Insights on the structure-activity relationship of peptides derived from Sticholysin II.

Sticholysin II (StII) is a pore-forming actinoporin from the sea anemone Stichodactyla helianthus. A mechanistic model of its action has been proposed: proteins bind to cell membrane, insert their N-termini into the lipid core and assemble into homo-tetramer pores responsible for host-cell death. Because very likely the first 10 residues of StII N-terminus are critical for membrane penetration, to dissect the molecular details of that functionality, we studied two synthetic peptides: StII1-30 and StII16-35 . They show diverse haemolytic and candidacidal activity that correlate with distinct orientations in SDS micelles. NMR shows that StII1-30 partly inserts into the micelle, while StII16-35 lays on the micelle surface. These results justify the diverse concentration dependence of their candidacidal activity supposing a different mechanism of action and providing new hints on StII lytic activity at molecular level. Biotechnological application of these peptides, focused on the development of therapeutic immunocomplexes, may be envisaged.

Looking over toxin-K(+) channel interactions. Clues from the structural and functional characterization of α-KTx toxin Tc32, a Kv1.3 channel blocker.

α-KTx toxin Tc32, from the Amazonian scorpion Tityus cambridgei, lacks the dyad motif, including Lys27, characteristic of the family and generally associated with channel blockage. The toxin has been cloned and expressed for the first time. Electrophysiological experiments, by showing that the recombinant form blocks Kv1.3 channels of olfactory bulb periglomerular cells like the natural Tc32 toxin, when tested on the Kv1.3 channel of human T lymphocytes, confirmed it is in an active fold. The nuclear magnetic resonance-derived structure revealed it exhibits an α/ß scaffold typical of the members of the α-KTx family. TdK2 and TdK3, all belonging to the same α-KTx 18 subfamily, share significant sequence identity with Tc32 but diverse selectivity and affinity for Kv1.3 and Kv1.1 channels. To gain insight into the structural features that may justify those differences, we used the recombinant Tc32 nuclear magnetic resonance-derived structure to model the other two toxins, for which no experimental structure is available. Their interaction with Kv1.3 and Kv1.1 has been investigated by means of docking simulations. The results suggest that differences in the electrostatic features of the toxins and channels, in their contact surfaces, and in their total dipole moment orientations govern the affinity and selectivity of toxins. In addition, we found that, regardless of whether the dyad motif is present, it is always a Lys side chain that physically blocks the channels, irrespective of its position in the toxin sequence.

The structure of the elicitor Cerato-platanin (CP), the first member of the CP fungal protein family, reveals a double ψß-barrel fold and carbohydrate binding.

Cerato-platanin (CP) is a secretion protein produced by the fungal pathogen Ceratocystis platani, the causal agent of the plane canker disease and the first member of the CP family. CP is considered a pathogen-associated molecular pattern because it induces various defense responses in the host, including production of phytoalexins and cell death. Although much is known about the properties of CP and related proteins as elicitors of plant defense mechanisms, its biochemical activity and host target(s) remain elusive. Here, we present the three-dimensional structure of CP. The protein, which exhibits a remarkable pH and thermal stability, has a double ψß-barrel fold quite similar to those found in expansins, endoglucanases, and the plant defense protein barwin. Interestingly, although CP lacks lytic activity against a variety of carbohydrates, it binds oligosaccharides. We identified the CP region responsible for binding as a shallow surface located at one side of the ß-barrel. Chemical shift perturbation of the protein amide protons, induced by oligo-N-acetylglucosamines of various size, showed that all the residues involved in oligosaccharide binding are conserved among the members of the CP family. Overall, the results suggest that CP might be involved in polysaccharide recognition and that the double ψß-barrel fold is widespread in distantly related organisms, where it is often involved in host-microbe interactions.

Stability improvement of the fatty acid binding protein Sm14 from S. mansoni by Cys replacement: structural and functional characterization of a vaccine candidate.

