Backbone dynamics of the antifungal Psd1 pea defensin and its correlation with membrane interaction by NMR spectroscopy

Abstract

Plant defensins are cysteine-rich cationic peptides, components of the innate immune system. The antifungal sensitivity of certain exemplars was correlated to the level of complex glycosphingolipids in the membrane of fungi strains. Psd1 is a 46 amino acid residue defensin isolated from pea seeds which exhibit antifungal activity. Its structure is characterized by the so-called cysteine-stabilized alpha/beta motif linked by three loops as determined by two-dimensional NMR. In the present work we explored the measurement of heteronuclear Nuclear Overhauser Effects, R1 and R2 (15)N relaxation ratios, and chemical shift to probe the backbone dynamics of Psd1 and its interaction with membrane mimetic systems with phosphatidylcholine (PC) or dodecylphosphocholine (DPC) with glucosylceramide (CMH) isolated from Fusarium solani. The calculated R2 values predicted a slow motion around the highly conserved among Gly12 residue and also in the region of the Turn3 His36-Trp38. The results showed that Psd1 interacts with vesicles of PC or PC:CMH in slightly different forms. The interaction was monitored by chemical shift perturbation and relaxation properties. Using this approach we could map the loops as the binding site of Psd1 with the membrane. The major binding epitope showed conformation exchange properties in the mus-ms timescale supporting the conformation selection as the binding mechanism. Moreover, the peptide corresponding to part of Loop1 (pepLoop1: Gly12 to Ser19) is also able to interact with DPC micelles acquiring a stable structure and in the presence of DPC:CMH the peptide changes to an extended conformation, exhibiting NOE mainly with the carbohydrate and ceramide parts of CMH.

Pliable natural biocide: Jaburetox is an intrinsically disordered insecticidal and fungicidal polypeptide derived from jack bean urease

“…Figure 3C shows that Jaburetox contains several potential disorder-based binding sites, such as a-MoRFs, general MoRFs, sites identified by MFSPSSMPRED and ANCHORindicated binding sites (AiBSs), which completely or partially overlap with each other. A larger agreement is observed among the various tools in the case of a potential binding site located in the N-terminal region (residues 1-21) compared to that found in the central portion (residues [43][44][45][46][47][48][49][50][51][52][53][54][55] or in the C-terminal region (residues [72][73][74][75][76][77][78][79][80][81]. This is probably due to the fact that the potential binding site at the N terminus has a stronger a-helical signal, which is recognized by the computational tools used in this study.…”

“…Some examples of antifungal polypeptides studied by NMR are Psd1, a 46-residue recombinant defensin from Pisum sativum [77,78], Rs-AFP1, a 51-residue defensin isolated from seeds of Raphanus sativus L. [79], Ib-AMP1, a 20-residue peptide derived from seeds of Impatiens balsamina [80], and EAFP2, a 41-residue polypeptide from Eucommia ulmoides [81]. Other plant-derived insecticides structurally characterized by [103].…”

Structural and Dynamic Insights of the Interaction between Tritrpticin and Micelles: An NMR Study

“…The HN coupling constants were also measured, but no significant differences were observed. 15 N-L 11 -TRP3 was also investigated in the presence of LLPC and LMPG micelles (Table S1). When compared to the free state, the relaxation parameter values changed substantially as a result of peptide-micelle interaction.…”

“…Chemical shift assignment and NOE peak list generation were performed using TopSpin software (Bruker). 15 N T 1 and 15 N T 2 relaxation times were measured from spectra with different relaxation delays: 50, 100, 200, 300, 400, 600 (triplicate), 800, 1000, and 1500 ms for T 1 15 N heteronuclear NOEs were determined from the ratio of peak intensities with and without saturation of the amide protons. Errors in the heteronuclear NOE values were calculated from peak intensities and noise levels in reference and saturated spectra.…”

“…AMPs frequently convert between different conformations in solution, acquiring more stable conformations upon binding to interfaces (1,(13)(14)(15)(16)(17)(18)(19). NMR is a powerful technique for studying such dynamic systems (1,10).…”