PsDef1

Plant defensins demonstrate high structural stability at extreme temperatures and pH values and, in general, are non-toxic to mammalian cells. These properties make them attractive candidates for use in biotechnology and biomedicine. Knowing the structure-function relationship is desirable to guide the design of plant defensin-based applications. Thus far, the broad range of biological activities was described only for one defensin from gymnosperms, the defensin PsDef1 from Scots pine. Here, we report that closely related defensin from the same taxonomy group, PsDef2, differing from PsDef1 by six amino acids, also possesses antimicrobial, antibacterial, and insect α-amylase inhibitory activities. We also report the solution structure and dynamics properties of PsDef2 assessed using a combination of experimental nuclear magnetic resonance (NMR) techniques. Lastly, we perform a comparative analysis of PsDef2 and PsDef1 gaining a molecular-level insight into their structure-dynamics-function relationship.

Plants have developed a complex defense response system against pests and pathogens. Defensins, produced by plants as part of their innate immune response, form the family of small, basic, cysteine-rich proteins with activity primarily directed against fungal pathogens. In addition, plant defensins can show antibacterial activity and protease and insect amylase inhibitory activities. However, in gymnosperms, only antifungal activity of defensins has been described thus far. Here, we report antibacterial and insect α-amylase inhibition activities for defensin PsDef1 from P. sylvestris, the first defensin from gymnosperms with a broad range of biological activities described. We also report the solution NMR structure of PsDef1 and its dynamics properties assessed by a combination of experimental NMR and computational techniques. Collectively, our data provide an insight into structure, dynamics, and functional properties of PsDef1 that could be common between defensins from this taxonomic group.

Defensins are part of the innate immune system in plants with activity against a broad range of pathogens, including bacteria, fungi and viruses. Several defensins from conifers, including Scots pine defensin 1 (Pinus sylvestris defensin 1, (PsDef1)) have shown a strong antifungal activity, however structural and physico-chemical properties of the family, needed for establishing the structure-dynamics-function relationships, remain poorly characterized. We use several spectroscopic and computational methods to characterize the structure, dynamics, and oligomeric state of PsDef1. The three-dimensional structure was modeled by comparative modeling using several programs (Geno3D, SWISS-MODEL, I-TASSER, Phyre(2), and FUGUE) and verified by circular dichroism (CD) and infrared (FTIR) spectroscopy. Furthermore, FTIR data indicates that the structure of PsDef1 is highly resistant to high temperatures. NMR diffusion experiments show that defensin exists in solution in the equilibrium between monomers and dimers. Four types of dimers were constructed using the HADDOCK program and compared to the known dimer structures of other plant defensins. Gaussian network model was used to characterize the internal dynamics of PsDef1 in monomer and dimer states. PsDef1 is a typical representative of P. sylvestris defensins and hence the results of this study are applicable to other members of the family.