The Schistosoma mansoni fatty acid binding protein (FABP), Sm14, is a vaccine candidate against, S. mansoni and F. hepatica. Previously, we demonstrated the importance of a correct fold to achieve protection in immunized animals after cercariae challenge [[10]. C.R.R. Ramos, R.C.R. Figueredo, T.A. Pertinhez, M.M. Vilar, A.L.T.O. Nascimento, M. Tendler, I. Raw, A. Spisni, P.L. Ho, Gene structure and M20T polymorphism of the Schistosoma mansoni Sm14 fatty acid-binding protein: structural, functional and immunoprotection analysis. J. Biol. Chem. 278 (2003) 12745-12751.]. Here we show that the reduction of vaccine efficacy over time is due to protein dimerization and subsequent aggregation. We produced the mutants Sm14-M20(C62S) and Sm14-M20(C62V) that, as expected, did not dimerize in SDS-PAGE. Molecular dynamics calculations and unfolding experiments highlighted a higher structural stability of these mutants with respect to the wild-type. In addition, we found that the mutated proteins, after thermal denaturation, refolded to their active native molecular architecture as proved by the recovery of the fatty acid binding ability. Sm14-M20(C62V) turned out to be the more stable form over time, providing the basis to determine the first 3D solution structure of a Sm14 protein in its apo-form. Overall, Sm14-M20(C62V) possesses an improved structural stability over time, an essential feature to preserve its immunization capability and, in experimentally immunized animals, it exhibits a protection effect against S. mansoni cercariae infections comparable to the one obtained with the wild-type protein. These facts indicate this protein as a good lead molecule for large-scale production and for developing an effective Sm14 based anti-helminthes vaccine.

The solution structure of the outer membrane lipoprotein OmlA from Xanthomonas axonopodis pv. citri reveals a protein fold implicated in protein-protein interaction.

The outer membrane lipoprotein A (OmlA) belongs to a family of bacterial small lipoproteins widely distributed across the beta and gamma proteobacteria. Although the role of numerous bacterial lipoproteins is known, the biological function of OmlA remains elusive. We found that in the citrus canker pathogen, Xanthomonas axonopodis pv. citri (X. citri), OmlA is coregulated with the ferric uptake regulator (Fur) and their expression is enhanced when X. citri is grown on citrus leaves, suggesting that these proteins are involved in plant-pathogen interaction. To gain insights into the function of OmlA, its conformational and dynamic features were determined by nuclear magnetic resonance. The protein has highly flexible N- and C- termini and a structurally well defined core composed of three beta-strands and two small alpha-helices, which pack against each other forming a two-layer alpha/beta scaffold. This protein fold resembles the domains of the beta-lactamase inhibitory protein BLIP, involved in protein-protein binding. In conclusion, the structure of OmlA does suggest that this protein may be implicated in protein-protein interactions required during X. citri infection.

Development of a bacterial cloning vector for expression of scorpion toxins for biotechnological studies.

Scorpion venoms contain toxic peptides that recognize K(+) channels of excitable and non-excitable cells. These toxins comprise three structurally distinct groups designated alpha-KTx, beta-KTx, and gamma-KTx. It is highly desirable to develop systems for the expression of these toxins for further physiological and structural studies. In this work, an expression vector (pTEV3) was constructed by inserting protein D (major capsid of phage lambda) and TEV protease recognition site into plasmid pET21d DNA sequences. Three alpha-KTx toxins (OsK2, PbTx1, and BmKK3) were cloned into vector pTEV3 and expressed as soluble fusion proteins. The fractions containing the purified fusion proteins (protein D-toxin) were treated with TEV protease to remove protein D. The resulting toxins were analyzed by MALDI-TOF Mass Spectrometry. The results showed that the vector is appropriate for the expression of the target toxins in soluble form and that ion exchange purification of these toxins by flow-through recovery is possible. Analysis by MALDI-TOF Mass Spectrometry of Osk2 demonstrated that this toxin was expressed in its native form, as suggested by the values expected for the presence of two disulfide bridges.

