Calcium Ion Gradients Modulate the Zinc Affinity and Antibacterial Activity of Human Calprotectin
Calprotectin (CP) is an antimicrobial protein produced and released by neutrophils that inhibits the growth of pathogenic microorganisms by sequestering essential metal nutrients in the extracellular space. In this work, spectroscopic and thermodynamic metal-binding studies are presented to delineat...
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| Format: | Article |
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American Chemical Society
2013
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| Online Access: | http://hdl.handle.net/1721.1/82571 https://orcid.org/0000-0002-6153-8803 |
| _version_ | 1826206899869057024 |
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| author | Brophy, Megan Brunjes Hayden, Joshua A. Nolan, Elizabeth M. |
| author2 | Massachusetts Institute of Technology. Department of Chemistry |
| author_facet | Massachusetts Institute of Technology. Department of Chemistry Brophy, Megan Brunjes Hayden, Joshua A. Nolan, Elizabeth M. |
| author_sort | Brophy, Megan Brunjes |
| collection | MIT |
| description | Calprotectin (CP) is an antimicrobial protein produced and released by neutrophils that inhibits the growth of pathogenic microorganisms by sequestering essential metal nutrients in the extracellular space. In this work, spectroscopic and thermodynamic metal-binding studies are presented to delineate the zinc-binding properties of CP. Unique optical absorption and EPR spectroscopic signatures for the interfacial His3Asp and His4 sites of human calprotectin are identified by using Co(II) as a spectroscopic probe. Zinc competition titrations employing chromophoric Zn(II) indicators provide a 2:1 Zn(II):CP stoichiometry, confirm that the His[subscript 3]Asp and His[subscript 4] sites of CP coordinate Zn(II), and reveal that the Zn(II) affinity of both sites is calcium-dependent. The calcium-insensitive Zn(II) competitor ZP4 affords dissociation constants of K[subscript d1] = 133 ± 58 pM and K[subscript d2] = 185 ± 219 nM for CP in the absence of Ca(II). These values decrease to K[subscript d1] ≤ 10 pM and K[subscript d2] ≤ 240 pM in the presence of excess Ca(II). The K[subscript d1] and K[subscript d2] values are assigned to the His[subscript 3]Asp and His[subscript 4] sites, respectively. In vitro antibacterial activity assays indicate that the metal-binding sites and Ca(II)-replete conditions are required for CP to inhibit the growth of both Gram-negative and -positive bacteria. Taken together, these data provide a working model whereby calprotectin responds to physiological Ca(II) gradients to become a potent Zn(II) chelator in the extracellular space. |
| first_indexed | 2024-09-23T13:40:41Z |
| format | Article |
| id | mit-1721.1/82571 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T13:40:41Z |
| publishDate | 2013 |
| publisher | American Chemical Society |
| record_format | dspace |
| spelling | mit-1721.1/825712022-10-01T16:25:13Z Calcium Ion Gradients Modulate the Zinc Affinity and Antibacterial Activity of Human Calprotectin Brophy, Megan Brunjes Hayden, Joshua A. Nolan, Elizabeth M. Massachusetts Institute of Technology. Department of Chemistry Brophy, Megan Brunjes Hayden, Joshua A. Nolan, Elizabeth M. Calprotectin (CP) is an antimicrobial protein produced and released by neutrophils that inhibits the growth of pathogenic microorganisms by sequestering essential metal nutrients in the extracellular space. In this work, spectroscopic and thermodynamic metal-binding studies are presented to delineate the zinc-binding properties of CP. Unique optical absorption and EPR spectroscopic signatures for the interfacial His3Asp and His4 sites of human calprotectin are identified by using Co(II) as a spectroscopic probe. Zinc competition titrations employing chromophoric Zn(II) indicators provide a 2:1 Zn(II):CP stoichiometry, confirm that the His[subscript 3]Asp and His[subscript 4] sites of CP coordinate Zn(II), and reveal that the Zn(II) affinity of both sites is calcium-dependent. The calcium-insensitive Zn(II) competitor ZP4 affords dissociation constants of K[subscript d1] = 133 ± 58 pM and K[subscript d2] = 185 ± 219 nM for CP in the absence of Ca(II). These values decrease to K[subscript d1] ≤ 10 pM and K[subscript d2] ≤ 240 pM in the presence of excess Ca(II). The K[subscript d1] and K[subscript d2] values are assigned to the His[subscript 3]Asp and His[subscript 4] sites, respectively. In vitro antibacterial activity assays indicate that the metal-binding sites and Ca(II)-replete conditions are required for CP to inhibit the growth of both Gram-negative and -positive bacteria. Taken together, these data provide a working model whereby calprotectin responds to physiological Ca(II) gradients to become a potent Zn(II) chelator in the extracellular space. National Institutes of Health (U.S.) (Office of the Director, NIH Grant DP2OD007045) Searle Scholars Program Massachusetts Institute of Technology. Center for Environmental Health Sciences (NIH Grant P30-ES002109) Massachusetts Institute of Technology. Dept. of Chemistry 2013-11-25T17:04:03Z 2013-11-25T17:04:03Z 2012-10 2012-08 Article http://purl.org/eprint/type/JournalArticle 0002-7863 1520-5126 http://hdl.handle.net/1721.1/82571 Brophy, Megan Brunjes, Joshua A. Hayden, and Elizabeth M. Nolan. “Calcium Ion Gradients Modulate the Zinc Affinity and Antibacterial Activity of Human Calprotectin.” Journal of the American Chemical Society 134, no. 43 (October 31, 2012): 18089-18100. https://orcid.org/0000-0002-6153-8803 en_US http://dx.doi.org/10.1021/ja307974e Journal of the American Chemical Society Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. application/pdf American Chemical Society PMC |
| spellingShingle | Brophy, Megan Brunjes Hayden, Joshua A. Nolan, Elizabeth M. Calcium Ion Gradients Modulate the Zinc Affinity and Antibacterial Activity of Human Calprotectin |
| title | Calcium Ion Gradients Modulate the Zinc Affinity and Antibacterial Activity of Human Calprotectin |
| title_full | Calcium Ion Gradients Modulate the Zinc Affinity and Antibacterial Activity of Human Calprotectin |
| title_fullStr | Calcium Ion Gradients Modulate the Zinc Affinity and Antibacterial Activity of Human Calprotectin |
| title_full_unstemmed | Calcium Ion Gradients Modulate the Zinc Affinity and Antibacterial Activity of Human Calprotectin |
| title_short | Calcium Ion Gradients Modulate the Zinc Affinity and Antibacterial Activity of Human Calprotectin |
| title_sort | calcium ion gradients modulate the zinc affinity and antibacterial activity of human calprotectin |
| url | http://hdl.handle.net/1721.1/82571 https://orcid.org/0000-0002-6153-8803 |
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