The disordered plant dehydrin Lti30 protects the membrane during water-related stress by cross-linking lipids

Abstract

Dehydrins are intrinsically disordered proteins, generally expressed in plants as a response to embryogenesis and water-related stress. Their suggested functions are in membrane stabilization and cell protection. All dehydrins contain at least one copy of the highly conserved K-segment, proposed to be a membrane-binding motif. The dehydrin Lti30 (Arabidopsis thaliana) is up-regulated during cold and drought stress conditions and comprises six K-segments, each with two adjacent histidines. Lti30 interacts with the membrane electrostatically via pH-dependent protonation of the histidines. In this work, we seek a molecular understanding of the membrane interaction mechanism of Lti30 by determining the diffusion and molecular organization of Lti30 on model membrane systems by imaging total internal reflection- fluorescence correlation spectroscopy (ITIR-FCS) and molecular dynamics (MD) simulations. The dependence of the diffusion coefficient explored by ITIR-FCS together with MD simulations yields insights into Lti30 binding, domain partitioning, and aggregation. The effect of Lti30 on membrane lipid diffusion was studied on fluorescently labeled supported lipid bilayers of different lipid compositions at mechanistically important pH conditions. In parallel, we compared the mode of diffusion for short individual K-segment peptides. The results indicate that Lti30 binds the lipid bilayer via electrostatics, which restricts the mobility of lipids and bound protein molecules. At low pH, Lti30 binding induced lipid microdomain formation as well as protein aggregation, which could be correlated with one another. Moreover, at physiological pH, Lti30 forms nanoscale aggregates when proximal to the membrane suggesting that Lti30 may protect the cell by "cross-linking" the membrane lipids.

Keywords: Diffusion; K-segments; Lti30; disorder; fluorescence correlation spectroscopy (FCS); histidine; intrinsically disordered protein; lipid bilayer; membrane lipid; molecular dynamics; supported lipid bilayers.

Figure 1.

ITIR-FCS data of Lti30 binding with supported lipid bilayers. The effect of Lti30 on lipid mobility in supported lipid bilayers composed of different compositions and at pH 5.8, 6.3, 7.4, and 9.0 was measured. The measurements were performed at a region of 21 × 21 pixels in the form of 50,000 frames at a time exposure of 1 ms. The measurements were done up to 40 min after the addition of Lti30 over the membrane. Membrane lipid diffusion is quantified using two parameters diffusion coefficient (D) and diffusion law intercept (τ₀). The protein–to–lipid ratio used is 1:130. A, DOPC; B, DOPC:DOPS (4:1); C, DOPC:DPPC (1:1). Error bars are given as standard deviations (S.D.). The experiments have been repeated three times to ensure the reproducibility. The gray-shaded area in τ₀ graphs represents the range of margin of error in our setup (−0.1 s < 0 < 0.1 s) in which the particle is considered to undergo free diffusion. For representative raw data see Figs. S1–S3.

Figure 2.

Comparison of the diffusion of K-segment and His–K-segment on DOPC:DOPS (4:1) bilayer at pH 6.3 and 7.4. The protein–to–lipid ratio used was 1:130. A, diffusion coefficient (D) of fluorescently labeled K-segment/HH on DOPC:DOPS (4:1) bilayer. B, diffusion law intercept (τ₀) of fluorescently labeled K-segment/HH on the membrane. C, diffusion coefficient of membrane lipids bound to the peptide. Error bars for A–C are given as S.D. calculated from three independent experiments. D, diffusion law intercept (τ₀) of fluorescently labeled His–K-segment on the membrane at different spots (one representative set). Inset is the image of the DOPC:DOPS (4:1) membrane bound with rhodamine-labeled with His–K-segment at pH 6.3. Those spots are marked for which τ₀ values are reported. For representative raw data (images, ACF, and diffusion law plots) see Fig. S4.

Figure 3.

Visualization of Lti30 aggregates on DOPC:DOPS (4:1) membrane at pH 5.8, 6.3, 7.4, and 9.0 using thioflavin T dye on a TIRF microscope. First, Lti30 was incubated with the supported lipid bilayer for 60 min. Then, 5 μm of ThT dye was added, and images were recorded. Scale bar, 5 μm.

Figure 4.

Peptide–membrane interactions during simulations at pH 4. A, K-segment, and B, His–K-segment. Each panel corresponds to a particular membrane system: DOPC, DOPC:DOPS (4:1), or DOPC:DPPC (1:1). At the top of each panel, the final 0.2-μs averaged partial mass density of the peptide is shown (with respect to Z-normal and y axis), with the outer leaflet phosphates indicated by a dashed line. The final snapshot of the simulation is shown at the bottom of each panel (peptide shown in cartoon representation in orange, lipids shown in licorice representation, with carbons colored cyan, oxygens red, and nitrogens blue).

Figure 5.

Peptide–membrane interactions during simulations at pH 10. A, K-segment, and B, His–K-segment. Each panel corresponds to a particular membrane system: DOPC, DOPC:DOPS (4:1), or DOPC:DPPC (1:1). At the top of each panel, the final 0.2-μs averaged partial mass density of the peptide (with respect to Z-normal and y axis), with the outer leaflet phosphates indicated by a dashed line. The final snapshot of the simulation is shown at the bottom of each panel (peptide shown in cartoon representation in orange, lipids shown in licorice representation, with carbons colored cyan, oxygens red, and nitrogens blue).

Figure 6.

Per-residue peptide secondary structure propensity over the simulation time in lipid membranes. A, K-segment at acidic pH; B, His–K-segment at acidic pH; C, K-segment at basic pH; D, His–K-segment at basic pH.

Figure 7.

Interactions of individual K-segment and His–K-segment with lipid bilayers. A, distribution of the number of contacts between peptide and lipid phosphate or anionic lipid carboxylate groups (0.6 nm cutoff) are shown for the His–K-segment at acidic pH (data shown in solid format) and basic pH (data shown in dashed format). B, distribution of the number of contacts between peptide and lipid phosphate or anionic lipid carboxylate groups (0.6 nm cutoff) are shown for the K-segment at acidic pH.