Truncation of amidated fragment 33-61 of bovine alpha-hemoglobin: effects on the structure and anticandidal activity.

Peptides derived from endogenous hemoglobin play important biological roles in a variety of living systems. In previous works we showed that the fragment 33-61 of bovine alpha-hemoglobin (Hb33-61) and its C-terminus amidated analogue (Hb33-61a) exhibit antimicrobial activity and we determined the 3D structure of Hb33-61a bound to sodium dodecyl sulfate micelles. Here we report that Hb33-61a is lethal to Candida albicans at 6.25 microM probably through disruption of its plasma membrane. In addition, we show that, even when used at 50 microM, Hb33- 61a produces low hemolysis (16% +/- 3.0%). Recognizing that one of the key steps to study new compounds with potential pharmaceutical application is to identify the structural elements essential to express biological activity, we also investigated the anticandidal activity of Hb33- 61a fragments. The results indicated that Hb40-61a exhibits the same minimal inhibitory concentration as Hb33-61a, whereas Hb33-52a and Hb48-61a are significantly less active. Noteworthy, for all the peptides tested, we observed that C-terminus amidation produces a potentiation of their anticandidal activity and we associate that increased biological activity to a preferred structural and spatial organization of the C-terminal region favored by amidation. Finally, the data show that the most active peptides (Hb33-61a and Hb40-61a) are characterized by a central hinge joining the C-terminal region that presents, containing a beta-turn, followed by and a helical element, to the N-terminal region that presents only a beta-turn. We hypothesize that these two structured regions, by fluctuating independently in the lipid environment, may act in a coordinated fashion disrupting the yeast plasma membrane.

Interaction of local anesthetics with a peptide encompassing the IV/S4-S5 linker of the Na+ channel.

The peptide pIV/S4-S5 encompasses the cytoplasmic linker between helices S4-S5 in domain IV of the voltage-gated Na+ channel, residues 1644-1664. The interaction of two local anesthetics (LA), lidocaine and benzocaine, with pIV/S4-S5 has been studied by DOSY, heteronuclear NMR 1H-15N-HSQC spectroscopy and computational methods. DOSY indicates that benzocaine, a neutral ester, exhibits stronger interaction with pIV/S4-S5 than lidocaine, a charged amine-amide. Weighted average chemical shifts, Deltadelta(1H-15N), show that benzocaine affects residues L1653, M1655 and S1656 while lidocaine slightly perturbs residues I1646, L1649 and A1659, L1660, near the N- and C-terminus, respectively. Computational methods confirmed the stability of the benzocaine binding and the existence of two binding sites for lidocaine. Even considering that the approach of studying the peptide in the presence of a co-solvent (TFE/H2O, 30%/70% v/v) has an inherently limited implication, our data strongly support the existence of multiple LA binding sites in the IV/S4-S5 linker, as suggested in the literature. In addition, we consider that LA can bind to the S4-S5 linker with diverse binding modes and strength since this linker is part of the receptor for the "inactivation gate particle". Conditions for devising new functional studies, aiming to better understand Na+ channel functionality as well as the various facets of LA pharmacological activity are proposed in this work.

Lytic activity and structural differences of amphipathic peptides derived from trialysin.

