Abstract
Calreticulin is an abundant endoplasmic reticulum resident protein that fulfills at least two basic functions. Firstly, due to its ability to bind monoglucosylated high mannose oligosaccharides, calreticulin is a central component of the folding quality control system of glycoproteins. On the other hand, thanks to its capacity to bind high amounts of calcium, calreticulin is one of the main calcium buffers in the endoplasmic reticulum. This last activity resides on a highly negatively charged domain located at the C terminus. Interestingly, this domain has been proposed to regulate the intracellular localization of calreticulin. Structural information for this domain is currently scarce. Here we address this issue by employing a combination of biophysical techniques and molecular dynamics simulation. We found that calreticulin C-terminal domain at low calcium concentration displays a disordered structure, whereas calcium addition induces a more rigid and compact conformation. Remarkably, this change develops when calcium concentration varies within a range similar to that taking place in the endoplasmic reticulum upon physiological fluctuations. In addition, a much higher calcium concentration is necessary to attain similar responses in a peptide displaying a randomized sequence of calreticulin C-terminal domain, illustrating the sequence specificity of this effect. Molecular dynamics simulation reveals that this ordering effect is a consequence of the ability of calcium to bring into close proximity residues that lie apart in the primary structure. These results place calreticulin in a new setting in which the protein behaves not only as a calcium-binding protein but as a finely tuned calcium sensor.
Highlights
Within the endoplasmic reticulum (ER), CRT fulfills at least two important roles
Calcium binding increases CRT stability, as revealed by chaotrope-induced denaturation, thermal stability, and protease susceptibility experiments [23, 37,38,39]. These effects are probably a consequence of calcium binding to the N-terminal domain, because, in general, they are observed when calcium concentration varies within the micromolar range
It has been shown that calcium depletion or C-terminal domain removal induces a conformation that exhibits increased affinity for hydrophobic peptides [40]
Summary
Stokes Radius Measurements—The dependence of CRT hydrodynamic radius with calcium was studied using size-exclusion chromatography (SEC) and dynamic light scattering (DLS) In both experiments the protein was dissolved in 30 mM Tris-HCl, pH 7.5, 300 mM NaCl with the addition of the indicated concentration of CaCl2. CD spectra were corrected for protein concentration and expressed in molar ellipticity (per residue) []MRW ϭ /(10 ϫCϫlϫn), where is the observed ellipticity in millidegrees, C is the molar concentration, l the path length in centimeters, and n the number of peptide bonds. Each peptide was located in a cubic box (6.45 nm side) with periodic boundary conditions, and we employed the smooth particle mesh Ewald method to treat the long range electrostatic interactions [32] In these simulations Ca2ϩ and Naϩ concentrations were 24.8 mM and 74.4 mM, respectively, while the ClϪ concentration was 24.8 mM. Randomized peptides of the same amino acid composition were run as control samples
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