Structural and evolutionary insights into the multidomain galectin from the red abalone Haliotis rufescens with specificity for sulfated glycans.

Galectins are an evolutionarily ancient family of lectins characterized by their affinity for ß-galactosides and a conserved binding site in the carbohydrate recognition domain (CRD). These lectins are involved in multiple physiological functions, including the recognition of glycans on the surface of viruses and bacteria. This feature supports their role in innate immune responses in marine mollusks. Here, we identified and characterized a galectin, from the mollusk Haliotis rufescens (named HrGal), with four CRDs that belong to the tandem-repeat type. HrGal was purified by affinity chromatography in a galactose-agarose resin and exhibited a molecular mass of 64.11 kDa determined by MALDI-TOF mass spectrometry. The identity of HrGal was verified by sequencing, confirming that it is a 555 amino acid protein with a mass of 63.86 kDa. This protein corresponds to a galectin reported in GenBank with accession number AHX26603. HrGal is stable in the presence of urea, reducing agents, and ions such as Cu2+ and Zn2+. The recombinant galectin (rHrGal) was purified from inclusion bodies in the presence of these ions. A theoretical model obtained with the AlphaFold server exhibits four non-identical CRDs, with a ß sandwich folding and the representative motifs for binding ß-galactosides. This allows us to classify HrGal within the tandem repeat galectin family. On the basis of a phylogenetic analysis, we found that the mollusk sequences form a monophyletic group of tetradomain galectins unrelated to vertebrate galectins. HrGal showed specificity for galactosides and glucosides but only the sulfated sugars heparin and ι-carrageenan inhibited its hemagglutinating activity with a minimum inhibitory concentration of 4 mM and 6.25 X 10-5% respectively. The position of the sulfate groups seemed crucial for binding, both by carrageenans and heparin.

The K+-Dependent and -Independent Pyruvate Kinases Acquire the Active Conformation by Different Mechanisms.

Eukarya pyruvate kinases possess glutamate at position 117 (numbering of rabbit muscle enzyme), whereas bacteria have either glutamate or lysine. Those with E117 are K+-dependent, whereas those with K117 are K+-independent. In a phylogenetic tree, 80% of the sequences with E117 are occupied by T113/K114/T120 and 77% of those with K117 possess L113/Q114/(L,I,V)120. This work aims to understand these residues' contribution to the K+-independent pyruvate kinases using the K+-dependent rabbit muscle enzyme. Residues 117 and 120 are crucial in the differences between the K+-dependent and -independent mutants. K+-independent activity increased with L113 and Q114 to K117, but L120 induced structural differences that inactivated the enzyme. T120 appears to be key in folding the protein and closure of the lid of the active site to acquire its active conformation in the K+-dependent enzymes. E117K mutant was K+-independent and the enzyme acquired the active conformation by a different mechanism. In the K+-independent apoenzyme of Mycobacterium tuberculosis, K72 (K117) flips out of the active site; in the holoenzyme, K72 faces toward the active site bridging the substrates through water molecules. The results provide evidence that two different mechanisms have evolved for the catalysis of this reaction.

Energetic and structural effects of the Tanford transition on ligand recognition of bovine ß-lactoglobulin.

Bovine ß-lactoglobulin, an abundant protein in whey, is a promising nanocarrier for peroral administration of drug-like hydrophobic molecules, a process that involves transit through the different acidic conditions of the human digestive tract. Among the several pH-induced conformational rearrangements that this lipocalin undergoes, the Tanford transition is particularly relevant. This transition, which occurs with a midpoint around neutral pH, involves a conformational change of the E-F loop that regulates accessibility to the primary binding site. The effect of this transition on the ligand binding properties of this protein has scarcely been explored. In this study, we carried out an energetic and structural characterization of ß-lactoglobulin molecular recognition at pH values above and below the zone in which the Tanford transition occurs. The combined analysis of crystallographic, calorimetric, and molecular dynamics data sheds new light on the interplay between self-association, ligand binding, and the Tanford pre- and post-transition conformational states, revealing novel aspects underlying the molecular recognition mechanism of this enigmatic lipocalin.

