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. 2011 Mar 18;286(11):8771-85.
doi: 10.1074/jbc.M110.169193. Epub 2010 Dec 22.

Calreticulin Is a Thermostable Protein With Distinct Structural Responses to Different Divalent Cation Environments

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Free PMC article

Calreticulin Is a Thermostable Protein With Distinct Structural Responses to Different Divalent Cation Environments

Sanjeeva J Wijeyesakere et al. J Biol Chem. .
Free PMC article

Abstract

Calreticulin is a soluble calcium-binding chaperone of the endoplasmic reticulum (ER) that is also detected on the cell surface and in the cytosol. Calreticulin contains a single high affinity calcium-binding site within a globular domain and multiple low affinity sites within a C-terminal acidic region. We show that the secondary structure of calreticulin is remarkably thermostable at a given calcium concentration. Rather than corresponding to complete unfolding events, heat-induced structural transitions observed for calreticulin relate to tertiary structural changes that expose hydrophobic residues and reduce protein rigidity. The thermostability and the overall secondary structure content of calreticulin are impacted by the divalent cation environment, with the ER range of calcium concentrations enhancing stability, and calcium-depleting or high calcium environments reducing stability. Furthermore, magnesium competes with calcium for binding to calreticulin and reduces thermostability. The acidic domain of calreticulin is an important mediator of calcium-dependent changes in secondary structure content and thermostability. Together, these studies indicate interactions between the globular and acidic domains of calreticulin that are impacted by divalent cations. These interactions influence the structure and stability of calreticulin, and are likely to determine the multiple functional activities of calreticulin in different subcellular environments.

