Mapping the Ca(2+) induced structural change in calreticulin

J Proteomics. 2016 Jun 16;142:138-48. doi: 10.1016/j.jprot.2016.05.015. Epub 2016 May 16.

Abstract

Calreticulin is a highly conserved multifunctional protein implicated in many different biological systems and has therefore been the subject of intensive research. It is primarily present in the endoplasmatic reticulum where its main functions are to regulate Ca(2+) homeostasis, act as a chaperone and stabilize the MHC class I peptide-loading complex. Although several high-resolution structures of calreticulin exist, these only cover three-quarters of the entire protein leaving the extended structures unsolved. Additionally, the structure of calreticulin is influenced by the presence of Ca(2+). The conformational changes induced by Ca(2+) have not been determined yet as they are hard to study with traditional approaches. Here, we investigated the Ca(2+)-induced conformational changes with a combination of chemical cross-linking, mass spectrometry, bioinformatics analysis and modelling in Rosetta. Using a bifunctional linker, we found a large Ca(2+)-induced change to the cross-linking pattern in calreticulin. Our results are consistent with a high flexibility in the P-loop, a stabilization of the acidic C-terminal and a relatively close interaction of the P-loop and the acidic C-terminal.

Biological significance: The function of calreticulin, an endoplasmatic reticulin chaperone, is affected by fluctuations in Ca(2+)concentration, but the structural mechanism is unknown. The present work suggests that Ca(2+)-dependent regulation is caused by different conformations of a long proline-rich loop that changes the accessibility to the peptide/lectin-binding site. Our results indicate that the binding of Ca(2+) to calreticulin may thus not only just be a question of Ca(2+) storage but is likely to have an impact on the chaperone activity.

Keywords: BS(3) d(0); BS(3) d(4); Calreticulin; Chemical cross-linking; Mass spectrometry; MassAI software; Protein structure; Rosetta modelling.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Calcium / pharmacology*
  • Calcium-Binding Proteins / chemistry
  • Calreticulin / chemistry*
  • Calreticulin / isolation & purification
  • Computational Biology
  • Female
  • Humans
  • Mass Spectrometry
  • Molecular Chaperones / metabolism
  • Placenta / chemistry
  • Pregnancy
  • Protein Binding / drug effects
  • Protein Conformation / drug effects

Substances

  • Calcium-Binding Proteins
  • Calreticulin
  • Molecular Chaperones
  • calreticulin, human
  • Calcium