Preparation and Iron Redox Speciation Study of the Fe(II)-Binding Antimicrobial Protein Calprotectin

Hadley, Rose C.; Nolan, Elizabeth M.

doi:10.1007/978-1-4939-9030-6_25

Cited by 6 publications

(9 citation statements)

References 28 publications

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“…This Cys-null variant has been employed extensively in metal-binding and antimicrobial activity studies, and all available data indicate that it displays the same Ca 2+dependent heterotetramerization and transition-metal-binding properties as hCP. 10,37 We first measured HDX kinetics for CP-Ser at the peptide level in the absence and presence of excess Ca 2+ ions. By examining the S100A8 and S100A9 peptide fragments obtained following pepsin digestion, we identified peptide fragments corresponding to each of the four EF-hand domains and the tetramer interface.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…CP-Ser allows experiments to be conducted without the complications associated with disulfide bonding (e.g., no need for a reducing agent in the HDX or other measurement). This Cys-null variant has been employed extensively in metal-binding and antimicrobial activity studies, and all available data indicate that it displays the same Ca 2+ -dependent heterotetramerization and transition-metal-binding properties as hCP. , …”

Section: Resultsmentioning

confidence: 99%

“…CP-Ser was prepared as the apo-heterodimer and handled as previously described. 10,37 Hydrogen/Deuterium Exchange (HDX). The HDX experiment and data analysis procedures were similar to previously described protocols.…”

Section: ■ Summary and Outlookmentioning

confidence: 99%

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Calcium Binding to the Innate Immune Protein Human Calprotectin Revealed by Integrated Mass Spectrometry

et al. 2020

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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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Section: ■ Results and Discussionmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Calcium Binding to the Innate Immune Protein Human Calprotectin Revealed by Integrated Mass Spectrometry

et al. 2020

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“…After this incubation, the 57 Fe was completely dissolved. This stock was diluted to 500 μM in Milli-Q water, and its concentration was measured using a ferrozine assay, as described previously ( 62 ).…”

Section: Methodsmentioning

confidence: 99%

“…This stock was diluted to 500 µM in Milli-Q water and its concentration was measured using a ferrozine assay, as previously described. (61) P. aeruginosa PA14 was streaked from a freezer stocks onto a TSB 1.5% agarose plate and grown overnight (12-16h) at 37 °C. A single colony was used to inoculate 2 mL TSB and incubated overnight (12-16h) on a rotating wheel at 37 °C.…”

Section: Fe Uptake Assaymentioning

confidence: 99%

Heme protects Pseudomonas aeruginosa and Staphylococcus aureus from calprotectin-induced iron starvation

Zygiel

Obisesan

Nelson

et al. 2021

Journal of Biological Chemistry

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Pseudomonas aeruginosa and Staphylococcus aureus are opportunistic bacterial pathogens that cause severe infections in immunocompromised individuals and patients with cystic fibrosis. Both P. aeruginosa and S. aureus require iron to infect the mammalian host. To obtain iron, these pathogens may rely on siderophore-mediated ferric iron uptake, ferrous iron uptake, or heme uptake at different points during infection. The preferred iron source depends on environmental conditions, including the presence of iron-sequestering host-defense proteins. Here, we investigate how the presence of heme, a highly relevant iron source during infection, affects bacterial responses to iron withholding by the innate immune protein calprotectin (CP). Prior work has shown that P. aeruginosa is starved of iron in the presence of CP. We report that P. aeruginosa upregulates expression of heme uptake machinery in response to CP. Furthermore, we show that heme protects P. aeruginosa from CP-mediated inhibition of iron uptake and iron-starvation responses. We extend our study to a second bacterial pathogen, S. aureus , and demonstrate that CP also inhibits iron uptake and induces iron-starvation responses by this pathogen. Similarly to P. aeruginosa , we show that heme protects S. aureus from CP-mediated inhibition of iron uptake and iron-starvation responses. These findings expand our understanding of microbial responses to iron sequestration by CP and highlight the importance of heme utilization for bacterial adaptation to host iron-withholding strategies.

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The CRISPR-associated Cas4 protein from Leptospira interrogans demonstrate versatile nuclease activity

Dixit¹,

Anand²,

Hussain³

et al. 2021

Current Research in Microbial Sciences

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Preparation and Iron Redox Speciation Study of the Fe(II)-Binding Antimicrobial Protein Calprotectin

Cited by 6 publications

References 28 publications

Calcium Binding to the Innate Immune Protein Human Calprotectin Revealed by Integrated Mass Spectrometry

Calcium Binding to the Innate Immune Protein Human Calprotectin Revealed by Integrated Mass Spectrometry

Heme protects Pseudomonas aeruginosa and Staphylococcus aureus from calprotectin-induced iron starvation

The CRISPR-associated Cas4 protein from Leptospira interrogans demonstrate versatile nuclease activity

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