Figures

FIGURE 1.
FIGURE 1.
Structural features of calreticulin and calnexin. A, superimposition of the globular domains of calnexin (PDB code 1JHN) (3) and calreticulin (PDB code 3O0V) (4) comparing the putative high affinity calcium-binding sites of the two proteins (shown as sticks). Calcium ions from the two crystal structures are shown as spheres. B, schematic of calreticulin showing the locations of the glycan and high affinity calcium-binding sites of calreticulin. The globular, P-, and acidic domains of calreticulin are also shown. The long helix of calreticulin, which precedes the acidic domain, is indicated. C, structure of the globular domain of calreticulin (PDB code 3O0V (4); corresponding to mCRT residues 1–351, excluding the P-domain). The long C-terminal helix of calreticulin is indicated. An arrow denotes the location of residue 339, which truncates seven C-terminal residues of the visible helix. The mCRT(1–351) construct includes the entire visible helix and five C-terminal unstructured residues. Asp311, which is a component of the high affinity binding site of calreticulin, is labeled. The calcium atom bound to the high affinity site is indicated and rendered as a black sphere. Asp342, Asp345, and Glu346, which could contribute to the low affinity calcium-binding sites of mCRT(1–351) are labeled. Glu347, which could additionally contribute to low affinity calcium binding in mCRT(1–351), was not visible in the crystal structure of the globular domain of calreticulin (PDB code 3O0V) (4). The image was produced using PyMOL (DeLano Scientific, Palo Alto, CA). D, sequence alignment (with observed and predicted secondary structures) of calreticulin residues 318–399 (corresponding to the long C-terminal helix in the globular domain of calreticulin along with its acidic regions), and the C-terminal sequence of the luminal region of canine calnexin (CNX) (residues 444–483). The observed secondary structure content from the crystal structures of the globular domain of calreticulin (PDB code 3O0V) (4) and the luminal domain of calnexin (PDB code 1JHN) (3) are shown above the corresponding sequences in light gray. The predicted secondary structure content for calreticulin is shown below the sequence in dark gray. Secondary structure predictions were obtained using PSIPRED (28), with sequence alignments obtained using Clustal W2 (29). α-Helices are rendered as rectangles. Numbered arrows indicate the location of residues 339, 351, and 362 in the calreticulin sequence, which correspond to the C-terminal truncation mutants characterized in this study. Asn327, the putative glycosylation site of calreticulin, which remains non-glycosylated in murine fibroblasts unless exposed to calcium-depleting conditions, is shaded with a gray background. Asp342, Asp345, Glu346, and Glu347, which could contribute to low affinity calcium binding by mCRT(1–351) and mCRT(1–362), and Glu352, Glu353, Glu354, Glu355, Asp356, Glu361, and Glu362, which could additionally contribute to low affinity calcium binding by mCRT(1–362), are indicated in bold.
FIGURE 2.
FIGURE 2.
Calreticulin has high and low affinity calcium-binding sites within the globular and acidic domains. Isothermal titration calorimetry at 37 °C of calcium binding to the high affinity sites of apo-mCRT(WT) (A), apo-mCRT(1–339) (B), and apo-mCRT(ΔP) (C) by sequential injections of 3.3 or 1.7 μm CaCl2. D, isothermal titration calorimetry at 37 °C of calcium binding to the low affinity sites of mCRT(WT) using a starting calcium concentration of 50 μm (to saturate the high affinity site) followed by sequential injections of 33 μm CaCl2. The figure shows representative raw titration curves (above) and the corresponding curve fit (below). The calculated thermodynamic parameters for at least two replicates of each analyzed construct are reported in Table 1.
FIGURE 3.
FIGURE 3.
At any given concentration of calcium, the secondary structure composition of calreticulin shows little variation to 60 °C. Far-UV CD spectra for mCRT(WT) (A–C) or mCRT(1–339) (D–F) under apo, 50 μm or 500 μm CaCO3 conditions. Also shown for contrast (in panel C) is the far-UV CD spectrum (measured from 260 to 210 nm) of mCRT(WT) denatured with 4 m guanidine HCl. An arrow denotes the shoulder seen at 228 nm. G–I, overlay of near-UV CD spectra of mCRT(WT) and mCRT(1–339) in 20 mm HEPES (pH 7.5) and 10 mm NaCl buffer containing 0, 50 μm, or 500 μm CaCl2 as indicated at 30 (G), 40 (H), and 50 °C (I). Unlike its secondary structure composition, which is invariant at a given calcium concentration, mCRT looses structural rigidity upon heating from 40 to 50 °C (panels H and I). Data were collected using a Jasco J-715 spectropolarimeter and represent the average of 2 independent sets of scans.
FIGURE 4.
FIGURE 4.
