Clonagem de cDNAS codificando globulinas 7S (Vicilinas) de dois genótipos de CAUPI [Vigna unguiculata (L.) Walp] com resposta contrastante ao caruncho (Callosobruchus maculatus): simulações computacionais revelam como as vicilinas do CAUPI interagem com Quitina.

Abstract

Cowpea (Vigna unguiculata) is a legume that has great socioeconomic importance in the Brazilian Northeast. However, in the post-harvest phase, one of the problems faced by farmers is the infestation by Calosobruchus maculatus (Coleoptera: Bruchidae). This pest can cause significant damage to the seeds, reducing their nutritional content and economic value. Several studies correlate the resistance of some cowpea cultivars to one or more variants of vicillins, 7S globulins that are one of the main reserve proteins of these seeds. These variants bind strongly to the chitin of the peritrophic membrane of C. maculatus larvae, impairing the absorption of nutrients and, consequently, causing their mortality. On the other hand, varicose veins of cultivars susceptible to C. maculatus bind poorly to chitin. But so far, the structural differences that are at the origin of the differentiated interaction between the variants of vicillin and chitin are still unknown. For this reason, the main objective of the present study was to investigate the structural basis of this differentiated behavior of variants of 7S vicillins that could explain, at least in part, the resistance of seeds of some cowpea cultivars to C. maculatus. In this work, partial cDNA sequences coding for cowpea vicillin were obtained from developing seeds of the EPACE-10 genotypes (susceptible to C. maculatus) and IT81D-1053 (brucellosis resistant) genotypes by PCR amplification, cloning and sequencing. The deduced amino acid sequences of the cDNA sequences were then compared to verify possible differences that could exist between the variants of 7S vicilins. Three-dimensional molecular models of cowpea vicillin, obtained by homology modeling, showed the typical characteristics of the domain of the members of the superfamily of the termites. Computational simulations using docking techniques and molecular dynamics revealed that each trimer of vicillin contained three chitin binding sites, each located at the apex of the triangle-shaped oligomer, which is characteristic of 7S globulins. The interaction models predicted that the carbohydrate molecules, embedded in the identified sites, were stabilized mainly by hydrogen bonds, many of them mediated by water molecules, a common structural feature observed in a large variety of proteins that bind to carbohydrates. In addition, many of the residues involved in the chitin binding sites of cowpea vicillin are conserved in other 7S globulins. Using a quantum mechanical-based method, the free energies of binding of chito-oligosaccharide molecules to the β-vignin oligomers were calculated, ranging from -690.6 kcal / mol (GlcNAc oligomers bound to β-vignin trimer R2) to -531.3 kcal / mol (chitin fragments bound to the β-vignin oligomer R3). These results agree with previous studies that demonstrated that cowpea vicillins purified from seeds of C. maculatus susceptible or resistant genotypes have the ability to bind in vitro to chitin and in vivo to chitin structures of the midgut of larvae. In addition, the conservation in other 7S globulins of many of the residues constituting the β-vignin chitin binding site as predicted in this paper also supports previous hypotheses that have shown that the vicillins of other legume species can also bind to chitin. The results of the present work provide the first description of the molecular mechanism involved in the interaction between cowpea and chitin vicillins.