF-hand coordi- nation loops of FH8 have two (Arg16 and Lys21) and four (Lys52, Lys54, Lys57 and Lys61) positively charged amino acids, representing considerable electro- static force repulsion, particularly for site II, when Ca 2+ approaches the loops. This is probably one of the main reasons for the low affinity observed for FH8. In fact, it has been reported that increasing the negative charge in the loop by replacing some amino acids with Asp increases the Ca 2+ content, even if the residue is not in one of the coordinating positions; in contrast, removal of negatively charged side chains causes a decrease in Ca 2+ affinity [26,27]. Curiously, the other fluke CaBPs (see Fig. 2) do not contain this positively charged stretch of amino acids, and it would be interesting to compare their respective Ca 2+ affini- ties, but no data are currently available. Besides FH8, several other low-affinity EF-hand proteins (K d =10 3 –10 4 m )1 ) have been described [14,28–30], including a-spectrin [31] and multiple AB CD Fig. 7. (A) ANS fluorescence spectroscopy. Changes in ANS fluorescence emission indicate a Ca 2+ -dependent increase in hydrophobic resi- due exposure. ANS emission in the presence of Ca 2+ -loaded FH8 (20 mM Ca 2+ , circles) was 2.8-fold more intense than in its apo state (1 m M EDTA, crosses). The addition of ANS to FH8 did not result in a significant change in fluorescence as compared with ANS only (not shown). (B) The changes in ANS fluorescence are specific for Ca 2+ ; no changes in ANS fluorescence emission were observed with 200 mM NaCl and 20 mM Mg 2+ (C) Chemical cross-linking of FH8. Purified recombinant FH8 was cross-linked using BS3 and the indicated Ca 2+ con- centrations. The lower lane corresponds to FH8 monomer, and the upper lane corresponds to FH8 dimer. (D) Titration of FH8 with Ca 2+ , using ANS fluorescence as reporter. Data were fitted to the Hill equation. FH8 from Fasciola hepatica H. Fraga et al. 5078 FEBS Journal 277 (2010) 5072–5085 ª 2010 The Authors Journal compilation ª 2010 FEBS members of the S100 [28] and CREC families [32]. Like FH8, all of these proteins can be found extra- cellularly or in the endoplasmic reticulum secretory pathways, where the Ca 2+ concentration is high. Secondary structure and thermal stability Information related to protein secondary structure and stability in the presence and absence of Ca 2+ was obtained by CD spectroscopy. The spectra are pre- sented in Fig. 8, and they reveal that FH8 has a signif- icant content of ordered secondary structure. The far-UV CD spectrum of FH8 at 20 °C (Fig. 8A), contains the shapes and amplitudes characteristic of proteins with a high percentage of helical structure. In the presence of 20 mm Ca 2+ , no significant changes in the CD spectrum were observed. This is in contrast to what is seen with the typical sensor proteins, CaM and TnC, which display shifts in the CD spectrum as a result of the reorganization of the helical packing within the protein [33]. However, CD studies on the N-terminal half of TnC also do not demonstrate changes upon the addition of Ca 2+ [34]. The CD spectra for FH8 also show that Ca 2+ bind- ing results in stabilization of the protein structure. Indeed, as shown in Fig. 8B, the thermal stability of the Ca 2+ -loaded FH8 is very high, and FH8 therefore behaves like the Ca 2+ -loaded states of other EF-hand CaBPs, namely CaM, TnC and calbindin D 9K . In all of these proteins, the denaturation temperatures of the Ca 2+ -loaded states are so high that they are not exper- imentally accessible [35,36]. Figure 8b shows that there is no observable loss in FH8 secondary structure up to 98 °C. The FH8 apo state is less stable, and we were able to determine its melting point (T m )as 74.0 ± 0.3 °C (Fig. 8C). Although not comparable to its Ca 2+ -loaded state, apo-FH8 is still a remarkably stable protein. It is known that the two EF-hand domains are sta- bilized by backbone hydrogen bonds connecting the Ca 2+ -binding loops in a short stretch of antiparallel b-sheet, as well as by numerous hydrophobic contacts between the helices. In the Ca 2+ -loaded state, the CaBPs are further stabilized by Ca 2+ ligand