Modelling study of DefSm-D, a plant defensin from flowers of Silybum marianum (Asteraceae)

AGUSTINA FERNÁNDEZ; MARIA LAURA COLOMBO; SANDRA ELIZABETH VAIRO CAVALLI; CONSTANZA LIGGIERI; LAURA SUSANA BAKÁS

Salto Grande

Congreso; Latin American Crosstalk In Biophysics And Physiology; 2015

SBF y SAB

Plant defensins are a family of cationicantimicrobial peptides ubiquitously expressed, involved in innate immunedefense. They are small cysteine rich peptides withhighly variable sequence but with a conserved structure. Sequencediversity appears to be correlated with differential functions [1].The objectives of this study were to build a 3D model and to identify theputative function of a defensin cloned from flowers of Silybum marianum (L.) Gaertn. (Asteraceae). The ultimate goal ofthis work is the rational design of new proteins with novel function orbehaviour. Total RNA was isolated from immature flowers harvested in spring. cDNAwas synthesized by retrotranscription of total RNA and used as PCR templatewith specific primers. Full length cDNA was generated by 5?RACE. The amplifiedproduct of 774 bp was cloned, analysed by restriction mapping and sequenced. In silico analysis of deduced amino acidsequence enabled the identification of a signal peptide, a defensin domain (DefSm-D,Fig. B) and a C-terminal proline-rich region. A structural model of DefSm-D(Fig. A) was built using MODELLER v9.14 based on the crystal structure of Artv1of Artemisia vulgaris as a template(PDB: 2KPY), found with HHPred fold assignment method. The best model was assessed for quality using both energetic and structural criteria. Alsofunctional prediction analysis was performed using ProFunc server and theobtained model as input. Typical defensin folding pattern was observed. Threedisulphide bridges between six cysteine residues give the cysteine-stabilized αβmotif (CSαβ). The conserved g-coreregion (a hairpin loop connecting β-strands2 and 3), presumably linked with antifungal activity, was also presented in DefSm-D.From functional analysis significant results were obtained for extracelularcomponent; defense, stress, and biotic stimulus response; killing of cells fromanother organism and antifungal response.