Mapping the Ca2+ induced structural change in calreticulin

Sanne Grundvad Boelt, Christoffer Norn, Morten Ib Rasmussen, Ingemar André, Evaldas Čiplys, Rimantas Slibinskas, Gunnar Houen, Peter Højrup

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

UNLABELLED: 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.

Original languageEnglish
JournalJournal of Proteomics
Volume142
Pages (from-to)138-148
ISSN1874-3919
DOIs
Publication statusPublished - 16. Jun 2016

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Calreticulin
Reticulin
Reticulum
Peptides
Biological systems
Bioinformatics
Computational Biology
Lectins
Proline
Mass spectrometry
Conformations
Proteins
Homeostasis
Stabilization
Binding Sites
Research

Keywords

  • Journal Article

Cite this

Boelt, S. G., Norn, C., Rasmussen, M. I., André, I., Čiplys, E., Slibinskas, R., ... Højrup, P. (2016). Mapping the Ca2+ induced structural change in calreticulin. Journal of Proteomics, 142, 138-148. https://doi.org/10.1016/j.jprot.2016.05.015
Boelt, Sanne Grundvad ; Norn, Christoffer ; Rasmussen, Morten Ib ; André, Ingemar ; Čiplys, Evaldas ; Slibinskas, Rimantas ; Houen, Gunnar ; Højrup, Peter. / Mapping the Ca2+ induced structural change in calreticulin. In: Journal of Proteomics. 2016 ; Vol. 142. pp. 138-148.
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Boelt, SG, Norn, C, Rasmussen, MI, André, I, Čiplys, E, Slibinskas, R, Houen, G & Højrup, P 2016, 'Mapping the Ca2+ induced structural change in calreticulin', Journal of Proteomics, vol. 142, pp. 138-148. https://doi.org/10.1016/j.jprot.2016.05.015

Mapping the Ca2+ induced structural change in calreticulin. / Boelt, Sanne Grundvad; Norn, Christoffer; Rasmussen, Morten Ib; André, Ingemar; Čiplys, Evaldas; Slibinskas, Rimantas; Houen, Gunnar; Højrup, Peter.

In: Journal of Proteomics, Vol. 142, 16.06.2016, p. 138-148.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Mapping the Ca2+ induced structural change in calreticulin

AU - Boelt, Sanne Grundvad

AU - Norn, Christoffer

AU - Rasmussen, Morten Ib

AU - André, Ingemar

AU - Čiplys, Evaldas

AU - Slibinskas, Rimantas

AU - Houen, Gunnar

AU - Højrup, Peter

N1 - Copyright © 2016 Elsevier B.V. All rights reserved.

PY - 2016/6/16

Y1 - 2016/6/16

N2 - UNLABELLED: 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.

AB - UNLABELLED: 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.

KW - Journal Article

U2 - 10.1016/j.jprot.2016.05.015

DO - 10.1016/j.jprot.2016.05.015

M3 - Journal article

C2 - 27195812

VL - 142

SP - 138

EP - 148

JO - Journal of Proteomics

JF - Journal of Proteomics

SN - 1874-3919

ER -

Boelt SG, Norn C, Rasmussen MI, André I, Čiplys E, Slibinskas R et al. Mapping the Ca2+ induced structural change in calreticulin. Journal of Proteomics. 2016 Jun 16;142:138-148. https://doi.org/10.1016/j.jprot.2016.05.015