RESUMEN

Wheat germ agglutinin (WGA) demonstrates potential as an oral delivery agent owing to its selective binding to carbohydrates and its capacity to traverse biological membranes. In this study, we employed differential scanning calorimetry and molecular dynamics simulations to comprehensively characterize the thermal unfolding process of both the complete lectin and its four isolated domains. Furthermore, we present the nuclear magnetic resonance structures of three domains that were previously lacking experimental structures in their isolated forms. Our results provide a collective understanding of the energetic and structural factors governing the intricate unfolding mechanism of the complete agglutinin, shedding light on the specific role played by each domain in this process. The analysis revealed negligible interdomain cooperativity, highlighting instead significant coupling between dimer dissociation and the unfolding of the more labile domains. By comparing the dominant interactions, we rationalized the stability differences among the domains. Understanding the structural stability of WGA opens avenues for enhanced drug delivery strategies, underscoring its potential as a promising carrier throughout the gastrointestinal environment.

Asunto(s)

Estabilidad Proteica , Aglutininas del Germen de Trigo , Rastreo Diferencial de Calorimetría , Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular , Dominios Proteicos , Aglutininas del Germen de Trigo/química

RESUMEN

We report the characterization of the three-dimensional structure of the CEMP1-p1 peptide [MGTSSTDSQQAQHRRCSTSN: corresponding to residues 1-20 of the N-terminus of cementum protein 1 (CEMP1)]. This peptide imitates the capacity of CEMP1 to stimulate hydroxyapatite (HA) crystal nucleation and growth, and promotes the differentiation of periodontal ligament cells into a cementoblastic phenotype. Additionally, in experimental models of critical-sized calvarial defects in Wistar rats, CEMP1-p1 has shown osteogenic properties that enhanced the physiological deposition and maturation of newly formed bone. In this work, studies of CEMP1-p1 by circular dichroism (CD) and nuclear magnetic resonance (NMR) were performed in trifluoroethanol D2 (TFED2) and aqueous solution to determine the 3D structure of the peptide. Using the 3D model, experimental data from HA crystals formation and calcium fluorescence emission, we explain the biological mechanisms involved in CEMP1-p1 activity to promote calcium recruitment and its affinity to HA crystals. This information is valuable because it proposes, for the first time, a plausible molecular mechanism during the mineralization process, from a specific cementum protein-derived peptide.

Asunto(s)

Calcio , Cemento Dental , Ratas , Animales , Ratas Wistar , Péptidos/química , Espectroscopía de Resonancia Magnética

RESUMEN

Potassium channels play a key role in regulating many physiological processes, thus, alterations in their proper functioning can lead to the development of several diseases. Hence, the search for compounds capable of regulating the activity of these channels constitutes an intense field of investigation. Potassium scorpion toxins are grouped into six subfamilies (α, ß, γ, κ, δ, and λ). However, experimental structures and functional analyses of the long chain ß-KTx subfamily are lacking. In this study, we recombinantly produced the toxins TcoKIK and beta-KTx14.3 present in the venom of Tityus costatus and Lychas mucronatus scorpions, respectively. The 3D structures of these ß-KTx toxins were determined by nuclear magnetic resonance. In both toxins, the N-terminal region is unstructured, while the C-terminal possesses the classic CSα/ß motif. TcoKIK did not show any clear activity against frog Shaker and human KCNQ1 potassium channels; however, beta-KTx14.3 was able to block the KCNQ1 channel. The toxin-channel interaction mode was investigated using molecular dynamics simulations. The results showed that this toxin could form a stable network of polar-to-polar and hydrophobic interactions with KCNQ1, involving key conserved residues in both molecular partners. The discovery and characterization of a toxin capable of inhibiting KCNQ1 pave the way for the future development of novel drugs for the treatment of human diseases caused by the malfunction of this potassium channel. STATEMENT OF SIGNIFICANCE: Scorpion toxins have been shown to rarely block human KCNQ1 channels, which participate in the regulation of cardiac processes. In this study, we obtained recombinant beta-KTx14.3 and TcoKIK toxins and determined their 3D structures by nuclear magnetic resonance. Electrophysiological studies and molecular dynamics models were employed to examine the interactions between these two toxins and the human KCNQ1, which is the major driver channel of cardiac repolarization; beta-KTx14.3 was found to block effectively this channel. Our findings provide insights for the development of novel toxin-based drugs for the treatment of cardiac channelopathies involving KCNQ1-like channels.

