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*

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Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Abstrakt

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).

OriginalsprogEngelsk
Artikelnummer140710
TidsskriftBiochimica et Biophysica Acta - Proteins and Proteomics
Vol/bind1869
Udgave nummer11
Antal sider18
ISSN1570-9639
DOI
StatusUdgivet - nov. 2021

Bibliografisk note

Funding Information:
This work was partially supported by the Research Council of Lithuania grants No. 09.3.3-LMT-K-712-12-0003 (to GH) and 01.2.2-LMT-K-718-03-0021 (to EČ). Kirsten Beth Hansen is thanked for excellent technical assistance. MM research is supported by the Canadian Institutes of Health Research and the Natural Sciences and Engineering Research Council of Canada .

Publisher Copyright:
© 2021 Elsevier B.V.

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