Preprint Article Version 2 Preserved in Portico This version is not peer-reviewed

Characterization of the Relationship between the Chaperone and Lipid-Binding Functions of the 70-Kda Heat-Shock Protein, HspA1A

Larissa Smulders
ORCID logo ,
Amanda J. Daniels
,
Caroline P. Plescia
,
Devon Berger
,
Robert V. Stahelin
ORCID logo ,
Nikolas Nikolaidis
* ORCID logo
Version 1 : Received: 7 May 2020 / Approved: 9 May 2020 / Online: 9 May 2020 (04:48:12 CEST)
Version 2 : Received: 28 July 2020 / Approved: 29 July 2020 / Online: 29 July 2020 (12:18:02 CEST)

A peer-reviewed article of this Preprint also exists.

Smulders, L.; Daniels, A.J.; Plescia, C.B.; Berger, D.; Stahelin, R.V.; Nikolaidis, N. Characterization of the Relationship between the Chaperone and Lipid-Binding Functions of the 70-kDa Heat-Shock Protein, HspA1A. Int. J. Mol. Sci. 2020, 21, 5995. Smulders, L.; Daniels, A.J.; Plescia, C.B.; Berger, D.; Stahelin, R.V.; Nikolaidis, N. Characterization of the Relationship between the Chaperone and Lipid-Binding Functions of the 70-kDa Heat-Shock Protein, HspA1A. Int. J. Mol. Sci. 2020, 21, 5995.

Abstract

HspA1A, a molecular chaperone, translocates to the plasma membrane (PM) of stressed and cancer cells. This translocation results in HspA1A’s cell-surface presentation, which renders tumors radiation insensitive. To specifically inhibit the lipid-driven HspA1A’s PM translocation and devise new therapeutics it is imperative to characterize the unknown HspA1A’s lipid-binding regions and determine the relationship between the chaperone and lipid-binding functions. To elucidate this relationship, we determined the effect of phosphatidylserine (PS)-binding on the secondary structure and chaperone functions of HspA1A. Circular dichroism revealed that binding to PS resulted in minimal modification on HspA1A’s secondary structure. Measuring the release of inorganic phosphate revealed that PS-binding had no effect on HspA1A’s ATPase activity. In contrast, PS-binding showed subtle but consistent increases in HspA1A’s refolding activities. Furthermore, using a Lysine-71-Arginine mutation (K71A; a null-ATPase mutant) of HspA1A we show that although K71A binds to PS with affinities similar to the WT, the kinetics of the binding are significantly different, probably because of the mutant’s inability to achieve specific conformations. These observations suggest a two-step binding model that includes conformational changes and strongly support the notion that the chaperone and lipid-binding activities of HspA1A are dependent but the regions mediating these functions do not overlap. These findings provide the basis for future interventions to inhibit HspA1A’s PM-translocation in tumor cells, making them sensitive to radiation therapy.

Keywords

chaperone function; heat-shock proteins; lipid binding; phosphatidylserine; protein refolding

Subject

Biology and Life Sciences, Biochemistry and Molecular Biology

Copyright: This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Comments (1)

Comment 1
Received: 29 July 2020
Commenter: Nikolas Nikolaidis
Commenter's Conflict of Interests: Author
Comment: Updated results; addition of mutational and binding data; major text updates.
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