Calreticulin undergoes a loss of rigidity upon heating. Measurement (A and B) and quantification (C) of the TR50 of calreticulin for mCRT(WT) and mCRT(1–339) in 20 mm HEPES (pH 7.5) and 10 mm NaCl buffer containing 0, 50 μm, 500 μm, or 5 mm CaCl2 as indicated. Bar chart depicts mean TR50 ± S.E. (average of 2–6 independent analyses). D, in contrast to its tertiary structural rigidity, mCRT does not undergo a melting reaction (significant loss of secondary structure) upon heating from 20 to 90 °C as seen via the measurement of the far-UV CD signal for mCRT(WT) and mCRT(1–339) in 50 mm sodium phosphate (pH 7.5) and 500 mm NaF buffer containing 0 or 500 μm CaCO3 as indicated. Data represent the average of 2 independent scans. The reported p values indicate significant differences and were derived using two-tailed unpaired t tests. The p value for the difference in the TR50 of mCRT(WT) when going from 500 μm to 5 mm CaCl2 was 0.06. Unlike mCRT(WT), mCRT(1–339) showed no significant changes in TR50 (all p values > 0.3) associated with increasing [CaCl2]. Data were obtained using a Jasco J-715 spectropolarimeter.
FIGURE 5.
FIGURE 5.
Magnesium affects the tertiary structural rigidity of mCRT(WT) and mCRT(1–339) in a calcium-dependent manner. A and B, effects of 1 mm MgCl2 on rigidity of mCRT(WT). Measurement (A) and quantification (B) of TR50 values for mCRT(WT) in 20 mm HEPES (pH 7.5) and 10 mm NaCl buffer with or without 50 or 500 μm CaCl2 and in the presence or absence of 1 mm MgCl2. C and D, similar to panels A and B, but analyzing effects of 1 mm MgCl2 on rigidity of mCRT(1–339). Bar charts show mean TR50 ± S.E. (average of 2–6 independent analyses). Data derived in the absence of 1 mm MgCl2 were presented in Fig. 4. The reported p values indicate significant differences obtained from two-tailed unpaired t tests. As shown in panel D, mCRT(1–339) in its apo state did not show a significant decrease in TR50 in the presence of 1 mm MgCl2 (p = 0.78). Data were obtained using a Jasco J-715 spectropolarimeter.
FIGURE 6.
FIGURE 6.
The environmental calcium concentration impacts the secondary structure content of mCRT(WT), but not of mCRT(1–339), mCRT(1–351), or mCRT(1–362). Far-UV CD scans of mCRT(WT) (A), mCRT(1–339) (B), mCRT(1–351) (C), mCRT(1–362) (D), overlay of mCRT(WT) and mCRT(1–339) (E) in 0 and 50 μm CaCO3, overlay of mCRT(WT) and mCRT(1–351) in 0 and 50 μm CaCO3 (F), overlay of mCRT(1–351) and mCRT(1–339) in 50 μm CaCO3 (G), and overlay of mCRT(WT) and mCRT(1–362) in 0 and 50 μm CaCO3 (H). Proteins were in 50 mm sodium phosphate (pH 7.5) and 500 mm NaF with 50 μm, 500 μm, or 5 mm CaCO3 added as indicated. Data represent the averaged values of 4 independent sets of scans and were obtained using an Aviv 62DS spectropolarimeter.
FIGURE 7.
FIGURE 7.
The C terminus of the acidic region of calreticulin is required for calcium-dependent enhancements in thermostability over the ER range of calcium concentrations, whereas the globular domain mediates the reduction in thermostability of calreticulin at high calcium concentrations. Representative DSC scans (top panels) and quantifications of TTrans values (lower panels) for mCRT(WT) (A and E), mCRT(1–339) (B and F), mCRT(1–351) (C and G), and mCRT(1–362) (D and H) in 20 mm HEPES (pH 7.5) and 10 mm NaCl buffer containing 0, 50 μm, 500 μm, or 5 mm CaCl2 as indicated. Bar graphs represent mean TTrans ± S.E. (average of at least 3 independent sets of analyses). The p values shown on the bar graphs indicate significant differences obtained from paired t tests. Apo-mCRT(WT) was found to have a significantly lower TTrans value than apo-mCRT(1–351) (p = 0.05 in an unpaired t test) and mCRT(1–362) (p = 0.009 in an unpaired t test). The TTrans value for apo-mCRT(1–351) was not significantly lower than apo-mCRT(1–362) (p = 0.16 in an unpaired t test). The TTrans values for mCRT(1–339) under all tested [CaCl2] (0 μm, 50 μm, 500 μm, and 5 mm CaCl2) were significantly lower compared with other constructs at similar [CaCl2] (all p values < 0.009 in unpaired t tests). The p value for the difference in TTrans between apo-mCRT(1–339) versus mCRT(1–339) in 5 mm CaCl2 was 0.2 in an unpaired t test.
FIGURE 8.
FIGURE 8.
The C terminus of calreticulin becomes more exposed at high calcium concentrations. Fluorescence spectra of mCRT(WT) (A), mCRT(S391W) (B), and mCRT(A395W) (C) in 20 mm HEPES (pH 7.5) and 10 mm NaCl buffer with varying CaCl2 concentrations (0 μm, 50 μm, 500 μm, and 5 mm) depicting changes in intrinsic tryptophan fluorescence following excitation at 280 nm are shown. Data were collected in the range of 290–390 nm. Data represent the average of at least 2 independent sets of scans.
FIGURE 9.
FIGURE 9.
The acidic domain of calreticulin interacts with the globular domain and contributes to calcium dependence of the secondary structure content and thermostability of calreticulin. The long helix and acidic domain of calreticulin are depicted in dark gray. A, under calcium-depleted (apo) conditions, the presence of the N terminus of the acidic domain in mCRT(1–362) increases its secondary structure content stability and thermostability, suggesting that the N-terminal region of the acidic domain forms stabilizing interactions with the globular domain. B–D, the presence of the C-terminal residues of the acidic domain in mCRT(WT) destabilizes the globular domain, an effect that is mitigated upon occupancy of the calcium-binding sites of calreticulin. E, at high concentrations of calcium (5 mm), as the low affinity calcium-binding sites move toward full occupancy, independent disruption of electrostatic interactions within the globular domain initiates a global conformational change, including one that causes the C-terminal portion of the acidic domain of calreticulin to become more exposed to the external solvent environment.

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