Asunto(s)

Canales de Potasio , Venenos de Escorpión , Humanos , Canales de Potasio/metabolismo , Venenos de Escorpión/farmacología , Venenos de Escorpión/química , Secuencia de Aminoácidos , Canal de Potasio KCNQ1/genética , Simulación de Dinámica Molecular

RESUMEN

Peptide-based therapy against cancer is a field of great interest for biomedical developments. Since it was shown that SK3 channels promote cancer cell migration and metastatic development, we started using these channels as targets for the development of antimetastatic drugs. Particularly, tamapin (a peptide found in the venom of the scorpion Mesobuthus tamulus) is the most specific toxin against the SK2 channel currently known. Considering this fact, we designed diverse tamapin mutants based on three different hypotheses to discover a new potent molecule to block SK3 channels. We performed in vitro studies to evaluate this new toxin derivative inhibitor of cancer cell migration. Our results can be used to generate a new tamapin-based therapy against cancer cells that express SK3 channels.

RESUMEN

The genome of Entamoeba histolytica encodes approximately 50 Cysteine Proteases (CPs) whose activity is regulated by two Inhibitors of Cysteine Proteases (ICPs), EhICP1 and EhICP2. The main difference between both EhICPs is the acquisition of a 17 N-terminal targeting signal in EhICP2 and three exposed cysteine residues in EhICP1. The three exposed cysteines in EhICP1 potentiate the formation of cross-linking species that drive heterogeneity. Here we solved the NMR structure of EhICP1 using a mutant protein without accessible cysteines. Our structural data shows that EhICP1 adopts an immunoglobulin fold composed of seven ß-strands, and three solvent exposed loops that resemble the structures of EhICP2 and chagasin. EhICP1 and EhICP2 are able to inhibit the archetypical cysteine protease papain by intercalating their BC loops into the protease active site independently of the character of the residue (serine or threonine) responsible to interact with the active site of papain. EhICP1 and EhICP2 present signals of functional divergence as they clustered in different clades. Two of the three exposed cysteines in EhICP1 are located at the DE loop that intercalates into the CP substrate-binding cleft. We propose that the solvent exposed cysteines of EhICP1 play a role in regulating its inhibitory activity and that in oxidative conditions, the cysteines of EhICP1 react to form intra and intermolecular disulfide bonds that render an inactive inhibitor. EhICP2 is not subject to redox regulation, as this inhibitor does not contain a single cysteine residue. This proposed redox regulation may be related to the differential cellular localization between EhICP1 and EhICP2.

Asunto(s)

Entamoeba histolytica , Proteínas Protozoarias/química , Clonación Molecular , Inhibidores de Cisteína Proteinasa , Entamoeba histolytica/genética , Escherichia coli/genética , Mutagénesis Sitio-Dirigida , Papaína/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Soluciones