Trialysin is a pore-forming protein found in the saliva of Triatoma infestans (Hemiptera, Reduviidae), the insect vector of Chagas' disease. The protein is active against a broad range of cell types from bacteria to eukaryotic cells. Recognizing that the N-terminus of trialysin harbors the lytic motif [Amino, R., Martins, R. M., Procopio, J., Hirata, I. Y., Juliano, M. A., and Schenkman, S. (2002) J. Biol. Chem. 277, 6207-6213], we designed a set of peptides scanning this region to investigate the structural basis of its biological function. Peptides encompassing residues 1-32 (P6), 1-27 (P7), and 6-32 (P5) efficiently induced lysis of the protozoan parasite Trypanosoma cruzi and Escherichia coli in the 0.4-9.0 microM range, while much higher concentrations were required to cause hemolysis. Other more internal peptides, including peptide P2 (residues 21-47) and others up to residue 52, were less effective. P6 turned out to be the most active of all. P7 has a significantly higher activity than P5 against E. coli, while P5 has a hemolytic activity comparable to that of P6. CD spectroscopy showed that all tested peptides acquire a comparable helical content in solvent mixtures or in detergent micelles. The solution structure of P2 and P5-P7 was determined in a 30% trifluoroethanol/water mixture by nuclear magnetic resonance. All peptides exhibit a structure characterized by a central helical fold, and except for P2, which does not show a continuous hydrophobic surface, they are amphipathic. The structural models show that P5 and P7 extend their structural similarities with the most active peptide, P6, in either the C-terminus or the N-terminus. Amino acid substitutions in the N-terminus of P6 improved hemolysis but did not change the activity against T. cruzi. These results suggest that while amphipathicity is essential for the lytic activity, the selectivity of the active peptides for specific organisms appears to be associated with the structural features of their N- and C-termini.

Model peptides mimic the structure and function of the N-terminus of the pore-forming toxin sticholysin II.

To investigate the role of the N-terminal region in the lytic mechanism of the pore-forming toxin sticholysin II (St II), we studied the conformational and functional properties of peptides encompassing the first 30 residues of the protein. Peptides containing residues 1-30 (P1-30) and 11-30 (P11-30) were synthesized and their conformational properties were examined in aqueous solution as a function of peptide concentration, pH, ionic strength, and addition of the secondary structure-inducing solvent trifluoroethanol (TFE). CD spectra showed that increasing concentration, pH, and ionic strength led to aggregation of P1-30; as a consequence, the peptide acquired beta-sheet conformation. In contrast, P11-30 exhibited practically no conformational changes under the same conditions, remaining essentially structureless. Moreover, this peptide did not undergo aggregation. These differences clearly point to the modulating effect of the first 10 hydrophobic residues on the peptides aggregation and conformational properties. In TFE both the first ten hydrophobic peptides acquired alpha-helical conformation, albeit to a different extent, P11-30 displayed lower alpha-helical content. P1-30 presented a larger fraction of residues in alpha-helical conformation in TFE than that found in St II's crystal structure for that portion of the protein. Since TFE mimics the membrane environment, such increase in helical content could also occur upon toxin binding to membranes and represent a step in the mechanism of pore formation. The peptides conformational properties correlated well with their functional behavior. Thus, P1-30 exhibited much higher hemolytic activity than P11-30. In addition, P11-30 was able to block the toxin's hemolytic activity. The size of pores formed in red blood cells by P1-30 was estimated by measuring the permeability to PEGs of different molecular mass. The pore radius (0.95 +/- 0.01 nm) was very similar to that of the pore formed by the toxin. The results demonstrate that the synthetic peptide P1-30 is a good model of St II conformation and function and emphasize the contribution of the toxin's N-terminal region, and, in particular, the hydrophobic residues 1-10 to pore formation.

The micelle-bound structure of an antimicrobial peptide derived from the alpha-chain of bovine hemoglobin isolated from the tick Boophilus microplus.

