Membrane-Induced Folding of the Plant Stress Dehydrin Lti30

Abstract

Dehydrins are disordered proteins that are expressed in plants as a response to embryogenesis and water-related stress. The molecular function and structural action of the dehydrins are yet elusive, but increasing evidence points to a role in protecting the structure and functional dynamics of cell membranes. An intriguing example is the cold-induced dehydrin Lti30 that binds to membranes by its conserved K segments. Moreover, this binding can be regulated by pH and phosphorylation and shifts the membrane phase transition to lower temperatures, consistent with the protein's postulated function in cold stress. In this study, we reveal how the Lti30-membrane interplay works structurally at atomic level resolution in Arabidopsis (Arabidopsis thaliana). Nuclear magnetic resonance analysis suggests that negatively charged lipid head groups electrostatically capture the protein's disordered K segments, which locally fold up into α-helical segments on the membrane surface. Thus, Lti30 conforms to the general theme of structure-function relationships by folding upon binding, in spite of its disordered, atypically hydrophilic and repetitive sequence signatures. Moreover, the fixed and well-defined structure of the membrane-bound K segments suggests that dehydrins have the molecular prerequisites for higher level binding specificity and regulation, raising new questions about the complexity of their biological function.

Figure 1.

Schematic image of disordered Lti30 binding to lipid (PC:PG) vesicles. Blue represents a positive charge and red represents a negative charge. A, Sequence of Lti30 with K segments (boldface) and the positions of the K segments matching the K^Lti30 peptide (gray boxes). B, Sequence of the K^Lti30 peptide. C, Binding equilibrium between Lti30 and lipid vesicles, where attraction is influenced by charge. Binding is promoted by protonation of the flanking His pairs (Eriksson et al., 2011) and down-regulated by phosphorylation of the K segment Thr residues (Eriksson et al., 2011). Upon Lti30 binding, the membrane phase decreases 2.5°C and the vesicles cluster into macroscopic aggregates (Eriksson et al., 2011). For size reference, Lti30 has an extended length of 20 nm and the diameter of the vesicles is 100 nm. PC, Phosphatidylcholine; PG, phosphatidylglycerol.

Figure 2.

Stopped-flow data of Lti30 binding and clustering of PC:PG lipid vesicles (LUVs). Lti30 at 3.5 μm was rapidly mixed with 0.5 mm PC:PG vesicles at a 1:142 protein:lipid ratio. Binding of positively charged Lti30 promotes vesicle clustering, monitored by scattering at 400 nm. The process depends on the LUV net negative charge, which was varied between PG (1%–40%) and PC (99%–60%). A, Scattering at 400 nm. B, Absorbance at 400 nm. C, Scattering amplitude at 400 nm at 0.5 s, indicating two binding modes.

Figure 3.

Light microscopy analysis of the Lti30-induced clustering of PC:PG LUVs. Lti30 (14 μm) was used in the presence of LUVs (1.4 mm) with 1%, 3%, or 25% PG at pH 6.3. The protein:lipid ratio is 1:100. The schematic cartoons show a reductionist interpretation of the data: Lti30 (blue) and LUVs with increasing content of negatively charged PG (red). A, Lti30 and PC:PG (99:1): no visible clusters. B, Lti30 and PC:PG (97:3): small dispersed clusters less than 5 μm. C, Lti30 and PC:PG (75:25): clear macroscopic clusters larger than 5 μm (reduced magnification).

Figure 4.

Vesicle clustering by titration of various K-segments correlates to peptide net charge. Clustering of lipid vesicles was measured by absorbance at 400nm and peptide net charge was calculated at http://protcalc.sourceforge.net. A, Titration of different K-segments into 0.5 mM PC:PG vesicles (1:3) at pH 6.3 (blue lines) and pH 4.3 (red lines). The vesicle clustering follows peptide net charge. K^Lti30 represent the dominating type of K-segment in Lti30, K^C is the consensus K-segment and K^HHCHH is K^C with the addition of histidine flanks. K^C fail to bind vesicles regardless of pH, showing the regulatory impact of positively charged histidine flanks. B, Peptide sequences, with positively and negative residues in blue and red, respectively.

Figure 5.

FTIR spectra of Lti30 only and mixed with PC:PG vesicles. A, Second derivative infrared absorption spectra of Lti30 alone in the amide I region at pH 5 (black) and pH 8.6 (red). B and C, Corresponding spectra of vesicles alone (red line) and the Lti30-vesicle mix (black line) at pH 5 and 8.6, respectively.

Figure 6.

Structural analysis of membrane-bound K^Lti30 peptide by NMR. A, The five best structures of bicelle-bound K^Lti30 peptide from ¹H-NMR constraints, where the central nine residues of the peptide adopt a fixed α-helix, whereas the N and C termini are more disordered. B, Secondary structure propensity (SSP) from 1Ha, 1HN, and 1Hb chemical shifts, where positive values show the induced α-helix in the central part of the peptide. Negative secondary structure propensity values indicate extended conformation in the N-terminal region of the peptide. C, Determined NOE connectivity between peptide residues along the K^Lti30. The ordered n+3 couplings within the residue segment Gly-7 to Gln-15 are the hallmark of the α-helical structure.

Figure 7.

Atomic resolution structure of the membrane-binding domain of Lti30. A to C, Atomic model of the membrane-binding K^Lti30 segment of Lti30, in side, diagonal, and front views, respectively. Positively charged residues are in blue, negatively charged residues are in red, and the hydrophobic side of the amphipathic α-helix faces downward. D, NMR diffusion data of K^Lti30 alone, bicelles alone, and K^Lti30 mixed with bicelles, verifying K^Lti30 micelle binding. a.u., Arbitrary units. E, Model of the positioning of the His-flanked K^Lti30 segment into a lipid bilayer (PC:PG 98:2 mix), using periodic boundary conditions. Consistent with the binding data in Figures 2 to 5, the positive side chains H2, H3, K5, and K6 coordinate the negative charge of a PG head group.