RESUMEN

Cl13 is a toxin purified previously from the venom of the Mexican scorpion Centruroides limpidus. This toxin affects the function of voltage gated Na+-channels, human subtypes Nav1.4, Nav1.5 and Nav1.6 in a similar manner as other known ß-toxins from scorpion venoms. Here, we report a correction of the primary structure of Cl13, previously published. The peptide does contain 66 amino acids, but residue 58 is a tryptophan and the last C-terminal amino acid is an amidated lysine, instead of arginine. The main contribution of this communication is the determination of the 3D-structure of Cl13, by solution NMR, showing that Cl13 has the classical cysteine-stabilized α/ß (CSα/ß) folding. It has a triple stranded antiparallel beta sheet commonly present in scorpion sodium channel ß-toxins. In addition, we report and discuss a comparison of Cl13 structure with two other toxins (Cn2 and Css2) from scorpions of the same genus Centruroides, which shows important surface similarities with the structure reported here. Finally, the lack of neutralization of Cl13 toxin by two single-chain antibody fragments (scFvs), named LR and 10FG2, which are capable of neutralizing various toxins from Mexican scorpions, is revised. In particular, 10FG2 is capable of neutralizing toxins Cll1 and Cll2 of the same scorpion C. limpidus. The reasons why LR and 10FG2 are unable of neutralizing Cl13 toxin are discussed.

Asunto(s)

Venenos de Escorpión/química , Secuencia de Aminoácidos , Animales , Cisteína , Espectroscopía de Resonancia Magnética , México , Escorpiones

RESUMEN

Wheat germ agglutinin (WGA), a chitin binding lectin, has attracted increasing interest because of its unique characteristics such as conformational stability, binding specificity and transcytosis capacity. To pave the way for the study of the molecular basis of WGA's structural stability and binding capacity, as well as to facilitate its use in biomedical and biotechnological developments, we produced recombinant WGA and its 4 isolated hevein-like domains in a bacterial system. All the proteins were expressed as fusion constructs linked to a thioredoxin domain, which was enzymatically or chemically released. The structural and ligand-binding properties of recombinant WGA were similar to the wild lectin. The 4 isolated domains folded and were ligand-binding competent, indicating that each domain constitutes an independent folding unity. The biophysical characterization of the recombinant domains sheds new light on the intricate folding and binding behavior of this emblematic lectin.

Asunto(s)

Péptidos Catiónicos Antimicrobianos/química , Fenómenos Biofísicos , Lectinas de Plantas/química , Triticum/química , Aglutininas del Germen de Trigo/química , Péptidos Catiónicos Antimicrobianos/genética , Células Germinativas/química , Lectinas de Plantas/genética , Triticum/genética , Aglutininas del Germen de Trigo/genética

RESUMEN

Animal venoms are rich sources of ligands for studying ion channels and other pharmacological targets. Proteomic analyses of the soluble venom from the Mexican scorpion Vaejovis mexicanus smithi showed that it contains more than 200 different components. Among them, a 36-residue peptide with a molecular mass of 3864 Da (named Vm24) was shown to be a potent blocker of Kv1.3 of human lymphocytes (K(d) ∼ 3 pM). The three-dimensional solution structure of Vm24 was determined by nuclear magnetic resonance, showing the peptide folds into a distorted cystine-stabilized α/ß motif consisting of a single-turn α-helix and a three-stranded antiparallel ß-sheet, stabilized by four disulfide bridges. The disulfide pairs are formed between Cys6 and Cys26, Cys12 and Cys31, Cys16 and Cys33, and Cys21 and Cys36. Sequence analyses identified Vm24 as the first example of a new subfamily of α-type K(+) channel blockers (systematic number α-KTx 23.1). Comparison with other Kv1.3 blockers isolated from scorpions suggests a number of structural features that could explain the remarkable affinity and specificity of Vm24 toward Kv1.3 channels of lymphocytes.