Hemoglobin is known to be a source of peptides involved in several functions. The peptide FLSFPTTKTYFPHFDLSHGSAQVKGHGAK (Hb33-61) is a proteolytic product of the bovine hemoglobin alpha-chain found in the gut content of the cattle tick, Boophilus microplus, and it possesses antimicrobial activity. Since in the past we showed that the amidated form of Hb33-61, Hb33-61a, is active against a few Gram-positive bacteria and fungi strains at micromolar concentration [Fogaca et al. (1999) J. Biol. Chem. 274, 25330-25334], we have been prompted to shed more light on its functional and structural features. Here we show that the peptide is able to disrupt the bacterial membrane ofMicrococcus luteus A270. As for its structure, it has a random conformation in water, and it does not interact with zwitterionic micelles. On the other hand, it binds to negatively charged micelles acquiring a finite structural organization. The 3D structure of Hb33-61a bound to SDS micelles exhibits a nonconventional conformation for an antimicrobial peptide. The backbone is characterized by the presence of a beta-turn in the N-terminus and by a beta-turn followed by a alpha-helical stretch in the C-terminus. A hinge, whose spatial organization is stabilized by side-chain-side-chain interactions, joins these two regions. Interestingly, it preserves structural features present in the corresponding segment of the bovine hemoglobin alpha-chain. Hb33-61a does not possess a well-defined amphipathic nature, and H/D exchange experiments show that while the C-terminal region is embedded in the SDS micelle, one face of the N-terminal half is partly exposed to the solvent.

Differential effects of uncharged aminoamide local anesthetics on phospholipid bilayers, as monitored by 1H-NMR measurements.

We have collected evidences of a "transient site" for the local anesthetics (LA) lidocaine, etidocaine, bupivacaine and mepivacaine in sonicated egg phosphatidylcholine (EPC) vesicles. The effects of the uncharged anesthetic species at a fixed LA/EPC ratio inside the bilayer were measured by chemical shifts (C.S.) and longitudinal relaxation times (T(1)) of the lipid hydrogens. Two sort of changes were detected: (I) decrease, indicating specific orientation of the LA aromatic ring (measured as up-field C.S. changes by the short-range ring-current effect) and less rotational freedom (smaller T(1) values) for EPC hydrogens such as the two glycerol-CH(2) and the choline-CH(2) bound to the PO(4-) group, probably due to the nearby presence of the LA; (II) increase, indicating the aromatic ring is now perpendicular to the orientation observed before (causing down-field changes in C.S.) and larger T(1) values for all the choline and glycerol hydrogens, as a result of LA insertion behind these well-organized bilayer regions. The less hydrophobic, linear and nonlinear (lidocaine and mepivacaine, respectively) aminoamide analogs provide similar effects-described in I; their hydrophobic counterparts (etidocaine and bupivacaine) also produced comparable effects (depicted in II). The preferential positioning and orientation of each LA inside the bilayer is then determined by its hydrophobic and steric properties. We propose that this "transient site" in the lipid milieu exists also in biological membranes, where it can modulates the access of the uncharged LA species to its site(s) of action in the voltage-gated sodium channel.

Modeling the Trypanosoma cruzi Tc85-11 protein and mapping the laminin-binding site.

Trypanosoma cruzi expresses a set of glycoproteins encoded by the gp85/trans-sialidase gene superfamily. In this report a structure model is proposed for a cloned member of the superfamily, the Tc85-11 protein. The structure consists of an N-terminus beta-propeller and a C-terminus beta-sandwich interconnected by an alpha-helix. The recombinant protein, corresponding to the N-domain (Tc85-N), binds to laminin in a selective manner. Six synthetic 20-mer peptides from the N-domain adhere onto the surface of LLC-MK(2) cells and two of these peptides specifically inhibit the Tc85-N/laminin interaction, indicating that they are the laminin-binding sites of the molecule. Thus, Tc85-11 and other related members of the family appear to be good candidates to play an important role in T. cruzi infection via a laminin mediated host-parasite interaction.

How C-terminal carboxyamidation alters the biological activity of peptides from the venom of the eumenine solitary wasp.

