Mapping human calreticulin regions important for structural stability

Evaldas Čiplys, Tautvydas Paškevičius, Eimantas Žitkus, Juras Bielskis, Raimundas Ražanskas, Tomas Šneideris, Vytautas Smirnovas, Algirdas Kaupinis, David J. Tester, Michael J. Ackerman, Peter Højrup, Marek Michalak, Gunnar Houen, Rimantas Slibinskas*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Calreticulin (CALR) is a highly conserved multifunctional chaperone protein primarily present in the endoplasmic reticulum, where it regulates Ca2+ homeostasis. Recently, CALR has gained special interest for its diverse functions outside the endoplasmic reticulum, including the cell surface and extracellular space. Although high-resolution structures of CALR exist, it has not yet been established how different regions and individual amino acid residues contribute to structural stability of the protein. In the present study, we have identified key residues determining the structural stability of CALR. We used a Saccharomyces cerevisiae expression system to express and purify 50 human CALR mutants, which were analysed for several parameters including secretion titer, melting temperature (Tm), stability and oligomeric state. Our results revealed the importance of a previously identified small patch of conserved surface residues, amino acids 166–187 (“cluster 2”) for structural stability of the human CALR protein. Two residues, Tyr172 and Asp187, were critical for maintaining the native structure of the protein. Mutant D187A revealed a severe drop in secretion titer, it was thermally unstable, prone to degradation, and oligomer formation. Tyr172 was critical for thermal stability of CALR and interacted with the third free Cys163 residue. This illustrates an unusual thermal stability of CALR dominated by Asp187, Tyr172 and Cys163, which may interact as part of a conserved structural unit. Besides structural clusters, we found a correlation of some measured parameter values in groups of CALR mutants that cause myeloproliferative neoplasms (MPN) and in mutants that may be associated with sudden unexpected death (SUD).

Original languageEnglish
Article number140710
JournalBiochimica et Biophysica Acta - Proteins and Proteomics
Volume1869
Issue number11
Number of pages18
ISSN1570-9639
DOIs
Publication statusPublished - Nov 2021

Bibliographical note

Publisher Copyright:
© 2021 Elsevier B.V.

Keywords

  • Calreticulin
  • Mutants
  • Secretion
  • Structural stability
  • Thermal stability

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