Asunto(s)

Canal de Potasio Kv1.3/antagonistas & inhibidores , Bloqueadores de los Canales de Potasio/farmacología , Venenos de Escorpión/química , Venenos de Escorpión/farmacología , Linfocitos T/efectos de los fármacos , Secuencias de Aminoácidos , Animales , Disulfuros/química , Evaluación Preclínica de Medicamentos/métodos , Humanos , Espectroscopía de Resonancia Magnética , Ratones , Modelos Moleculares , Péptidos/síntesis química , Péptidos/química , Péptidos/farmacología , Filogenia , Conformación Proteica , Venenos de Escorpión/síntesis química , Escorpiones/química

RESUMEN

The three-dimensional structures of the long-chain mammalian scorpion ß-toxin CssII from Centruroides suffusus suffusus and of its recombinant form, HisrCssII, were determined by NMR. The neurotoxin CssII (nCssII) is a 66 amino acid long peptide with four disulfide bridges; it is the most abundant and deadly toxin from the venom of this scorpion. Both native and recombinant CssII structures were determined by nuclear magnetic resonance using a total of 828 sequential distance constraints derived from the volume integration of the cross peaks observed in 2D NOESY spectra. Both nCssII and HisrCssII structures display a mixed α/ß fold stabilized by four disulfide bridges formed between pairs of cysteines: C1-C8, C2-C5, C3-C6, and C4-C7 (the numbers indicate the relative positions of the cysteine residues in the primary structure), with a distortion induced by two cis-prolines in its C-terminal part. The native CssII electrostatic surface was compared to both the recombinant one and to the Cn2 toxin, from the scorpion Centruroides noxius, which is also toxic to mammals. Structural features such N- and C-terminal differences could influence toxin specificity and affinity towards isoforms of different sub-types of Na(v) channels.

Asunto(s)

Canal de Sodio Activado por Voltaje NAV1.5/química , Neurotoxinas/química , Venenos de Escorpión/química , Escorpiones/química , Potenciales de Acción/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Células CHO , Cricetinae , Cisteína/química , Disulfuros , Escherichia coli/genética , Humanos , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Canal de Sodio Activado por Voltaje NAV1.5/genética , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Neurotoxinas/genética , Neurotoxinas/aislamiento & purificación , Neurotoxinas/toxicidad , Técnicas de Placa-Clamp , Prolina/química , Pliegue de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/toxicidad , Venenos de Escorpión/genética , Venenos de Escorpión/aislamiento & purificación , Venenos de Escorpión/toxicidad , Escorpiones/patogenicidad , Soluciones , Electricidad Estática , Transfección

RESUMEN

Cn12 isolated from the venom of the scorpion Centruroides noxius has 67 amino-acid residues, closely packed with four disulfide bridges. Its primary structure and disulfide bridges were determined. Cn12 is not lethal to mammals and arthropods in vivo at doses up to 100 microg per animal. Its 3D structure was determined by proton NMR using 850 distance constraints, 36 phi angles derived from 36 coupling constants obtained by two different methods, and 22 hydrogen bonds. The overall structure has a two and half turn alpha-helix (residues 24-32), three strands of antiparallel beta-sheet (residues 2-4, 37-40 and 45-48), and a type II turn (residues 41-44). The amino-acid sequence of Cn12 resembles the beta scorpion toxin class, although patch-clamp experiments showed the induction of supplementary slow inactivation of Na(+) channels in F-11 cells (mouse neuroblastoma N18TG-2 x rat DRG2), which means that it behaves more like an alpha scorpion toxin. This behaviour prompted us to analyse Na(+) channel binding sites using information from 112 Na(+) channel gene clones available in the literature, focusing on the extracytoplasmic loops of the S5-S6 transmembrane segments of domain I and the S3-S4 segments of domain IV, sites considered to be responsible for binding alpha scorpion toxins.

Asunto(s)

Estructura Secundaria de Proteína , Venenos de Escorpión/metabolismo , Escorpiones/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Humanos , Enlace de Hidrógeno , Espectroscopía de Resonancia Magnética , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Técnicas de Placa-Clamp , Filogenia , Unión Proteica , Estructura Terciaria de Proteína , Ratas , Venenos de Escorpión/química , Venenos de Escorpión/clasificación , Venenos de Escorpión/genética , Alineación de Secuencia , Canales de Sodio/química , Canales de Sodio/metabolismo