Crystal structure of a C-type lysozyme from Litopenaeus vanamei exhibiting a high binding constant to its chitotriose inhibitor.

Although information about invertebrate lysozymes is scarce, these enzymes have been described as components of the innate immune system, functioning as antibacterial proteins. Here we describe the first thermodynamic and structural study of a new C-type lysozyme from a Pacific white shrimp Litopenaeus vannamei (LvL), which has shown high activity against both Gram (+) and Gram (-) bacteria including Vibrio sp. that is one of the most severe pathogens in penaeid shrimp aquaculture. Compared with hen egg-white lysozyme, its sequence harbors a seven-residue insertion from amino acid 97 to 103, and a nine-residue extension at the C-terminus only found in penaeid crustaceans, making this enzyme one of the longest lysozyme reported to date. LvL was crystallized in the presence and absence of chitotriose. The former crystallized as a monomer in space group P61 and the latter in P212121 with two monomers in the asymmetric unit. Since the enzyme crystallized at a pH where lysozyme activity is deficient, the ligand could not be observed in the P61 structure; therefore, we performed a docking simulation with chitotriose to compare with the hen egg lysozyme crystallized in the presence of the ligand. Remarkably, additional amino acids in LvL caused an increase in the length of α-helix H4 (residues 97-103) that is directly related to ligand recognition. The Ka for chitotriose (4.1 × 105 M-1), as determined by Isothermal Titration Calorimetry, was one order of magnitude higher than those for lysozymes from hen and duck eggs. Our results revealed new interactions of chitiotriose with residues in helix H4.

Structure and conformational stability of the triosephosphate isomerase from Zea mays. Comparison with the chemical unfolding pathways of other eukaryotic TIMs.

We studied the structure, function and thermodynamic properties for the unfolding of the Triosephosphate isomerase (TIM) from Zea mays (ZmTIM). ZmTIM shows a catalytic efficiency close to the diffusion limit. Native ZmTIM is a dimer that dissociates upon dilution into inactive and unfolded monomers. Its thermal unfolding is irreversible with a Tm of 61.6 ±â€¯1.4 °C and an activation energy of 383.4 ±â€¯11.5 kJ mol-1. The urea-induced unfolding of ZmTIM is reversible. Transitions followed by catalytic activity and spectroscopic properties are monophasic and superimposable, indicating that ZmTIM unfolds/refolds in a two-state behavior with an unfolding ΔG°(H20) = 99.8 ±â€¯5.3 kJ mol-1. This contrasts with most other studied TIMs, where folding intermediates are common. The three-dimensional structure of ZmTIM was solved at 1.8 Å. A structural comparison with other eukaryotic TIMs shows a similar number of intramolecular and intermolecular interactions. Interestingly the number of interfacial water molecules found in ZmTIM is lower than those observed in most TIMs that show folding intermediates. Although with the available data, there is no clear correlation between structural properties and the number of equilibrium intermediates in the unfolding of TIM, the identification of such structural properties should increase our understanding of folding mechanisms.

Reversibility and two state behaviour in the thermal unfolding of oligomeric TIM barrel proteins.

Temperature is one of the main variables that modulate protein function and stability. Thermodynamic studies of oligomeric proteins, the dominant protein natural form, have been often hampered because irreversible aggregation and/or slow reactions are common. There are no reports on the reversible equilibrium thermal unfolding of proteins composed of (ß/α)8 barrel subunits, albeit this "TIM barrel" topology is one of the most abundant and versatile in nature. We studied the eponymous TIM barrel, triosephosphate isomerase (TIM), belonging to five species of different bacterial taxa. All of them were found to be catalytically efficient dimers. The three-dimensional structure of four enzymes was solved at high/medium resolution. Irreversibility and kinetic control were observed in the thermal unfolding of two TIMs, while for the other three the thermal unfolding was found to follow a two-state equilibrium reversible process. Shifts in the global stability curves of these three proteins are related to the organismal temperature range of optimal growth and modulated by variations in maximum stability temperature and in the enthalpy change at that temperature. Reversibility appears to correlate with the low isoelectric point, the absence of a residual structure in the unfolded state, small cavity volume in the native state, low conformational stability and a low melting temperature. Furthermore, the strong coupling between dimer dissociation and monomer unfolding may reduce aggregation and favour reversibility. It is therefore very thought-provoking to find that a common topological ensemble, such as the TIM barrel, can unfold/refold in the Anfinsen way, i.e. without the help of the cellular machinery.

