Cellular Chaperone Function of Intrinsically Disordered Dehydrin ERD14

Abstract

Disordered plant chaperones play key roles in helping plants survive in harsh conditions, and they are indispensable for seeds to remain viable. Aside from well-known and thoroughly characterized globular chaperone proteins, there are a number of intrinsically disordered proteins (IDPs) that can also serve as highly effective protecting agents in the cells. One of the largest groups of disordered chaperones is the group of dehydrins, proteins that are expressed at high levels under different abiotic stress conditions, such as drought, high temperature, or osmotic stress. Dehydrins are characterized by the presence of different conserved sequence motifs that also serve as the basis for their categorization. Despite their accepted importance, the exact role and relevance of the conserved regions have not yet been formally addressed. Here, we explored the involvement of each conserved segment in the protective function of the intrinsically disordered stress protein (IDSP) A. thaliana's Early Response to Dehydration (ERD14). We show that segments that are directly involved in partner binding, and others that are not, are equally necessary for proper function and that cellular protection emerges from the balanced interplay of different regions of ERD14.

Keywords: CD spectroscopy; LEA protein; dehydrin; early response to dehydration; heat stress; intrinsic structural disorder; plant chaperone; structure-function relationship.

Figure 1

Conserved segments, charge distribution, in silico, and experimental α-helical propensities of the ERD14 protein. Conserved regions: Ka-, Kb-, Kc- (blue), S- (yellow), Chp- (green), and H-segment (red) indicated in the sequence of ERD14. In addition to individual charges above the regions, net charge/number of charged side chains/length of regions summed up. Underlined letters show residues with α-helical propensity calculated from nuclear magnetic resonance (NMR) chemical shifts [9,16] by δ2D method [17]. Below the residues, H indicates the α-helical propensity estimated by in silico PredictProtein analysis [18,19,20].

Figure 2

Overview of the viability assay. After a standard induction period, cells were stressed by a heat shock of 50 °C for 15 min (final set-up, after optimization of the stress conditions by screening at different temperatures for different time length shown at the left bottom panel). Viability of stressed and non-stressed samples were compared based on their cell growth after appropriate dilution in fresh medium (Calibration—grey curves). Cell growth was followed by absorbance at 600 nm. Half-time of growth curves varies linearly, with dilution plotted on a logarithmic scale, i.e., it is informative of the concentration of viable cells in the sample. For our experiments, we have set the OD at 0.6 as this period of growth by convention and applied serial dilutions to obtain the calibration curve and calculate the relative survival ratios of the different samples (NS: non-stressed samples, S: stressed samples). Based on this, we can compare the viability of cells exposed to the same stress conditions but expressing various constructs (colored curves).

Figure 3

Conserved regions of ERD14 contribute to cell protection. (A) Schematic representation of the conserved regions within ERD14. (B) Survival rate (viability) is reduced by the applied heat stress to 26.2% (compared to non-stressed cells) for non-transformed cells, 36.8% for cells containing an empty vector, and about 39% in the case of overexpression of control proteins: GST (38.4%) and IDP calpastatin (38.7%). Overexpression of ERD14 increases the survival rate to 74.5%. Removing the conserved regions by randomizing the amino acid sequence (Full-Scr) abolished the protective effect (viability 38.9%). (C) Protective effect of a series of mutants in which conserved regions of the protein have been individually deleted. Data represent mean ± SEM and the results of at least 18 parallels for each construct. Significant differences (p < 0.05) compared to WT ERD14 are labeled with asterisks (*).

Figure 4

CD measurements of single deletion mutants and Full-Scr construct of ERD14 with TFE induced secondary structure changes. (A) Circular dichroism spectra of the ∆Ka-, ∆Kb-, ∆Kc-, ∆S-, ∆H-, ∆Chp-, and Full-Scr (FS) constructs of ERD14, recorded in 10 mM Na-phosphate buffer (pH 7.4), indicating similar, mainly disordered, structures, which is demonstrated by a characteristic minimum below 200 nm and low amplitude around 220 nm. (B) CD spectra of the different constructs of ERD14 recorded in Na-phosphate buffer containing 30% TFE. There are significant differences in the extent of the induced secondary structure, as shown by the spectral differences between the variants. (C) Comparison of the CD spectra of the FS ERD14 variant in native (dark blue) and in 30% TFE containing (light blue) buffer, indicating minor effects of TFE on the structure of the variant with scrambled sequence. (D) The degrees of formation of α-helices (blue areas) obtained with the δ2D method [17] using Nuclear Magnetic Resonance Chemical shifts mapped to the WT ERD14 sequence, and its graphical representation inserted above the diagram shows good accordance with the in-silico estimated α-helix content [20] (boxes at the top).

Figure 5

Interplay of different conserved elements in ERD14. (A) Protective effect of double- and triple deletion mutants, in which two or three segments are deleted (K-segments Ka, Kb, and Kc, as well as Chp-, S-, and H-segments; cf. sequence: Supplementary Table S1). Data represent mean ± SEM and the results of at least 18 parallels for each construct. Significant differences (p < 0.05) compared to WT ERD14 are labeled with asterisks (*). (B) Protective effect of scrambled mutants, in which either the full sequence is scrambled (Full-Scr), or particular binding regions are kept intact (e.g., Kc in Scr-Kc) and the rest of the sequence is randomized. Data represent mean ± SEM and the results of at least 18 parallels for each construct. Significant differences (p < 0.05) compared to WT ERD14 are labeled with asterisks (*). (C) The relative % contributions of motifs of ERD14 (Ka, S, Chp, Kb, H, and Kc) and their combinations (e.g., KcS is combined Kc and S) are quantified, as given in Materials and Methods (Supplementary Table S2). Their % contributions to the activity above the given background, WT ERD14 and Full-Scr ERD14, either alone or in combinations, are presented in a matrix. Numbers highlighted in color demonstrate that effects are usually higher in the WT than in the scrambled background.