title: Mapping the Ca(2+) induced structural change in calreticulin.
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.
  
  
  
  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.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK 5230 Odense, Denmark; Department of Autoimmunology and Biomarkers, Statens Serum Institut, Artillerivej 5, DK 2300 Copenhagen, Denmark.
authors.forename: Sanne Grundvad
authors.initials: SG
authors.lastname: Boelt
authors.affiliation: Department of Biochemistry and Structural Biology, Lund University, Paradisgatan 2, SE 221 00 Lund, Sweden.
authors.forename: Christoffer
authors.initials: C
authors.lastname: Norn
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK 5230 Odense, Denmark.
authors.forename: Morten Ib
authors.initials: MI
authors.lastname: Rasmussen
authors.affiliation: Department of Biochemistry and Structural Biology, Lund University, Paradisgatan 2, SE 221 00 Lund, Sweden.
authors.forename: Ingemar
authors.initials: I
authors.lastname: André
authors.affiliation: Department of Eukayote Gene Engineering, Institute of Biotechnology, Vilnius University, V. Graičiūno St, LT 02241 Vilnius, Lithuania.
authors.forename: Evaldas
authors.initials: E
authors.lastname: Čiplys
authors.affiliation: Department of Eukayote Gene Engineering, Institute of Biotechnology, Vilnius University, V. Graičiūno St, LT 02241 Vilnius, Lithuania.
authors.forename: Rimantas
authors.initials: R
authors.lastname: Slibinskas
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK 5230 Odense, Denmark; Department of Autoimmunology and Biomarkers, Statens Serum Institut, Artillerivej 5, DK 2300 Copenhagen, Denmark.
authors.forename: Gunnar
authors.initials: G
authors.lastname: Houen
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK 5230 Odense, Denmark. Electronic address: php@bmb.sdu.dk.
authors.forename: Peter
authors.initials: P
authors.lastname: Højrup
mesh_terms: D002118:Calcium / Q000494:pharmacology*; D002135:Calcium-Binding Proteins / Q000737:chemistry; D037282:Calreticulin / Q000737:chemistry* / Q000302:isolation & purification; D019295:Computational Biology; D005260:Female; D006801:Humans; D013058:Mass Spectrometry; D018832:Molecular Chaperones / Q000378:metabolism; D010920:Placenta / Q000737:chemistry; D011247:Pregnancy; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects
chemical_list: C576483:CALR protein, human; D002135:Calcium-Binding Proteins; D037282:Calreticulin; D018832:Molecular Chaperones; D002118:Calcium
keywords: BS(3) d(0); BS(3) d(4); Calreticulin; Chemical cross-linking; Mass spectrometry; MassAI software; Protein structure; Rosetta modelling