Enhanced antimicrobial activity of novel synthetic peptides derived from vejovine and hadrurin.

BACKGROUND: Microbial antibiotic resistance is a challenging medical problem nowadays. Two scorpion peptides displaying antibiotic activity: hadrurin and vejovine were taken as models for the design of novel shorter peptides with similar activity. METHODS: Using the standard Fmoc-based solid phase synthesis technique of Merrifield twelve peptides (18 to 29 amino acids long) were synthesized, purified and assayed against a variety of multi-drug resistant Gram-negative bacteria from clinical isolates. Hemolytic and antiparasitic activities of the peptides and their possible interactions with eukaryotic cells were verified. Release of the fluorophore calcein from liposomes treated with these peptides was measured. RESULTS: A peptide with sequence GILKTIKSIASKVANTVQKLKRKAKNAVA), and three analogs: Δ(Α29), Δ(K12-Q18; Ν26-Α29), and K4N Δ(K12-Q18; Ν26-Α29) were shown to inhibit the growth of Gram-negative (E. coli ATCC25922) and Gram-positive bacteria (S. aureus), as well as multi-drug resistant (MDR) clinical isolated. The antibacterial and antiparasitic activities were found with peptides at 0.78 to 25µM and 5 to 25µM concentration, respectively. These peptides have low cytotoxic and hemolytic activities at concentrations significantly exceeding their minimum inhibitory concentrations (MICs), showing values between 40 and 900µM for their EC50, compared to the parent peptides vejovine and hadrurin that at the same concentration of their MICs lysed more than 50% of human erythrocytes cells. CONCLUSIONS: These peptides promise to be good candidates to combat infections caused by Gram-negative bacteria from nosocomial infections. GENERAL SIGNIFICANCE: Our results confirm that well designed synthetic peptides can be an alternative for solving the lack of effective antibiotics to control bacterial infections.

Structural analysis of the endogenous glycoallergen Hev b 2 (endo-ß-1,3-glucanase) from Hevea brasiliensis and its recognition by human basophils.

Endogenous glycosylated Hev b 2 (endo-ß-1,3-glucanase) from Hevea brasiliensis is an important latex allergen that is recognized by IgE antibodies from patients who suffer from latex allergy. The carbohydrate moieties of Hev b 2 constitute a potentially important IgE-binding epitope that could be responsible for its cross-reactivity. Here, the structure of the endogenous isoform II of Hev b 2 that exhibits three post-translational modifications, including an N-terminal pyroglutamate and two glycosylation sites at Asn27 and at Asn314, is reported from two crystal polymorphs. These modifications form a patch on the surface of the molecule that is proposed to be one of the binding sites for IgE. A structure is also proposed for the most important N-glycan present in this protein as determined by digestion with specific enzymes. To analyze the role of the carbohydrate moieties in IgE antibody binding and in human basophil activation, the glycoallergen was enzymatically deglycosylated and evaluated. Time-lapse automated video microscopy of basophils stimulated with glycosylated Hev b 2 revealed basophil activation and degranulation. Immunological studies suggested that carbohydrates on Hev b 2 represent an allergenic IgE epitope. In addition, a dimer was found in each asymmetric unit that may reflect a regulatory mechanism of this plant defence protein.

