Revealing the properties of plant defensins through dynamics

Abstract

Defensins are potent, ancient natural antibiotics that are present in organisms ranging from lower organisms to humans. Although the structures of several defensins have been well characterized, the dynamics of only a few have been studied. This review discusses the diverse dynamics of two plant defensins for which the structure and dynamics have been characterized, both in the free state and in the presence of target membranes. Multiple motions are observed in loops and in secondary structure elements and may be related to twisting or breathing of the α-helix and β-sheet. This complex behavior is altered in the presence of an interface and is responsive to the presence of the putative target. The stages of membrane recognition and disruption can be mapped over a large time scale range, demonstrating that defensins in solution exist as an ensemble of different conformations, a subset of which is selected upon membrane binding. Therefore, studies on the dynamics have revealed that defensins interact with membranes through a mechanism of conformational selection.

Figure 1

The characterization of pico- to millisecond timescales using spin relaxation measurements can probe internal motions of the bond vector. Model-free analysis of relaxation data (longitudinal relaxation (R₁) and transverse relaxation (R₂)) yield an order parameter (S²) that describes the amplitude of motion, a constant (τ_eff) describing its timescale [37,38,39] (a) and R_ex that describes the additional exchange contribution in R₂ (b). A representative relaxation dispersion curve demonstrating the dependence of R_ex that can be used to extract the populations (p), exchange rates (k) and chemical shifts (ω) of ground and exited states (see the equation) [40]. The curve was fitted using CPMGFit program (www.palmer.hs.columbia.edu) [41] (c).

Figure 2

Common membrane mimetic systems and sequence alignment of plant defensins used in this study to probe events of membrane recognition. The resulting NMR spectrum of a given nucleus depends on the affinity of the membrane-defensin complex. In a fast exchange regime, the spectrum exhibits sharp lines. If the interaction is too strong (high affinity complexes), the spectrum exhibits broad NMR lines (a). Comparison of the primary sequence of representative plant defensins. Conserved cysteine residues are colored in red and the conserved glycine residue is highlighted in blue. The γ-core motif (GXC or CXG-C) is indicated by a blue box [61] (b).

Figure 3

Complex motions of Sd5 probed by backbone relaxation parameters. Residues exhibiting fast motion (ps-ns) are probed using both micelles and vesicles, but those involved in conformational exchange are probed only in the presence of CMH. The regions mapped in membrane recognition with different dynamic properties are highlighted in purple, green and yellow.

Figure 4

Proposed model for the Sd5 interaction surface with a membrane mimetic. In a conformational selection mechanism, the binding competent conformation (blue, P) preexistts in solution prior to the addition of the ligand (L). Residues affected by the interactions with PC:CMH vesicles are colored in purple (the specific binding site), and DPC micelles are colored in yellow and green (the nonspecific binding site). The component of the fungal membrane, glucosylceramide (CMH), is highlighted in red.

Figure 5

Dynamics properties of the plant defensins Sd5 and Psd1. Quantitative analysis of exchange dynamics in Sd5. ¹⁵N-NMR relaxation dispersion curves were measured at 800 and 600 MHz. Side chains of the residues with amide backbone conformational exchange measured by NMR relaxation dispersion are colored magenta in the NMR structure of Sd5 (PDB code 2KSK) (a). Ribbon representation of Psd1 highlighting the side chains of the residues in conformational exchange in blue and the flexible hinge Gly12 in cyan. Gly12 is the only residue with decreased order parameter, which indicates thermal flexibility (PDB code 1JKZ) (b).