Folding and Lipid Composition Determine Membrane Interaction of the Disordered Protein COR15A

Abstract

Plants from temperate climates, such as the model plant Arabidopsis thaliana, are challenged with seasonal low temperatures that lead to increased freezing tolerance in fall in a process termed cold acclimation. Among other adaptations, this involves the accumulation of cold-regulated (COR) proteins, such as the intrinsically disordered chloroplast-localized protein COR15A. Together with its close homolog COR15B, it stabilizes chloroplast membranes during freezing. COR15A folds into amphipathic α-helices in the presence of high concentrations of low-molecular-mass crowders or upon dehydration. Under these conditions, the (partially) folded protein binds peripherally to membranes. In our study, we have used coarse-grained molecular dynamics simulations to elucidate the details of COR15A-membrane binding and its effects on membrane structure and dynamics. Simulation results indicate that at least partial folding of COR15A and the presence of highly unsaturated galactolipids in the membranes are necessary for efficient membrane binding. The bound protein is stabilized on the membrane by interactions of charged and polar amino acids with galactolipid headgroups and by interactions of hydrophobic amino acids with the upper part of the fatty acyl chains. Experimentally, the presence of liposomes made from a mixture of lipids mimicking chloroplast membranes induces additional folding in COR15A under conditions of partial dehydration, in agreement with the simulation results.

Structural properties and cellular expression of AfrLEA6, a group 6 late embryogenesis abundant protein from embryos of Artemia franciscana

“…At concentrations that are physiologically relevant to these embryos in the hydrated state (≥ 340 mM), trehalose has been shown to drive folding equilibrium towards the native state of globular proteins (Auton et al 2011;Xie and Timasheff 1997). LEA protein folding in the presence of molecular crowding agents at high concentration (≥ 2 M) has been successfully demonstrated (Bremer et al 2017;Navarro-Retamal et al 2018). However, our CD spectra gathered for AfrLEA2, AfrLEA3m, and AfrLEA6 in solutions of trehalose across physiological concentrations indicate no impact of trehalose on the structure of LEA proteins or BSA.…”

“…While many questions still exist in terms of the varied functions of LEA proteins, in vitro and in vivo studies have contributed insights into their roles (for reviews, see Hand et al 2011;Hincha and Thalhammer 2012;Janis et al 2018a;Tunnacliffe and Wise 2007). For example, LEA proteins have been shown to protect lipid bilayers of various compositions during drying and freezing (Hundertmark et al 2011;Navarro-Retamal et al 2018;Steponkus et al 1998;Thalhammer et al 2014;Tolleter et al 2007Tolleter et al , 2010, preserve the activity of target enzymes (Boswell et al 2014a;Goyal et al 2005;Grelet et al 2005;Popova et al 2015), and prevent protein aggregation ("molecular shielding"; Boucher et al 2010;Goyal et al 2005;Yuen et al 2019). The gain of secondary structure (α-helix, β-sheet, turns) that accompanies the dehydration of some LEA proteins may allow them to perform separate/additional roles in the dried state (Goyal et al 2003).…”

Similar Yet Different–Structural and Functional Diversity among Arabidopsis thaliana LEA_4 Proteins

“…We have provided evidence that COR15A and COR15B protect chloroplast and plasma membranes during freezing in vivo, but do not stabilize chloroplast enzymes under these conditions [19]. The association of COR15A with membranes requires the transition from a random coil to an at least partially α-helical structure [19,32,34]. LEA7, another A. thaliana LEA_4 protein from a different clade, on the other hand, stabilizes the enzyme lactate dehydrogenase as well as enzymes in the A. thaliana soluble leaf proteome during freezing and drying [35].…”

“…A similar prediction pattern, combining high disorder probability with a high propensity for α-helix formation, has been reported for LEA proteins previously by Janis et al [40], who suggested that such a prediction pattern might indicate folding potential. We have previously shown that COR15A folds into amphipathic α-helices in response to reduced water availability [14,31], which drives membrane association and stabilization [32,41]. This structural element requires a regular distribution of hydrophobic amino acids along the protein sequence, usually every N+3 and/or N+4 positions, separated by polar residues [42].…”

“…We have contributed several studies on the LEA_4 proteins COR15A, COR15B, LEA25 and LEA11, all stemming from the same homology clade [19,[30][31][32][33]. All are essentially IDPs in the fully hydrated state, show a coil-helix transition in response to desiccation and partial dehydration and stabilize liposomes modeling the lipid composition of inner chloroplast membranes during a freeze/thaw cycle.…”