Regulation of sporulation initiation by NprR and its signaling peptide NprRB: molecular recognition and conformational changes.

NprR belongs to the RNPP family of quorum-sensing receptors, a group of intracellular regulators activated directly by signaling oligopeptides in Gram-positive bacteria. In Bacillus thuringiensis (Bt), nprR is located in a transcriptional cassette with nprRB that codes for the precursor of the signaling peptide NprRB. NprR is a transcriptional regulator activated by binding of reimported NprRB; however, several reports suggest that NprR also participates in sporulation but the mechanism is unknown. Our in silico results, based on the structural similarity between NprR from Bt and Spo0F-binding Rap proteins from Bacillus subtilis, suggested that NprR could bind Spo0F to modulate the sporulation phosphorelay in Bt. Deletion of nprR-nprRB cassette from Bt caused a delay in sporulation and defective trigger of the Spo0A∼P-activated genes spoIIA and spoIIIG. The DNA-binding domain of NprR was not necessary for this second function, since truncated NprRΔHTH together with nprRB gene was able to restore the sporulation wild type phenotype in the ΔnprR-nprRB mutant. Fluorescence assays showed direct binding between NprR and Spo0F, supporting that NprR is a bifunctional protein. To understand how the NprR activation by NprRB could result in two different functions, we studied the molecular recognition mechanism between the signaling peptide and the receptor. Using synthetic variants of NprRB, we found that SSKPDIVG displayed the highest affinity (Kd = 7.19 nM) toward the recombinant NprR and demonstrated that recognition involves conformational selection. We propose that the peptide concentration in the cell controls the oligomerization state of the NprR-NprRB complex for switching between its two functions.

A trimeric glycosylated GH45 cellulase from the red abalone (Haliotis rufescens) exhibits endo and exoactivity.

The red abalone (Haliotis rufescens) represents North America's most important aquaculture species. Its hepatopancreas is rich in cellulases and other polysaccharide-degrading enzymes, which provide it the remarkable ability to digest cellulose-rich macroalgae; nevertheless, its cellulolytic systems are poorly explored. This manuscript describes some functional and structural properties of an endogenous trimeric glycosylated endoglucanase from H. rufescens. The purified enzyme showed a molecular mass of 23.4 kDa determined by MALDI-TOF mass spectrometry, which behaved as a homotrimer in gel filtration chromatography and zymograms. According to the periodic acid-Schiff reagent staining, detecting sugar moieties in SDS-PAGE gel confirmed that abalone cellulase is a glycoprotein. Hydrolysis of cello-oligosaccharides and p-nitrophenyl-ß-D-glucopyranosides confirmed its endo/exoactivity. A maximum enzyme activity toward 0.5% (w/v) carboxymethylcellulose of 53.9 ± 1.0 U/mg was achieved at 45°C and pH 6.0. We elucidated the abalone cellulase primary structure using proteases and mass spectrometry methods. Based on these results and using a bioinformatic approach, we identified the gene encoding this enzyme and deduced its full-length amino acid sequence; the mature protein comprised 177 residues with a calculated molecular mass of 19.1 kDa and, according to sequence similarity, it was classified into the glycosyl-hydrolase family 45 subfamily B. An AlphaFold theoretical model and docking simulations with cellopentaose confirmed that abalone cellulase is a ß-sheet rich protein, as also observed by circular dichroism experiments, with conserved catalytic residues: Asp26, Asn109, and Asp134. Interestingly, the AlphaFold-Multimer analysis indicated a trimeric assembly for abalone cellulase, which supported our experimental findings. The discovery and characterization of these enzymes may contribute to developing efficient cellulose bioconversion processes for biofuels and sustainable bioproducts.

Protein tyrosine phosphatase 1B (PTP1B) function, structure, and inhibition strategies to develop antidiabetic drugs.