interac- tions, and are normally more stable [13,37]. Curiously, apo-FH8 is substantially more stable towards thermal denaturation than CaM or TnC (T m $ 55 °C) [35], although apo-calbindin D 9k does demonstrate even higher T m values (85 °C or higher [36]). Homology models The N-terminal fragment of CaM from Rattus norvegi- cus was used for homology modeling of FH8, as this is the only organism for which the X-ray crystallographic structures for the apo (Protein Data Bank: 1QX5) and Ca 2+ -loaded (Protein Data Bank: 3B32) states are available [38,39]. swiss-model in the alignment mode was used to produce the models for FH8 in the Ca 2+ - loaded and Ca 2+ -free conformations. The 69 amino acids of FH8 were modeled without any insertions or deletions included in the sequence alignment. Analyses of the models were performed by Anolea and Gromos [40], revealing a favorable energy environment for all of the amino acids, with the exception of a few resi- dues belonging to the Ca 2+ -binding sites. The final total energy for the models was approximately A B C Fig. 8. Far-UV spectra of FH8. (A) FH8 at 20 °C in the absence (thin line) and in the presence (thick line) of 20 m M Ca 2+ . (B) FH8 at 98 °C in the absence (thin line) and in the presence (thick line) of 20 m M Ca 2+ . (C) Thermal denaturation of the apo state of FH8 was followed by CD absorbance measurements at 210 nm. Unfolding was shown to be reversible, and T m was calculated on the assump- tion of a two-state model; details are given in Experimental procedures. H. Fraga et al. FH8 from Fasciola hepatica FEBS Journal 277 (2010) 5072–5085 ª 2010 The Authors Journal compilation ª 2010 FEBS 5079 )1750 kJÆmol )1 , according to swiss-model calcula- tions. Both models were checked with swiss-pdbviewer [41], in order to verify the conservation of structural features with a functional role, namely those implicated in Ca 2+ binding. The FH8 amino acid side chains directly implicated in Ca 2+ binding and that were different from those present in R. norvegicus corre- sponded to the amino acids that did not present a good local conformation when the quality of the models was evaluated. As the sequence alignments had shown that these few amino acids were identical to those present in TnC from Mus musculus (Protein Data Bank: 1A2X), we manually modeled the side chains of Asn17, Asn53 and Asp55, according to the orientations observed for TnC. The model structures in the open (Ca 2+ -loaded) and closed (apo) conformations are presented in Fig. 5. Closer views of Ca 2+ -binding sites I and II in the open conformation are also shown. In site II, Asp59 ()X position) is shown, as sequence alignments showed that, although it is different in CaM, it is identical in TnC and therefore could be modeled with a high level of confidence. As happens for TnC, Asp59 is probably hydrogen-bonded to a water molecule that belongs to the coordination sphere of Ca 2+ . FH8 has four helical regions, and the corresponding residues for the Ca 2+ - loaded ⁄ Ca 2+ -free conformations are as follows: A, 2–14 ⁄ 2–13; B, 24–34 ⁄ 24–33; C, 40–48 ⁄ 40–50; and D, 60–68 ⁄ 58–68. Ca 2+ -binding sites I and II have the appropriate amino acids and geometry to coordinate the metal ions, and are located in loop regions (15–26) and (51–62) that are flanked by helices on either side. In fact, the last three residues at the end of each loop form the first turn of exiting helices B for site I and D for site II. In the FH8 Ca 2+ -loaded protein, the antiparallel b-sheet that links Ca 2+ -binding sites I and II has only two hydrogen bonds, which are established between the main chain carbonyls and amides of Val22 and Leu58. In the case of the closed conformation, there is an additional hydrogen bond established between the carbonyl of Gly20 and the amide group of Leu60 (Fig. 5). If helices A and D are kept roughly with the same orientation in the images of Fig. 5, which represents both states, it can be seen that the helical packing is different. When Ca 2+ is bound, helices B and C open up slightly and expose a number of hydrophobic side chains, which were kept away from the solvent in the apo state. Figure 6 shows a comparison of the surface hydrophobicity for CaM and FH8 in the apo and Ca 2+ -bound states. Amino acids such as Val, Ile, Leu and Phe, which are very important in defining the protein hydrophobic regions, are in green, and the others are in gray. FH8 shows, in both states, larger hydrophobic surfaces than are seen in CaM. The sig- nificant exposed hydrophobic surface in the FH8 Ca 2+ -free state indicates a possible area for dimeriza- tion or a region of interaction with other proteins. As in CaM, the hydrophobic patch that becomes exposed upon Ca 2+ -binding is probably a region of interaction between FH8 and other proteins. Figures 5 and 6B also show the position of Cys36, whose oxidation we observed in SDS gels. It belongs to the B ⁄ C linker, a region that was proposed to be crucial for explaining the difference in behavior of the sensor and buffer proteins [15]. Moreover, Cys36, besides being very close to the protein exposed hydro- phobic patch, even in the apo form, is on the surface, and therefore may easily become oxidized through covalent binding to small or larger molecules, such as another FH8 macromolecule. However, if two FH8 macromolecules are covalently linked through the Cys36 residues, this complex will not acquire the dumbbell shape presented by CaM and TnC, both containing four EF-hand motifs, organized into two domains linked by a long helix. It has been reported that CaM has several Met resi- dues on the surface, which are close to each other in the apo conformation and not so close in the Ca 2+ - loaded (open) conformation, and these residues were considered to be important because, when they were mutated, activation of the ligands was impaired [42]. Moreover, they were proposed to have a role in stabi- lizing the open conformation [43]. FH8 has one unique Met (Met1), which sits at the N-terminal and obvi- ously will not be involved in ligand binding. Reinforcing the idea of the movement between heli- ces, we also found, in the modeled FH8 structure, sev- eral hydrophobic interactions between the amino acids of the four helices. Table 1 shows the hydrophobic interhelical contacts, and it is obvious that the interac- tions within the pairs of helices A ⁄ D and B ⁄ C are the least affected by Ca 2+ binding, whereas in the Table 1. The hydrophobic interhelical contacts present in FH8. Helices Ca 2+ -free state Ca 2+ -loaded state A ⁄ D Val7, Leu11 ⁄ Leu63, Val64, Leu67 Val7, Leu10, Leu11 ⁄ Leu60, Leu63, Leu67 A ⁄ B Val7, Leu10, Leu11 ⁄ Phe30 Val13 ⁄ Phe30 B ⁄ C Ala24, Leu27 ⁄ Ile43, Phe46, Ile47 Ala24, Leu27 ⁄ Ile43, Phe46 B ⁄ D Leu27, Phe30 ⁄ Leu63, Leu67 C ⁄ D Phe46, His50 ⁄ Leu63, Ile66 Phe46 ⁄ Ile66 FH8 from Fasciola hepatica H. Fraga et al. 5080 FEBS Journal 277 (2010) 5072–5085 ª 2010 The Authors Journal compilation ª 2010 FEBS Ca 2+ -loaded state, the interhelical interfaces concern- ing B ⁄ D disappear completely, and they are reduced in number for A ⁄ B. In fact, the B ⁄ C pair of helices swings away from the A ⁄ D pair. This is in agreement with findings reported for CaM [43]. Figures 5 and 6 also show the positions of the two Phe residues that were responsible for the detection of structural modifications upon Ca 2+ binding by fluores- cence. Phe30 belongs to helix B and is close to binding site I, whereas Phe46 belongs to helix C and is part of the hydrophobic patch that becomes exposed when the protein has an open conformation. Our molecular models show that when Ca 2+ binds, helices B and C move in relation to their previous positions. In the apo state, Phe30 is surrounded by hydrophobic side chains belonging to helices A (Val7, Leu10, Leu11 and Leu14), B (Val22 and Leu27) and D (Leu63 and Leu67). Upon binding of Ca 2+ , Phe30 moves far away from helices A and D, and only Leu27 remains close to it. Phe46 in the closed conformation has several aromatic side chains around it, namely Leu27 from helix B, Ile43, Ile47 and His50 from helix C, and Leu58, Leu63 and Ile66 from helix D; however, in the open conformation, only Leu27 and Ile66 remain close to Phe46. These modifications explain the observed dif- ferences in Phe fluorescence for the Ca 2+ -loaded and Ca 2+ -free FH8 states. Discussion FH8 is an EF-hand CaBP with only one globular domain and the characteristics of a sensor protein, dis- playing increases in radius and hydrophobic residue exposure with Ca 2+ loading that are typical of this class of proteins. Interestingly, the only Cys residue present in FH8 is on the protein surface, where it is able to be oxidized and form the dimers that we observed in SDS gels. As Cys36 is located in the B ⁄ C linker, the three-dimensional arrangement of the FH8 dimer will be significantly different from the CaM or TnC overall structures. Curiously, Cys36 is strictly conserved in this group of helminth proteins, suggest- ing a specific role for this amino acid in these proteins. FH8, like CaM and TnC, contains a short B ⁄ C linker, a region that allows adjustment of the relative posi- tions of these two helices in different conformations. Interestingly, both CaM and TnC are also the proteins that exhibit the largest domain opening. As occurs for other members of the EF-hand family, FH8 demonstrates an unusual thermal stability (T m = 74.0 ± 0.3 °C), and in the Ca 2+ -loaded state, FH8 is even more stable, as it is further stabilized by Ca 2+ ligand interactions. FH8 displays a low affinity for Ca 2+ (K obs =10 4 m )1 ), probably because of charge repulsion between the metal ion and the binding site, as loop I has two positively charged side chains and loop II has four, which is an extremely high number in comparison with canonical CaBPs. This may be in accordance with its extracellular location (1.2 mm Ca 2+ ) and F. hepatica migration to the gallbladder, where the Ca 2+ concentration is partic- ularly high (18 mm). Interestingly, very high concentra- tions of Ca 2+ were reported for the Schistosoma glycocalyx, where the homologous SJ8 and SM8 proteins were found [11]. Ca 2+ and CaBP are well- known mediators of changes in cell morphology, and modifications in the glycocalyx have been suggested to be one mechanism for host immune system suppression by F. hepatica [44]. Using homology modeling, we were able to observe that the structural integrity of the two-EF-hand domain is maintained by a short stretch of antiparallel b-sheet connecting the Ca 2+ -binding loops and by numerous hydrophobic contacts between the helices. We confirmed that, like other sensor proteins, FH8 in the Ca 2+ -loaded state has a reduced number of interh- elical contacts relative to those present in the Ca 2+ -free state, allowing movement of the B⁄ C pair of helices relative to the A ⁄ D pair. Although the extent and nature of the Ca 2+ -induced conformational changes can only be determined through full structural characterization, FH8 repre- sents, in our opinion, a case study protein for the EF- hand family, owing to its solubility, size (69 amino acids) and sensor properties, which allow a good con- trast with the similar-sized calbindin D 9k (76 amino acids), the prototypical Ca 2+ buffer protein. In summary, we present here a new EF-hand protein that behaves like a sensor CaBP, displays low affinity for Ca 2+ , and is highly stable in its apo and Ca 2+ - loaded states. Our work will proceed with the determi- nation of the three-dimensional structures of FH8 in the apo and Ca 2+ -loaded states, using X-ray crystal- lography and ⁄ or NMR, and mutagenesis of key amino acids in order to study their influence on Ca 2+ affinity and conformational changes. This will contribute to a complete understanding of the main aspects that drive conformational changes and affinity in CaBPs. Experimental procedures Unless indicated otherwise, all reactions were performed at 22 °Cin10mm Tris (pH 8.0) containing 2 mm TECP, 1mm EDTA and 150 mm KCl. ANS, phenylmethanesulfo- nyl fluoride, Hepes, EDTA, ampicillin, Tris, BS3 and TECP were obtained from Sigma. pTYB1 plasmid, NdeI, SapIand H. Fraga et al. FH8 from Fasciola hepatica FEBS Journal 277 (2010) 5072–5085 ª 2010 The Authors Journal compilation ª 2010 FEBS 5081 [...]... 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Portugal Introduction Fasciola hepatica is a trematode parasite that is respon- sible for fascioliasis. Although traditionally regarded as a parasite of livestock,