Characterization of the Relationship between the Chaperone and Lipid-Binding Functions of the 70-kDa Heat-Shock Protein, HspA1A
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 therap...
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| Format: | Article |
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MDPI AG
2020-08-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/21/17/5995 |
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| author | Larissa Smulders Amanda J. Daniels Caroline B. Plescia Devon Berger Robert V. Stahelin Nikolas Nikolaidis |
| author_facet | Larissa Smulders Amanda J. Daniels Caroline B. Plescia Devon Berger Robert V. Stahelin Nikolas Nikolaidis |
| author_sort | Larissa Smulders |
| collection | DOAJ |
| description | 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-Alanine mutation (K71A; a null-ATPase mutant) of HspA1A we show that although K71A binds to PS with affinities similar to the wild-type (WT), the mutated protein associates with lipids three times faster and dissociates 300 times faster than the WT HspA1A. These observations suggest a two-step binding model including an initial interaction of HspA1A with lipids followed by a conformational change of the HspA1A-lipid complex, which accelerates the binding reaction. Together these findings strongly support the notion that the chaperone and lipid-binding activities of HspA1A are dependent but the regions mediating these functions do not overlap and provide the basis for future interventions to inhibit HspA1A’s PM-translocation in tumor cells, making them sensitive to radiation therapy. |
| first_indexed | 2024-03-10T17:06:18Z |
| format | Article |
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| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T17:06:18Z |
| publishDate | 2020-08-01 |
| publisher | MDPI AG |
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| series | International Journal of Molecular Sciences |
| spelling | doaj.art-d3cedf6296854142b0fe303bb04b77262023-11-20T10:48:13ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672020-08-012117599510.3390/ijms21175995Characterization of the Relationship between the Chaperone and Lipid-Binding Functions of the 70-kDa Heat-Shock Protein, HspA1ALarissa Smulders0Amanda J. Daniels1Caroline B. Plescia2Devon Berger3Robert V. Stahelin4Nikolas Nikolaidis5Department of Biological Science, Center for Applied Biotechnology Studies, and Center for Computational and Applied Mathematics, College of Natural Sciences and Mathematics, California State University Fullerton, Fullerton, CA 92834-6850, USADepartment of Biological Science, Center for Applied Biotechnology Studies, and Center for Computational and Applied Mathematics, College of Natural Sciences and Mathematics, California State University Fullerton, Fullerton, CA 92834-6850, USADepartment of Medicinal Chemistry and Molecular Pharmacology and the Purdue University Center for Cancer Research, Purdue University, West Lafayette, IN 47907, USADepartment of Biological Science, Center for Applied Biotechnology Studies, and Center for Computational and Applied Mathematics, College of Natural Sciences and Mathematics, California State University Fullerton, Fullerton, CA 92834-6850, USADepartment of Medicinal Chemistry and Molecular Pharmacology and the Purdue University Center for Cancer Research, Purdue University, West Lafayette, IN 47907, USADepartment of Biological Science, Center for Applied Biotechnology Studies, and Center for Computational and Applied Mathematics, College of Natural Sciences and Mathematics, California State University Fullerton, Fullerton, CA 92834-6850, USAHspA1A, 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-Alanine mutation (K71A; a null-ATPase mutant) of HspA1A we show that although K71A binds to PS with affinities similar to the wild-type (WT), the mutated protein associates with lipids three times faster and dissociates 300 times faster than the WT HspA1A. These observations suggest a two-step binding model including an initial interaction of HspA1A with lipids followed by a conformational change of the HspA1A-lipid complex, which accelerates the binding reaction. Together these findings strongly support the notion that the chaperone and lipid-binding activities of HspA1A are dependent but the regions mediating these functions do not overlap and provide the basis for future interventions to inhibit HspA1A’s PM-translocation in tumor cells, making them sensitive to radiation therapy.https://www.mdpi.com/1422-0067/21/17/5995chaperone functionheat-shock proteinslipid bindingphosphatidylserineprotein refolding |
| spellingShingle | Larissa Smulders Amanda J. Daniels Caroline B. Plescia Devon Berger Robert V. Stahelin Nikolas Nikolaidis Characterization of the Relationship between the Chaperone and Lipid-Binding Functions of the 70-kDa Heat-Shock Protein, HspA1A International Journal of Molecular Sciences chaperone function heat-shock proteins lipid binding phosphatidylserine protein refolding |
| title | Characterization of the Relationship between the Chaperone and Lipid-Binding Functions of the 70-kDa Heat-Shock Protein, HspA1A |
| title_full | Characterization of the Relationship between the Chaperone and Lipid-Binding Functions of the 70-kDa Heat-Shock Protein, HspA1A |
| title_fullStr | Characterization of the Relationship between the Chaperone and Lipid-Binding Functions of the 70-kDa Heat-Shock Protein, HspA1A |
| title_full_unstemmed | Characterization of the Relationship between the Chaperone and Lipid-Binding Functions of the 70-kDa Heat-Shock Protein, HspA1A |
| title_short | Characterization of the Relationship between the Chaperone and Lipid-Binding Functions of the 70-kDa Heat-Shock Protein, HspA1A |
| title_sort | characterization of the relationship between the chaperone and lipid binding functions of the 70 kda heat shock protein hspa1a |
| topic | chaperone function heat-shock proteins lipid binding phosphatidylserine protein refolding |
| url | https://www.mdpi.com/1422-0067/21/17/5995 |
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