Tyrosine protein phosphatase non-receptor type 1 (PTP1B; also known as protein tyrosine phosphatase 1B) is a member of the protein tyrosine phosphatase (PTP) family and is a soluble enzyme that plays an essential role in different physiological processes, including the regulation of metabolism, specifically in insulin and leptin sensitivity. PTP1B is crucial in the pathogenesis of type 2 diabetes mellitus and obesity. These biological functions have made PTP1B validated as an antidiabetic and anti-obesity, and potentially anticancer, molecular target. Four main approaches aim to inhibit PTP1B: orthosteric, allosteric, bidentate inhibition, and PTPN1 gene silencing. Developing a potent and selective PTP1B inhibitor is still challenging due to the enzyme's ubiquitous expression, subcellular location, and structural properties. This article reviews the main advances in the study of PTP1B since it was first isolated in 1988, as well as recent contextual information related to the PTP family to which this protein belongs. Furthermore, we offer an overview of the role of PTP1B in diabetes and obesity, and the challenges to developing selective, effective, potent, bioavailable, and cell-permeable compounds that can inhibit the enzyme.

Ligand binding and self-association cooperativity of ß-lactoglobulin.

Unlike most small globular proteins, lipocalins lack a compact hydrophobic core. Instead, they present a large central cavity that functions as the primary binding site for hydrophobic molecules. Not surprisingly, these proteins typically exhibit complex structural dynamics in solution, which is intricately modified by intermolecular recognition events. Although many lipocalins are monomeric, an increasing number of them have been proven to form oligomers. The coupling effects between self-association and ligand binding in these proteins are largely unknown. To address this issue, we have calorimetrically characterized the recognition of dodecyl sulfate by bovine ß-lactoglobulin, which forms weak homodimers at neutral pH. A thermodynamic analysis based on coupled-equilibria revealed that dimerization exerts disparate effects on the ligand-binding capacity of ß-lactoglobulin. Protein dimerization decreases ligand affinity (or, reciprocally, ligand binding promotes dimer dissociation). The two subunits in the dimer exhibit a positive, entropically driven cooperativity. To investigate the structural determinants of the interaction, the crystal structure of ß-lactoglobulin bound to dodecyl sulfate was solved at 1.64 Å resolution.

Liver exhibits thermal variations according to the stage of fibrosis progression: A novel use of modulated-differential scanning calorimetry for research in hepatology.

AIM: Liver fibrosis results in a disproportion of the hepatic composition and architecture, characterized by a progressive accumulation of fibrillar proteins at the liver parenchyma. Modulated-differential scanning calorimetry (mDSC) is an experimental methodology able to determine the specific thermal signature from any biological substance, based on the variation in heat flow and heat capacity. As these physicochemical properties are directly influenced by compositional and structural changes, we decided to study the thermal behavior of the liver during fibrosis using mDSC. METHODS: Liver fibrosis was induced in rats by bile duct ligation or carbon tetrachloride administration. Degree of liver fibrosis was determined by histological examination using the Masson-trichrome stain, accompanied by hepatic expression of α-smooth muscle actin. The thermal analysis was performed in a modulated-differential scanning calorimeter using 20 mg of fresh liver mass. RESULTS: The liver showed a characteristic thermal signature in control animals, which progressively differed among mild (F1), moderate (F2) and advanced (F3-F4) liver fibrosis. For heat flow, the hepatic thermal signature from F3-F4 rats exhibited significant differences when compared with F1, F2 and controls. In terms of heat capacity, liver specimens provided a specific thermal signature for each stage of disease, characterized by a transition temperature onset at 95°C for controls, whereas in F1, F2 and F3-F4 animals this temperature significantly decreased to 93°C, 84°C and 75°C, respectively. CONCLUSION: Because the liver shows a differential thermal signature according to the degree of fibrosis, mDSC could be a novel tool in the study of liver fibrosis progression.

Molecular Basis of Plant Profilins' Cross-Reactivity.

