The Unstructured N-terminal Region of Arabidopsis Group 4 Late Embryogenesis Abundant (LEA) Proteins Is Required for Folding and for Chaperone-like Activity under Water Deficit

Cesar L Cuevas-Velazquez,Gloria Saab-Rincón,José Luis Reyes,Alejandra A Covarrubias

doi:10.1074/jbc.m116.720318

Cesar L Cuevas-Velazquez, Gloria Saab-Rincón + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m116.720318

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Abstract

Late embryogenesis abundant (LEA) proteins are a conserved group of proteins widely distributed in the plant kingdom that participate in the tolerance to water deficit of different plant species. In silico analyses indicate that most LEA proteins are structurally disordered. The structural plasticity of these proteins opens the question of whether water deficit modulates their conformation and whether these possible changes are related to their function. In this work, we characterized the secondary structure of Arabidopsis group 4 LEA proteins. We found that they are disordered in aqueous solution, with high intrinsic potential to fold into α-helix. We demonstrate that complete dehydration is not required for these proteins to sample ordered structures because milder water deficit and macromolecular crowding induce high α-helix levels in vitro, suggesting that prevalent conditions under water deficit modulate their conformation. We also show that the N-terminal region, conserved across all group 4 LEA proteins, is necessary and sufficient for conformational transitions and that their protective function is confined to this region, suggesting that folding into α-helix is required for chaperone-like activity under water limitation. We propose that these proteins can exist as different conformers, favoring functional diversity, a moonlighting property arising from their structural dynamics.

Highlights

Low water availability caused by different environmental conditions such as drought, or low temperatures represents a vulnerable situation for many forms of life, for plants
Most late embryogenesis abundant (LEA) proteins show high hydrophilicity, high content of small amino acids, and absence or deficit of hydrophobic residues, properties that are extended to a larger set of proteins called hydrophilins, which have been found in species from the three domains of life and that accumulate under water deficit [2, 4]
We demonstrate that even though Arabidopsis members of subgroups 4A (AtLEA4-2) and 4B (AtLEA4-5) LEA proteins are structurally disordered in solution, low osmotic potentials and macromolecular crowding can induce significant levels of ␣-helix, in the conserved AtLEA4-5 N-terminal region, whereas the C-terminal region displays high structural disorder

Summary

Experimental Procedures

In Silico Analyses—AtLEA4 proteins were aligned using T-Coffee multiple sequence alignment. Because pTrc99A:AtLEA4-1, pTrc99A:AtLEA4-2, and pTrc99A: AtLEA4-578–158 did not lead to a successful protein expression in bacteria, instead corresponding ORFs were inserted into the pTYB11 vector to obtain them as intein fusion proteins (IMPACT-CN expression system; New England Biolabs Inc.) To this end, AtLEA4-1 and AtLEA4-2 coding sequences were amplified from pJET1.2 intermediary plasmids using specific oligonucleotides containing SapI and PstI restriction sites: 5Ј-GGTGGTTGCTCTTCCAACATGCAATCGGCGAAACAGAAG-3Ј and 5Ј-GGTGGTCTGCAGTCATTAGTAGTGATGATGATTATGATGTCC-3Ј for AtLEA4-1 and, 5Ј-GGTGGTTGCTCTTCCAACATGCAGTCGGCGAAGG-3Ј and 5ЈGGTGGTCTGCAGTCATTAGATCTGTCCCGGCG-3Ј for AtLEA4-2. LDH from rabbit muscle (Roche) was diluted to 250 nM (monomer) as the final concentration, in the presence or absence of the corresponding test protein (AtLEA4-2, AtLEA4-5, AtLEA4-51–77, or AtLEA4578–158) in buffer of 25 mM Tris-HCl, pH 7.5. LDH activity was measured as described above These assays were reproduced using protein samples from at least three independent purification batches. Significant differences were calculated with Tukey’s multiple comparison post-test (p Ͻ 0.01)

Results

Aqueous solution

Discussion