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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.
Afiliación
  • Adhikari J; Department of Chemistry, Washington University at St. Louis, St. Louis, Missouri 63130, United States.
  • Stephan JR; Department of Chemistry, Massachusetts Institute of Technology, Boston, Massachusetts 02139, United States.
  • Rempel DL; Department of Chemistry, Washington University at St. Louis, St. Louis, Missouri 63130, United States.
  • Nolan EM; Department of Chemistry, Massachusetts Institute of Technology, Boston, Massachusetts 02139, United States.
  • Gross ML; Department of Chemistry, Washington University at St. Louis, St. Louis, Missouri 63130, United States.
J Am Chem Soc ; 142(31): 13372-13383, 2020 08 05.
Article en En | MEDLINE | ID: mdl-32589841
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.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Calcio / Complejo de Antígeno L1 de Leucocito / Inmunidad Innata Tipo de estudio: Prognostic_studies Límite: Humans Idioma: En Revista: J Am Chem Soc Año: 2020 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Calcio / Complejo de Antígeno L1 de Leucocito / Inmunidad Innata Tipo de estudio: Prognostic_studies Límite: Humans Idioma: En Revista: J Am Chem Soc Año: 2020 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos