Báo cáo khoa học: FH8 – a small EF-hand protein from Fasciola hepatica docx

Sequence analysis revealed that FH8 has two EF-hand Ca2+-binding motifs, and our experimental data show that the protein binds Ca2+ and that this induces conformational alterations, thus

Hugo Fraga1, Tiago Q Faria2, Filipe Pinto1, Agostinho Almeida3, Rui M M Brito2,4and

Ana M Damas1,5

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 Cieˆncias Biome´dicas de Abel Salazar, University of Porto, Portugal

Introduction

Fasciola hepaticais 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

Ca2+-binding motifs, and our experimental data show that the protein binds Ca2+ and that this induces conformational alterations, thus causing

it to behave like a sensor protein Moreover, FH8 displays low affinity for

Ca2+ (Kobs= 104M )1) and is highly stable in its apo and Ca2+-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 Ca2+ 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;

Tm, melting point; TnC, troponin C.

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 secdetec-tions 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 Ca2+, 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 Ca2+ 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

Ca2+ However, the composition and length of the

Ca2+-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 Ca2+ -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: Ca2+ sen-sors, represented by CaM and TnC, and Ca2+buffers, such as calbindin D9k and parvalbumin The sensor proteins display Ca2+-dependent conformational changes, whereas Ca2+ buffers, which are involved in

Ca2+ signal modulation, undergo minimal structural changes upon Ca2+binding It was reported that when

Ca2+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 D9k[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 Ca2+-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

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 Ca2+ 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 D9k, as a model for the buffer CaBPs They contain one (calbindin D9k) or two (CaM and TnC) globular domains, each of them with two EF-hand Ca2+-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 D9k 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 Ca2+-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 D9k has 14 amino acids in the corresponding loop

In most EF-hand proteins, Ca2+ 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 Ca2+ 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

Ca2+ 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 Ca2+-binding loop are helical, and form the first turn of the exiting helix

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 m M

EDTA or 5 m M Ca 2+

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 Ca2+-binding

sites are very similar to those in CaM and TnC, but

not to those in calbindin D9k

The conserved amino acids that coordinate Ca2+

(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 Ca2+affinity, whereas mutations

at other Ca2+-coordinating positions do not have such

drastic consequences [18,19] As reports on structural

and biochemical data indicate that the mechanisms of

Ca2+-induced conformational changes in CaM and

TnC are similar [20], we foresee that the Asn⁄ Asp

modifications will not impair Ca2+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 Ca2+ coordination, it is possible that the positively charged side chain will influence Ca2+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 Ca2+sensor protein CaM together with the C-terminal fragments of TnC and Ca2+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 Ca2+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 D9Kproteins are colored blue and have the following meaning:

‘*’, identical in all sequences in the alignment; ‘:’, conserved substitutions; ‘.’, semiconserved substitutions.

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 correspsec-onds to four amino acids of

the first helix plus the Ca2+-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

Ca2+-binding affinity

Ca2+-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 Ca2+ This

was initially tested using the PAGE mobility assay, as

it has been reported that, in the presence of Ca2+,

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 Ca2+

(Fig 1B) This preliminary result, demonstrating the

functionality of FH8 EF-hand motifs, was further

substantiated with an equilibrium dialysis experiment

with Ca2+ (5 mm), where a ratio of 2.0 ± 0.3 Ca2+

per FH8 macromolecule was observed, indicating that

both Ca2+-binding sites are functional

As FH8 was able to bind Ca2+, possible structural

modifications associated with Ca2+ 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 Ca2+ binding [21] Accordingly, Phe fluorescence was mea-sured in the presence of EDTA and Ca2+, and we observed an increase of 30% in the Ca2+-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

Ca2+binding (Figs 5 and 6) As can be seen inFig 6,

a hydrophobic patch becomes exposed upon Ca2+ binding in the case of CaM, and for FH8 the solvent-exposed hydrophobic area is also larger in the Ca2+ -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 Ca2+ binding [22] We measured the hydrodynamic radius (RH) of FH8 in the presence and absence of Ca2+ Despite the large standard deviation

of the results, it is clear that FH8 displays a larger RH

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 Ca2+(circles) and 3 m M EDTA (crosses) (B) R H

of FH8 in the presence of 20 m M Ca 2+ (circles) and 1 m M EDTA (crosses) determined by DLS FH8 shows an increase in R H that is consistent with a more elongated shape in its Ca2+-loaded state.

(2.7 ± 1.1 nm) in the Ca2+-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 Ca2+ coordination results in alterations in FH8 structure

After determining that FH8 undergoes conforma-tional changes upon Ca2+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 Ca2+ 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 Ca2+ sensors show strong enhancement of ANS fluorescence upon Ca2+ binding [15] Consistent with the previous hypothesis,

Ca2+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 Mg2+, demonstrating the specific-ity of the conformational change for Ca2+ (Fig 7B) The failure of Mg2+ 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

Ca2+, the primary amine cross-linker suberic acid bis(3-sulfo-N-hydroxysuccinimide) ester (BS3) was used

A

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 Ca2+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).

in the presence of different concentrations of Ca2+ 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 Ca2+above 1 mm

The conformational alterations induced by Ca2+

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 Ca2+ in

the presence of FH8 The Ca2+ 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 Kobs

of 590 ± 20 lm This result is in agreement with the

findings of experiments on several EF-hand CaBPs,

where cooperativity in Ca2+ binding was observed

However, the Kobs was unusually high in comparison

with the canonical proteins of the sensor family In

order to corroborate this result, equilibrium dialysis

with 250 lm Ca2+ was performed Consistent with

the low affinity suggested by the Ca2+titration curve,

a ratio of 0.2 Ca2+per FH8 was determined,

confirm-ing that FH8 has a low affinity for Ca2+ (data not

shown) In fact, EF-hand CaBPs have binding

con-stants for Ca2+ that span a wide range (103–109m)1),

with no obvious correlation with the type or arrange-ment of the Ca2+ ligands [14], although it is known that exposure of hydrophobic residues results in a lower affinity for Ca2+, 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

Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ affini-ties, but no data are currently available

Besides FH8, several other low-affinity EF-hand proteins (Kd= 103–104m)1) have been described [14,28–30], including a-spectrin [31] and multiple

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 m M 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 m M

NaCl and 20 m M 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 Ca2+, using ANS fluorescence as reporter Data were fitted to the Hill equation.

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 Ca2+concentration is high

Secondary structure and thermal stability

Information related to protein secondary structure and

stability in the presence and absence of Ca2+ was

obtained by CD spectroscopy The spectra are

pre-sented inFig 8, and they reveal that FH8 has a

signif-icant content of ordered secondary structure The

far-UV CD spectrum of FH8 at 20C (Fig 8A),

contains the shapes and amplitudes characteristic of

proteins with a high percentage of helical structure In the presence of 20 mm Ca2+, 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 Ca2+[34]

The CD spectra for FH8 also show that Ca2+ bind-ing results in stabilization of the protein structure Indeed, as shown in Fig 8B, the thermal stability of the Ca2+-loaded FH8 is very high, and FH8 therefore behaves like the Ca2+-loaded states of other EF-hand CaBPs, namely CaM, TnC and calbindin D9K In all

of these proteins, the denaturation temperatures of the

Ca2+-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

98C The FH8 apo state is less stable, and we were able to determine its melting point (Tm) as 74.0 ± 0.3C (Fig 8C) Although not comparable to its Ca2+-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

Ca2+-binding loops in a short stretch of antiparallel b-sheet, as well as by numerous hydrophobic contacts between the helices In the Ca2+-loaded state, the CaBPs are further stabilized by Ca2+ ligand interac-tions, and are normally more stable [13,37] Curiously, apo-FH8 is substantially more stable towards thermal denaturation than CaM or TnC (Tm 55 C) [35], although apo-calbindin D9k does demonstrate even higher Tmvalues (85C or higher [36])

Homology models The N-terminal fragment of CaM from Rattus norvegi-cuswas 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

Ca2+-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 Ca2+ -loaded and Ca2+-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 Ca2+-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 Ca2+ (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.

)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 Ca2+ binding The FH8 amino acid side chains

directly implicated in Ca2+ 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 (Ca2+-loaded) and

closed (apo) conformations are presented in Fig 5

Closer views of Ca2+-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 Ca2+ FH8 has four helical

regions, and the corresponding residues for the Ca2+

-loaded⁄ Ca2+-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 Ca2+-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 Ca2+-loaded protein, the antiparallel

b-sheet that links Ca2+-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 Ca2+ 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

Ca2+-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

Ca2+-free state indicates a possible area for dimeriza-tion or a region of interacdimeriza-tion with other proteins As

in CaM, the hydrophobic patch that becomes exposed upon Ca2+-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 Ca2+ -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 Ca2+ 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

Ca2+-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 Ca2+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 Ca2+ 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 Ca2+, 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 Ca2+-loaded and

Ca2+-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 Ca2+ 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 conformaposi-tions

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

(Tm= 74.0 ± 0.3C), and in the Ca2+-loaded state,

FH8 is even more stable, as it is further stabilized by

Ca2+ligand interactions

FH8 displays a low affinity for Ca2+(Kobs= 104m)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 Ca2+

) and F hepatica migration to the gallbladder, where the Ca2+concentration is partic-ularly high (18 mm) Interestingly, very high concentra-tions of Ca2+ were reported for the Schistosoma glycocalyx, where the homologous SJ8 and SM8 proteins were found [11] Ca2+ 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 Ca2+-binding loops and by numerous hydrophobic contacts between the helices

We confirmed that, like other sensor proteins, FH8 in the Ca2+-loaded state has a reduced number of interh-elical contacts relative to those present in the

Ca2+-free state, allowing movement of the B⁄ C pair of helices relative to the A⁄ D pair

Although the extent and nature of the Ca2+-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 D9k (76 amino acids), the prototypical Ca2+buffer protein

In summary, we present here a new EF-hand protein that behaves like a sensor CaBP, displays low affinity for Ca2+, and is highly stable in its apo and Ca2+ -loaded states Our work will proceed with the determi-nation of the three-dimensional structures of FH8 in the apo and Ca2+-loaded states, using X-ray crystal-lography and⁄ or NMR, and mutagenesis of key amino acids in order to study their influence on Ca2+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

1 mm EDTA and 150 mm KCl ANS, phenylmethanesulfo-nyl fluoride, Hepes, EDTA, ampicillin, Tris, BS3 and TECP were obtained from Sigma pTYB1 plasmid, NdeI, SapI and