Alternative conformations of a group 4 Late Embryogenesis Abundant protein associated to its in vitro protective activity

Abstract

Late Embryogenesis Abundant (LEA) proteins are a group of intrinsically disordered proteins implicated in plant responses to water deficit. In vitro studies revealed that LEA proteins protect reporter enzymes from inactivation during low water availability. Group 4 LEA proteins constitute a conserved protein family, displaying in vitro protective capabilities. Under water deficiency or macromolecular crowding, the N-terminal of these proteins adopts an alpha-helix conformation. This region has been identified as responsible for the protein in vitro protective activity. This study investigates whether the attainment of alpha-helix conformation and/or particular amino acid residues are required for the in vitro protective activity. The LEA4-5 protein from Arabidopsis thaliana was used to generate mutant proteins. The mutations altered conserved residues, deleted specific conserved regions, or introduced prolines to hinder alpha-helix formation. The results indicate that conserved residues are not essential for LEA4-5 protective function. Interestingly, the C-terminal region was found to contribute to this function. Moreover, alpha-helix conformation is necessary for the protective activity only when the C-terminal region is deleted. Overall, LEA4-5 shows the ability to adopt alternative functional conformations under the tested conditions. These findings shed light on the in vitro mechanisms by which LEA proteins protect against water deficit stress.

Figure 1

Phylogenetic analysis of group 4 LEA proteins in plants. Schematic phylogenetic relationship between plant LEA4 proteins showing the clustering into two subgroups (LEA4-A and LEA4-B). LEA4 related proteins were also found in seedless species, exhibiting their ancestral origin (non-color branches). The Phytozome protein codes are shown at the end of each branch. Arabidopsis LEA4 proteins are highlighted with arrows.

Figure 2

Multiple sequence alignment (MSA) of the N-terminal region of 30 representative LEA4 proteins showing the closest phylogenetical relation to AtLEA4-5 protein. Alignment visualization was performed by Jalview software. Residues were colored as follows: red: negatively charged residues; blue: positively charged residues; green: polar uncharged residues; pink; residues with hydrophobic side chain; magenta: glycine and proline; orange: tyrosine and phenylalanine, and yellow: cysteine. Colored asterisks represent the location of the amino acid residues modified in the mutants generated from AtLEA4-5. Black: LEA4-5-NT1; blue: LEA4-5-NT2; green: LEA4-5-NT3; yellow: LEA4-5-NT4; red: LEA4-5-NT9P. Motifs 1, 2 and 3 were considered according to Battaglia et al.. The numbers in the upper part of the figure correspond to the AtLEA4-5 protein sequence. LEA4-5 protein from Arabidopsis is highlighted in a red box.

Figure 3

AtLEA4-5 mutants where the most LEA4 protein conserved residues were changed. (a) Schematic localization of the point mutations in each AtLEA4-5 mutant. (b) CD spectra of mutant proteins in 80% glycerol solution. Inset shows the corresponding differential spectra. [Φ] = Molar ellipticity. (c) Dichroweb analysis showing the fractions of different secondary structures in wild-type and mutant proteins. (d) Protecting activity of wild-type and mutant proteins in freeze–thaw assays using LDH as reporter enzyme. These data was obtained from three independent experiments with three technical replicates, using 10:1 LEA:LDH molar ratio. Error bars represent the standard deviations between samples. ns non statistically difference as compared to LEA4-5.

Figure 4

AtLEA4-5 deletion mutants. (a) Schematic description of the deletions generated from AtLEA4-5 protein. (b) CD spectra of the deletion mutant proteins in 80% ethylene glycol (EG) solution. Inset shows the corresponding differential spectra. [Φ] = Molar ellipticity. (c) Dichroweb analysis showing the fractions of different secondary structures in wild-type and mutant proteins. (d) Protecting activity of wild-type and mutant proteins in freeze–thaw assays using LDH as reporter enzyme. These data come from three independent experiments with three technical replicates, using 10:1 LEA:LDH molar ratio. Error bars represent the standard deviations between samples. ns non statistically difference as compared to LEA4-5. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 5

Impact of proline insertions in the N-terminal region and the complete AtLEA4-5 protein on their secondary structure and protective activity. (a) Schematic description of AtLEA4-5 mutants containing proline residues, and of two deletion mutants at the C-terminal end. (b) CD spectra of mutant proteins in 80% ethylene glycol (EG) solution. Inset shows the corresponding differential spectra. [Φ] = Molar ellipticity. (c) Dichroweb analysis showing the fractions of different secondary structures in wild-type and mutant proteins. (d) Protecting activity of wild-type and mutant proteins in freeze–thaw assays using LDH as reporter enzyme. These data come from three independent experiments with three technical replicates, using 10:1 LEA:LDH molar ratio. Error bars represent the standard deviations between samples. ns non statistically difference as compared to LEA4-5. ***p < 0.001, ****p < 0.0001.

Figure 6

Protecting activity upon freeze–thaw treatments at different molar ratios of wild-type and mutants of LEA4-5. (a) These data come from three independent experiments with three technical replicates, using the molar ratio (LEA:LDH) indicated in the graph. Error bars represent the standard deviations from the mean. The x-axis values are in log₁₀ scale. (b) Estimated molar ratio needed to attain 50% protection activity (MR₅₀).