The in vitro structure and functions of the disordered late embryogenesis abundant three proteins

Abstract

Late embryogenesis abundant (LEA) proteins are produced during seed embryogenesis and in vegetative tissue in response to various abiotic stressors. A correlation has been established between LEA expression and stress tolerance, yet their precise biochemical mechanism remains elusive. LEA proteins are very rich in hydrophilic amino acids, and they have been found to be intrinsically disordered proteins (IDPs) in vitro. Here, we perform biochemical and structural analyses of the four LEA3 proteins from Arabidopsis thaliana (AtLEA3). We show that the LEA3 proteins are disordered in solution but have regions with propensity for order. All LEA3 proteins were effective cryoprotectants of LDH in the freeze/thaw assays, while only one member, AtLEA3-4, was shown to bind Cu²⁺ and Fe³⁺ ions with micromolar affinity. As well, only AtLEA3-4 showed binding and a gain in α-helicity in the presence of the membrane mimic dodecylphosphocholine (DPC). We explored this interaction in greater detail using ¹⁵ N-heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance, and demonstrate that two sets of conserved motifs present in AtLEA3-4 are involved in the interaction with the DPC micelles, which themselves gain α-helical structure.

Keywords: circular dichroism (CD); intrinsically disordered protein; late embryogenesis abundant (LEA) protein; micelle; multifunctional protein; nuclear magnetic resonance (NMR); plant biochemistry; stress.

FIGURE 1

Sequence, disorder prediction and structure of AtLEA3 proteins. (a) Sequences of the four AtLEA3 proteins. Cyan, mitochondrial transport sequence; green, twin‐Arg motif; red, W‐motif; purple, DAELR motif. The black triangles indicate the Mitofates predicted start site of the mature protein for AtLEA3‐1, 3–2 and 3–4, while the yellow triangle indicates the cut‐site used to produce soluble AtLEA3‐3. (b) In silico disorder prediction using several disorder predictors. Disorder profiles representing per‐residue disorder predispositions of these proteins were generated by PONDR VLXT, PONDR VSL2, PONDR VL3, PONDR FIT, IUPred_short, and IUPred_long. Dashed cyan lines show the mean disorder propensity calculated for each protein by averaging disorder profiles of individual predictors. The predicted intrinsic disorder scores above 0.5 are considered to correspond to the disordered residues/regions, whereas regions with the disorder scores between 0.2 and 0.5 are considered flexible. The rectangular box at the top of each panel shows the architectures as described in A). The black triangles indicate the Mitofates predicted start site of the mature protein for AtLEA3‐1, 3–2 and 3–4, while the yellow triangle indicates the cut‐site used to produce soluble AtLEA3‐3. (c) CD spectra of AtLEA3 proteins in 10 mM Tris, pH 8.0 alone in solution

FIGURE 2

Metal binding does not affect the structure of AtLEA3‐4. CD spectra of AtLEA3‐4 protein (at pH 7.4 and pH 5.5) in the presence and absence of 1 mM Cu²⁺ (pH 7.4) or 1 mM Fe³⁺ (pH 5.5). The figure legend is shown in the insert

FIGURE 3

Copper and iron bind AtLEA3‐4 with low affinity. (a) The thermogram of 2 mM Cu²⁺ (10 mM Tris, 100 mM NaCl, pH 7.4) titrated into 50 μM AtLEA3‐4 over 20 injections. (b) The best model for Cu²⁺ binding to AtLEA3‐4 protein using the ITC data. (c) The thermogram of 2 mM Fe³⁺ (10 mM Tris, 100 mM NaCl, pH 5.5) titrated into 50 μM AtLEA3‐4 over 20 injections. (d) The best model for Fe³⁺ binding to AtLEA3‐4 protein. Control experiments corrected for the heat effect of the dilution of metal into buffer alone

FIGURE 4

LDH Cryoprotection by AtLEA3 Proteins. The protection of LDH activity after freezing and thawing of the enzyme was examined in the presence and absence of several proteins (AtLEA3‐1, AtLEA3‐2, AtLEA3‐3, AtLEA3‐4, BSA, and lysozyme) over a range of concentrations. The activity of unfrozen LDH is defined as 100%. Error bars represent the standard deviation of n = 3 for all additives, while the lines represent fits to Equation 1. The figure legend is shown as an inset in the figure

FIGURE 5

CD spectra of AtLEA3 proteins in the presence of DPC micelles. (a) CD spectra of AtLEA3 proteins in 10 mM Tris, pH 8.0 alone (black) and in the presence of 10 mM DPC (red). The legends are shown as insets in the panels. (b) The [θ]₂₂₂ signal of AtLEA3‐4 protein was monitored in the presence of an increasing amount of DPC. DPC, dodecylphosphocholine

FIGURE 6

NMR analysis of DPC Binding to AtLEA3‐4. (a) ¹⁵N‐HSQC of AtLEA3‐4 alone (cyan) and in the presence of various concentrations of DPC (color gradient) up to 100 mM (red). Peak assignments are shown as the residue number followed by the one letter amino acid code. (b) Weighted chemical shift differences between bound and unbound forms of AtLEA3‐4 protein. The dashed line presents one SD of the chemical shifts above zero. Red asterisks indicate peaks undergoing slow or intermediate exchange. The rectangular box at the top of each panel shows the architectures showing the following motifs in AtLEA3‐4: first W‐motif (light red), first DAELR motif (light purple), second W‐motif (dark red) and second DAELR motif (dark purple). DPC, dodecylphosphocholine; NMR, nuclear magnetic resonance

FIGURE 7

DPC binding affinity and residual structure of AtLEA3‐4. (a) Binding curves of the AtLEA3‐4‐micelle interactions using three different residues as indicated in the insert. The symbols represent the chemical shift change while the lines represent the fitted binding curve. (b) Residual structure of AtLEA3‐4 protein alone. (c) Residual structure of AtLEA3‐4 protein in the presence of 100 mM DPC. The probability of secondary structure on per residues basis was calculated using δ2Δ (56). The predicted secondary structure legend is shown in the panel insert. DPC, dodecylphosphocholine