Calcium Binding to the Innate Immune Protein Human Calprotectin Revealed by Integrated Mass Spectrometry
Copyright © 2020 American Chemical Society. Although knowledge of the coordination chemistry and metal-withholding function of the innate immune protein human calprotectin (hCP) has broadened in recent years, understanding of its Ca2+-binding properties in solution remains incomplete. In particular,...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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American Chemical Society (ACS)
2022
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| Online Access: | https://hdl.handle.net/1721.1/141192.2 |
| _version_ | 1811096498397511680 |
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| author | Adhikari, Jagat Stephan, Jules R. Rempel, Don L. Nolan, Elizabeth M. Gross, Michael L. |
| author2 | Massachusetts Institute of Technology. Department of Chemistry |
| author_facet | Massachusetts Institute of Technology. Department of Chemistry Adhikari, Jagat Stephan, Jules R. Rempel, Don L. Nolan, Elizabeth M. Gross, Michael L. |
| author_sort | Adhikari, Jagat |
| collection | MIT |
| description | Copyright © 2020 American Chemical Society. Although knowledge of the coordination chemistry and metal-withholding function of the innate immune protein human calprotectin (hCP) has broadened in recent years, understanding of its Ca2+-binding properties in solution remains incomplete. In particular, the molecular basis by which Ca2+ binding affects structure and enhances the functional properties of this remarkable transition-metal-sequestering protein has remained enigmatic. To achieve a molecular picture of how Ca2+ binding triggers hCP oligomerization, increases protease stability, and enhances antimicrobial activity, we implemented a new integrated mass spectrometry (MS)-based approach that can be readily generalized to study other protein-metal and protein-ligand interactions. Three MS-based methods (hydrogen/deuterium exchange MS kinetics; protein-ligand interactions in solution by MS, titration, and H/D exchange (PLIMSTEX); and native MS) provided a comprehensive analysis of Ca2+ binding and oligomerization to hCP without modifying the protein in any way. Integration of these methods allowed us to (i) observe the four regions of hCP that serve as Ca2+-binding sites, (ii) determine the binding stoichiometry to be four Ca2+ per CP heterodimer and eight Ca2+ per CP heterotetramer, (iii) establish the protein-to-Ca2+ molar ratio that causes the dimer-to-tetramer transition, and (iv) calculate the binding affinities associated with the four Ca2+-binding sites per heterodimer. These quantitative results support a model in which hCP exists in its heterodimeric form and is at most half-bound to Ca2+ in the cytoplasm of resting cells. With release into the extracellular space, hCP encounters elevated Ca2+ concentrations and binds more Ca2+ ions, forming a heterotetramer that is poised to compete with microbial pathogens for essential metal nutrients. |
| first_indexed | 2024-09-23T16:44:38Z |
| format | Article |
| id | mit-1721.1/141192.2 |
| institution | Massachusetts Institute of Technology |
| language | English |
| last_indexed | 2024-09-23T16:44:38Z |
| publishDate | 2022 |
| publisher | American Chemical Society (ACS) |
| record_format | dspace |
| spelling | mit-1721.1/141192.22024-06-04T19:16:36Z Calcium Binding to the Innate Immune Protein Human Calprotectin Revealed by Integrated Mass Spectrometry Adhikari, Jagat Stephan, Jules R. Rempel, Don L. Nolan, Elizabeth M. Gross, Michael L. Massachusetts Institute of Technology. Department of Chemistry Copyright © 2020 American Chemical Society. Although knowledge of the coordination chemistry and metal-withholding function of the innate immune protein human calprotectin (hCP) has broadened in recent years, understanding of its Ca2+-binding properties in solution remains incomplete. In particular, the molecular basis by which Ca2+ binding affects structure and enhances the functional properties of this remarkable transition-metal-sequestering protein has remained enigmatic. To achieve a molecular picture of how Ca2+ binding triggers hCP oligomerization, increases protease stability, and enhances antimicrobial activity, we implemented a new integrated mass spectrometry (MS)-based approach that can be readily generalized to study other protein-metal and protein-ligand interactions. Three MS-based methods (hydrogen/deuterium exchange MS kinetics; protein-ligand interactions in solution by MS, titration, and H/D exchange (PLIMSTEX); and native MS) provided a comprehensive analysis of Ca2+ binding and oligomerization to hCP without modifying the protein in any way. Integration of these methods allowed us to (i) observe the four regions of hCP that serve as Ca2+-binding sites, (ii) determine the binding stoichiometry to be four Ca2+ per CP heterodimer and eight Ca2+ per CP heterotetramer, (iii) establish the protein-to-Ca2+ molar ratio that causes the dimer-to-tetramer transition, and (iv) calculate the binding affinities associated with the four Ca2+-binding sites per heterodimer. These quantitative results support a model in which hCP exists in its heterodimeric form and is at most half-bound to Ca2+ in the cytoplasm of resting cells. With release into the extracellular space, hCP encounters elevated Ca2+ concentrations and binds more Ca2+ ions, forming a heterotetramer that is poised to compete with microbial pathogens for essential metal nutrients. 2022-06-13T14:58:33Z 2022-03-15T17:58:19Z 2022-06-13T14:58:33Z 2020-06 2019-11 2022-03-15T17:49:33Z Article http://purl.org/eprint/type/JournalArticle 0002-7863 1520-5126 https://hdl.handle.net/1721.1/141192.2 Adhikari, Jagat, Stephan, Jules R, Rempel, Don L, Nolan, Elizabeth M and Gross, Michael L. 2020. "Calcium Binding to the Innate Immune Protein Human Calprotectin Revealed by Integrated Mass Spectrometry." Journal of the American Chemical Society, 142 (31). en http://dx.doi.org/10.1021/jacs.9b11950 Journal of the American Chemical Society Creative Commons Attribution-Noncommercial-Share Alike http://creativecommons.org/licenses/by-nc-sa/4.0/ application/octet-stream American Chemical Society (ACS) PMC |
| spellingShingle | Adhikari, Jagat Stephan, Jules R. Rempel, Don L. Nolan, Elizabeth M. Gross, Michael L. Calcium Binding to the Innate Immune Protein Human Calprotectin Revealed by Integrated Mass Spectrometry |
| title | Calcium Binding to the Innate Immune Protein Human Calprotectin Revealed by Integrated Mass Spectrometry |
| title_full | Calcium Binding to the Innate Immune Protein Human Calprotectin Revealed by Integrated Mass Spectrometry |
| title_fullStr | Calcium Binding to the Innate Immune Protein Human Calprotectin Revealed by Integrated Mass Spectrometry |
| title_full_unstemmed | Calcium Binding to the Innate Immune Protein Human Calprotectin Revealed by Integrated Mass Spectrometry |
| title_short | Calcium Binding to the Innate Immune Protein Human Calprotectin Revealed by Integrated Mass Spectrometry |
| title_sort | calcium binding to the innate immune protein human calprotectin revealed by integrated mass spectrometry |
| url | https://hdl.handle.net/1721.1/141192.2 |
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