Metal-binding polymorphism in late embryogenesis abundant protein AtLEA4-5, an intrinsically disordered protein

Abstract

Late embryogenesis abundant (LEA) proteins accumulate in plants during adverse conditions and their main attributed function is to confer tolerance to stress. One of the deleterious effects of the adverse environment is the accumulation of metal ions to levels that generate reactive oxygen species, compromising the survival of cells. AtLEA4-5, a member of group 4 of LEAs in Arabidopsis, is an intrinsically disordered protein. It has been shown that their N-terminal region is able to undergo transitions to partially folded states and prevent the inactivation of enzymes. We have characterized metal ion binding to AtLEA4-5 by circular dichroism, electronic absorbance spectroscopy (UV-vis), electron paramagnetic resonance, dynamic light scattering, and isothermal titration calorimetry. The data shows that AtLEA4-5 contains a single binding site for Ni(II), while Zn(II) and Cu(II) have multiple binding sites and promote oligomerization. The Cu(II) interacts preferentially with histidine residues mostly located in the C-terminal region with moderate affinity and different coordination modes. These results and the lack of a stable secondary structure formation indicate that an ensemble of conformations remains accessible to the metal for binding, suggesting the formation of a fuzzy complex. Our results support the multifunctionality of LEA proteins and suggest that the C-terminal region of AtLEA4-5 could be responsible for antioxidant activity, scavenging metal ions under stress conditions while the N-terminal could function as a chaperone.

Keywords: Fuzzy complex; Intrinsically disordered proteins; Metal binding; Protein self-assembly.

Figure 1. Sequence alignment for group 4 LEA proteins from Arabidopsis thaliana: AtLEA4-5, AtLEA4-1, and AtLEA4-2.

According to the nomenclature of LEA proteins, numbers 1, 2, and 5 indicate the chromosome where their corresponding genes are localized. Fully conserved residues are contained in black boxes, positive charged residues are in blue, negative charged residues are in red, glycines are in green and histidines are in purple. Their amino terminal region is highly conserved, whereas their carboxyl terminal region is more variable, with 54.64% sequence identity between AtLEA4-1 and AtLEA4-2, 29.69% between AtLEA4-1 and AtLEA4-5, and 26.09% between AtLEA4-2 and AtLEA4-5.

Figure 2. Effect of metal ions on AtLEA4-5 secondary structure.

CD spectra of AtLEA4-5 titrated with (A) Cu(II), (B) Zn(II) and (C) Ni(II). Spectra of AtLEA4-5 in the absence (black line) or presence of 1, 2, 3, 4, and 7 equivalents of the metal ion (light to dark blue). The profiles did not undergo any significant variation after the addition of Cu(II), Zn(II) or Ni(II), suggesting that none of the metal ions induce changes in the AtLEA4-5 secondary structure under these conditions.

Figure 3. Cu(II)-AtLEA4-5 complex formation by electronic absorption.

(A) UV–vis and (B) CD spectra of AtLEA4-5 in the absence (black line) or presence of 1, 2, 3, 4, and 7 equivalents of Cu(II) (light to dark blue). The arrows indicate the three ligand to metal charge transfer (LMCT) bands and the d–d bands. These bands correspond to transitions from the histidine imidazole group to copper and backbone deprotonated amides to copper.

Figure 4. Cu(II)-AtLEA4-5 complex formation by EPR.

EPR spectra of AtLEA4-5 in the absence (black line) or presence of 1, 2, 3, 4, and 7 equivalents of Cu(II) (light to dark blue). Two sets of signals were measured with g_ll = 2.2177 and A_ll = 188.5 and g_ll = 2.261 and A_ll = 182, corresponding to a 4N and N3O equatorial coordination, respectively. These results indicate that there are at least two species of the Cu(II)-AtLEA4-5 complex with two different equatorial coordination modes.

Figure 5. Protein oligomerization induced by metal binding.

Correlation function of AtLEA4-5 in the absence (black line) or presence of 1, 2, 3, and 4 equivalents of Cu(II) (light to dark blue). The data were used to obtain translational diffusion coefficients by the cumulant and distribution methodologies (Fig. S5). Measurements of AtLEA4-5 in the presence of different Cu(II) concentrations yielded a shift to the right, indicating a size increase due to oligomerization induced by metal binding.

Figure 6. Metal binding by ITC.

Isothermal titration calorimetry of AtLEA4-5 bound to (A) Ni(II), (B) Cu(II), and (C) Zn(II). The left side shows the experimental isothermic titrations; while the right side shows the reaction heat. In all cases the solid line represents the best fit to a binding model. (A) Ni(II) was best fitted to a single binding site model with a 1:1 stoichiometry. (B) and (C) Cu(II) and (D) and (E) Zn(II) exhibited a complex behavior involving both exothermic and endothermic processes and were fitted to multiple binding sites models. These transitions indicate the presence of at least two processes accounting for the different heat reactions.

Figure 7. Metal binding model.

(A) An ensemble of conformations is accessible for binding. (B) Metals interact with moderate affinity mainly with His residues, with multiple forms and multiple binding sites, without forming a unique well-defined binding site. (C) As metal ion concentration increases their binding to the protein induces the formation of oligomeric species.