Profilins are ubiquitous allergens with conserved structural elements. Exposure to profilins from different sources leads to IgE-cross-reactivity and the pollen-latex-food syndrome. Monoclonal antibodies (mAbs) that cross-react with plant profilins and block IgE-profilin interactions are relevant for diagnosis, epitope mapping, and specific immunotherapy. We generated IgGs mAbs, 1B4, and 2D10, against latex profilin (anti-rHev b 8) that inhibit the interaction of IgE and IgG4 antibodies from sera of latex- and maize-allergic patients by 90% and 40%, respectively. In this study, we evaluated 1B4 and 2D10 recognition towards different plant profilins, and mAbs recognition of rZea m 12 mutants by ELISAs. Interestingly, 2D10 highly recognized rArt v 4.0101 and rAmb a 8.0101, and to a lesser extent rBet v 2.0101, and rFra e 2.2, while 1B4 showed recognition for rPhl p 12.0101 and rAmb a 8.0101. We demonstrated that residue D130 at the α-helix 3 in profilins, which is part of the Hev b 8 IgE epitope, is essential for the 2D10 recognition. The structural analysis suggests that the profilins containing E130 (rPhl p 12.0101, rFra e 2.2, and rZea m 12.0105) show less binding with 2D10. The distribution of negative charges on the profilins' surfaces at the α-helices 1 and 3 is relevant for the 2D10 recognition, and that may be relevant to explain profilins' IgE cross-reactivity.

Substrate binding in the allosteric site mimics homotropic cooperativity in the SIS-fold glucosamine-6-phosphate deaminases.

Glucosamine-6-phosphate (GlcN6P) deaminases from Escherichia coli (EcNagBI) and Shewanella denitrificans (SdNagBII) are special examples of what constitute nonhomologous isofunctional enzymes due to their convergence, not only in catalysis, but also in cooperativity and allosteric properties. Additionally, we found that the sigmoidal kinetics of SdNagBII cannot be explained by the existing models of homotropic activation. This study describes the regulatory mechanism of SdNagBII using enzyme kinetics, isothermal titration calorimetry (ITC), and X-ray crystallography. ITC experiments revealed two different binding sites with distinctive thermodynamic signatures: a single binding site per monomer for the allosteric activator N-acetylglucosamine 6-phosphate (GlcNAc6P) and two binding sites per monomer for the transition-state analog 2-amino-2-deoxy-D-glucitol 6-phosphate (GlcNol6P). Crystallographic data demonstrated the existence of an unusual allosteric site that can bind both GlcNAc6P and GlcNol6P, implying that the homotropic activation of this enzyme arises from the occupation of the allosteric site by the substrate. In this work we describe the presence of this novel allosteric site in the SIS-fold deaminases, which is responsible for the homotropic and heterotropic activation of SdNagBII by GlcN6P and GlcNAc6P, respectively. This study unveils an original mechanism to generate a high degree of homotropic activation in SdNagBII, mimicking the allosteric and cooperative properties of hexameric EcNagBI but with a reduced number of subunits.

Thermodynamic and kinetic analysis of the LAO binding protein and its isolated domains reveal non-additivity in stability, folding and function.

Substrate-binding proteins (SBPs) are used by organisms from the three domains of life for transport and signalling. SBPs are composed of two domains that collectively trap ligands with high affinity and selectivity. To explore the role of the domains and the integrity of the hinge region between them in the function and conformation of SBPs, here, we describe the ligand binding, conformational stability and folding kinetics of the Lysine Arginine Ornithine (LAO) binding protein from Salmonella thiphimurium and constructs corresponding to its two independent domains. LAO is a class II SBP formed by a continuous and a discontinuous domain. Contrary to the expected behaviour based on their connectivity, the discontinuous domain shows a stable native-like structure that binds l-arginine with moderate affinity, whereas the continuous domain is barely stable and shows no detectable ligand binding. Regarding folding kinetics, studies of the entire protein revealed the presence of at least two intermediates. While the unfolding and refolding of the continuous domain exhibited only a single intermediate and simpler and faster kinetics than LAO, the folding mechanism of the discontinuous domain was complex and involved multiple intermediates. These findings suggest that in the complete protein the continuous domain nucleates folding and that its presence funnels the folding of the discontinuous domain avoiding nonproductive interactions. The strong dependence of the function, stability and folding pathway of the lobes on their covalent association is most likely the result of the coevolution of both domains as a single unit.

