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

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 calciumdepleting 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.

Glycan-dependent and -independent Interactions Contribute to Cellular Substrate Recruitment by Calreticulin

“…Isothermal Titration Calorimetry (ITC)-ITC measurements (at 25°C) for calreticulin interacting with G1M3 were undertaken as described earlier (24). ITC runs were performed with calreticulin at a concentration of 50 M in 20 mM HEPES (pH 7.5), 10 mM NaCl, and 0.5 mM CaCl 2 .…”

“…Protein Purifications-Calreticulin and ERp57 were purified via nickel affinity chromatography as described previously (17,24). The secondary structure profiles of the calreticulin constructs were assessed via far-UV circular dichroism spectroscopy as described earlier (24).…”

“…The secondary structure profiles of the calreticulin constructs were assessed via far-UV circular dichroism spectroscopy as described earlier (24). Recombinant human ␤2-microglobulin (␤2M) was purified from Escherichia coli inclusion bodies as described previously (25,26).…”

“…A calreticulin construct with a N-terminal maltosebinding protein (MBP) tag (MBP-CRT) was generated using ligation-independent cloning to insert human CRT(WT) into the pMCSG9 vector (22) as described earlier (17,23). mCRT(K70C), mCRT(H128C), and the mCRT(E110C/E245C) double mutant were made in the background of mCRT(C146G) using the QuikChange II site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA) as described earlier (24) with mCRT in the pMCSG7 vector.…”

“…DSF thermostability data were analyzed using an asymmetric five-parameter dose-response curve (detailed in Ref. 24) to obtain the melting point (T m ) of each protein. Paired t tests were used to assess significance of the change in T m associated with the presence of glycans.…”