Inflammatory peptides display different types of post-transcriptional modifications, such as C-terminal amidation, that alter their biological activity. Here we describe the structural and molecular dynamics features of the mast cell degranulating peptide, eumenine mastoparan-AF (EMP-AF-NH(2)), found in the venom of the solitary wasp, and of its carboxyl-free C-terminal form (EMP-AF-COO(-)) characterized by a reduced activity. Circular dichroism indicates that both peptides switch from a random coil conformation in water to a helical structure in TFE and SDS micelles. NMR data, in 30% TFE, reveal that the two peptides fold into an alpha-helix spanning most of their length, while they differ in terms of molecular rigidity. To understand the origins of the conformational flexibility observed in the case of EMP-AF-COO(-), a 5 ns MD simulation was carried out for each peptide, in an explicit water/TFE environment. The results show that the two peptides differ in an H-bond between Leu14 NH(2) and the backbone carbonyl of Ile11. The loss of that H-bond in EMP-AF-COO(-) leads to a significant modification of its structural dynamics. In fact, as evidenced by essential dynamics analysis, while EMP-AF-NH(2) exists mainly as a rigid structure, EMP-AF-COO(-) presents two helical stretches that fluctuate in some sort of independent fashion. We conclude that the diverse biological activity of the two peptides is not simply due to the reduction of the net positive charge, as generally suggested, but also to a structural perturbation of the amphipathic alpha-helix that affects their ability to perturb the cell membrane.

The NMR-derived solution structure of a new cationic antimicrobial peptide from the skin secretion of the anuran Hyla punctata.

Amphibian skin secretions constitute an important source of molecules for antimicrobial drug research in order to combat the increasing resistance of pathogens to conventional antibiotics. Among the various types of substances secreted by the dermal granular amphibian glands, there is a wide range of peptides and proteins, often displaying potent antimicrobial activities and providing an effective defense system against parasite infection. In the present work, we report the NMR solution structure and the biological activity of a cationic 14-residue amphiphilic alpha-helical polypeptide named Hylaseptin P1 (HSP1), isolated from the skin secretion of the hylid frog Hyla punctata. The peptide antimicrobial activity was verified against Candida albicans, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, whereas no significant lytic effect was detected toward red or white blood cells.

Cloning and expression of calglandulin, a new EF-hand protein from the venom glands of Bothrops insularis snake in E. coli.

The EF-hand protein family is comprised of many proteins with conserved Ca(2+)-binding motifs with important biological roles in intracellular communication. During the generation of Expressed Sequence Tags (ESTs) from the venom glands of the Viperidae snake Bothrops insularis, we identified a cDNA coding for a putative Ca(2+) binding protein with four EF-hand motifs, named here calglandulin. The deduced amino acid sequence displayed the greatest sequence similarity with calmodulin (59%), followed by troponin-C (52%). The encoded polypeptide was first expressed in Escherichia coli as a 6XHis-tagged fusion protein. The expressed protein was purified by Ni(2+)-charged affinity chromatography and circular dichroism (CD) spectroscopy confirmed the prevalence of alpha-helix as observed in calmodulin/calmodulin-like proteins. A polyclonal antiserum was generated in mice using this recombinant calglandulin. To investigate the tissue-specific biological occurrence of this protein, this antiserum was used in Western blot experiments, which revealed an immunoreactive band in samples of venom gland extracts from different snakes, but not in the crude venom or in brain, heart and other tissues. This exclusive occurrence suggests a specialized function of calglandulin in snake venom glands.

Plasmodium falciparum histidine-rich protein II binds to actin, phosphatidylinositol 4,5-bisphosphate and erythrocyte ghosts in a pH-dependent manner and undergoes coil-to-helix transitions in anionic micelles.