Effects of a buried cysteine-to-serine mutation on yeast triosephosphate isomerase structure and stability.

All the members of the triosephosphate isomerase (TIM) family possess a cystein residue (Cys126) located near the catalytically essential Glu165. The evolutionarily conserved Cys126, however, does not seem to play a significant role in the catalytic activity. On the other hand, substitution of this residue by other amino acid residues destabilizes the dimeric enzyme, especially when Cys is replaced by Ser. In trying to assess the origin of this destabilization we have determined the crystal structure of Saccharomyces cerevisiae TIM (ScTIM) at 1.86 Å resolution in the presence of PGA, which is only bound to one subunit. Comparisons of the wild type and mutant structures reveal that a change in the orientation of the Ser hydroxyl group, with respect to the Cys sulfhydryl group, leads to penetration of water molecules and apparent destabilization of residues 132-138. The latter results were confirmed by means of Molecular Dynamics, which showed that this region, in the mutated enzyme, collapses at about 70 ns.

A native IgE in complex with profilin provides insights into allergen recognition and cross-reactivity.

Allergies have become a rising health problem, where plentiful substances can trigger IgE-mediated allergies in humans. While profilins are considered minor allergens, these ubiquitous proteins are primary molecules involved in cross-reactivity and pollen-food allergy syndrome. Here we report the first crystal structures of murine Fab/IgE, with its chains naturally paired, in complex with the allergen profilin from Hevea brasiliensis (Hev b 8). The crystallographic models revealed that the IgE's six complementarity-determining regions (CDRs) interact with the allergen, comprising a rigid paratope-epitope surface of 926 Å2, which includes an extensive network of interactions. Interestingly, we also observed previously unreported flexibility at Fab/IgE's elbow angle, which did not influence the shape of the paratope. The Fab/IgE exhibits a high affinity for Hev b 8, even when using 1 M NaCl in BLI experiments. Finally, based on the encouraging cross-reactivity assays using two mutants of the maize profilin (Zea m 12), this antibody could be a promising tool in IgE engineering for diagnosis and research applications.

Determining the molecular mechanism of inactivation by chemical modification of triosephosphate isomerase from the human parasite Giardia lamblia: a study for antiparasitic drug design.

Giardiasis, the most prevalent intestinal parasitosis in humans, is caused by Giardia lamblia. Current drug therapies have adverse effects on the host, and resistant strains against these drugs have been reported, demonstrating an urgent need to design more specific antigiardiasic drugs. ATP production in G. lamblia depends mainly on glycolysis; therefore, all enzymes of this pathway have been proposed as potential drug targets. We previously demonstrated that the glycolytic enzyme triosephosphate isomerase from G. lamblia (GlTIM), could be completely inactivated by low micromolar concentrations of thiol-reactive compounds, whereas, in the same conditions, the activity of human TIM (HuTIM) was almost unaltered. We found that the chemical modification (derivatization) of at least one Cys, of the five Cys residues per monomer in GlTIM, causes this inactivation. In this study, structural and functional studies were performed to describe the molecular mechanism of GlTIM inactivation by thiol-reactive compounds. We found that the Cys222 derivatization is responsible for GlTIM inactivation; this information is relevant because HuTIM has a Cys residue in an equivalent position (Cys217). GlTIM inactivation is associated with a decrease in ligand affinity, which affects the entropic component of ligand binding. In summary, this work describes a mechanism of inactivation that has not been previously reported for TIMs from other parasites and furthermore, we show that the difference in reactivity between the Cys222 in GlTIM and the Cys217 in HuTIM, indicates that the surrounding environment of each Cys residue has unique structural differences that can be exploited to design specific antigiardiasic drugs.