The recombinant histidine-rich protein II (HRPII) from Plasmodium falciparum was shown to bind actin and phosphatidylinositol 4,5-bisphosphate (PIP(2)) in vitro in a pH-dependent manner, very similar to hisactophilin, an actin-binding protein from ameba. Binding of HRPII to actin and PIP(2) occurred at pH 6.0 and 6.5, but not above pH 7.0. Circular dichroism (CD) spectroscopy confirmed that HRPII interacts with actin at pH below 7.0, as judged by the changes induced in the secondary structure of the HRPII/actin mixture. Further CD analysis demonstrated that HRPII adopts a predominantly alpha-helical conformation with anionic micelles of PIP(2) and SDS, but not with neutral micelles of phosphatidylcholine (PC), a feature that is common to many actin-binding proteins involved in cytoskeleton remodeling. Similarly to hisactophilin, a GFP-HRPII fusion protein shuttled from the cytoplasm to the nucleus of HeLa cells as the cellular pH was lowered from 8.0 to 6.0. HeLa cells transfected with the HRPII gene showed increased levels of histidine-rich proteins (HRPs) in the soluble cell fraction at pH 8.0. At pH 6.0, however, HRPs were detected mainly in the insoluble cell fraction. Interestingly, we found that HRPII binds to human erythrocyte membranes at pH 6.0 and 6.5 but not at pH above 7.0. Our results point to remarkable similarities between HRPII, hisactophilin, and actin-binding proteins. Possible roles of the HRPII during Plasmodium infection are discussed in the light of these findings.

Introduction of a chemical constraint in a short peptide derived from human acidic fibroblast growth factor elicits mitogenic structural determinants.

Fibroblast growth factors (FGFs) are regulatory proteins associated with a number of physiological and pathological states. On the basis of data suggesting a functional role for specific regions of human acidic FGF (aFGF), a linear peptide encompassing residues 99-108 (peptide1) and its cyclic analogue (peptide 2) were synthesized and their functional and structural features were investigated. While peptide 1 is inactive on Balb/c 3T3 fibroblasts, peptide 2 is mitogenic with ED(50) of approximately 50 microM. Moreover, peptide 1 is not able to inhibit the binding of human aFGF to cellular receptors whereas peptide 2 exhibits significant inhibitory activity. The NMR-derived solution conformers indicated the presence, only in peptide 2, of structural elements that we believe are related to its ability to emulate the biological activity of the native protein. These results suggest that the expression of mitogenic activity in short peptides, besides the presence of specific amino acids, requires the existence of stable structural features. In addition, they indicate that the introduction of chemical restraints in peptides can provide novel possibilities for the development of receptor agonists or antagonists.

Gene structure and M20T polymorphism of the Schistosoma mansoni Sm14 fatty acid-binding protein. Molecular, functioanl, and immunoprotection analysis.

The Schistosoma mansoni Sm14 antigen belongs to the fatty acid-binding protein family and is considered a vaccine candidate against at least two parasite worms, Fasciola hepatica and S. mansoni. Here the genomic sequence and the polymorphism of Sm14 have been characterized for the first time. We found that the conserved methionine at position 20 is polymorphic, being exchangeable with threonine (M20T). To evaluate the function of the amino acid residue at this position, we have also constructed the mutant Sm14-A20 besides the two native isoforms (Sm14-M20 and Sm14-T20). The three purified recombinant His(6)-tagged Sm14 proteins (rSm14-M20, rSm14-T20, and rSm14-A20) present a predominant beta-barrel structure as shown by CD spectroscopy. Thermal and urea unfolding studies evidenced a higher structural stability of rSm14-M20 over the other forms (rSm14-M20>rSm14-T20>rSm14-A20). All of the Sm14 proteins were able to bind 11-(dansylamino)undecanoic acid (DAUDA) without substantial difference in the binding affinity. However, rSm14-M20 exhibited a higher affinity for natural fatty acids than the rSm14-T20 and rSm14-A20 proteins as judged by competitive experiments against DAUDA (rSm14-M20>rSm14-T20>rSm14-A20). The rSm14-M20 or rSm14-T20 isoforms but not the rSm14-A20 mutant was able to induce significant protection against S. mansoni cercariae challenge in immunized mice. The level of protection efficacy correlates with the extent of structure stability of the recombinant Sm14 isoforms and mutant.