Bioinorganic Chemistry of the Human Host-Defense Protein Calprotectin
Thesis: Ph. D. in Biological Chemistry, Massachusetts Institute of Technology, Department of Chemistry, 2015.
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| Format: | Thesis |
| Language: | eng |
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Massachusetts Institute of Technology
2015
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| Online Access: | http://hdl.handle.net/1721.1/98823 |
| _version_ | 1811094806898671616 |
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| author | Brophy, Megan Brunjes |
| author2 | Elizabeth M. Nolan. |
| author_facet | Elizabeth M. Nolan. Brophy, Megan Brunjes |
| author_sort | Brophy, Megan Brunjes |
| collection | MIT |
| description | Thesis: Ph. D. in Biological Chemistry, Massachusetts Institute of Technology, Department of Chemistry, 2015. |
| first_indexed | 2024-09-23T16:05:26Z |
| format | Thesis |
| id | mit-1721.1/98823 |
| institution | Massachusetts Institute of Technology |
| language | eng |
| last_indexed | 2024-09-23T16:05:26Z |
| publishDate | 2015 |
| publisher | Massachusetts Institute of Technology |
| record_format | dspace |
| spelling | mit-1721.1/988232019-04-11T09:44:19Z Bioinorganic Chemistry of the Human Host-Defense Protein Calprotectin Brophy, Megan Brunjes Elizabeth M. Nolan. Massachusetts Institute of Technology. Department of Chemistry. Massachusetts Institute of Technology. Department of Chemistry. Chemistry. Thesis: Ph. D. in Biological Chemistry, Massachusetts Institute of Technology, Department of Chemistry, 2015. Vita. Cataloged from PDF version of thesis. Includes bibliographical references. The human innate immune system responds to bacterial and fungal pathogens by releasing the metal-chelating protein calprotectin (CP) at sites of infection and in the upper layers of the epidermis. CP is a Mn(II)- and Zn(ll)-binding protein. The work described in this thesis elucidates the metal-binding properties of CP, and correlates these properties with in vitro growth inhibition of bacteria and fungi. We report that the metal-binding properties of CP are modulated by Ca(ll), and we propose a working model in which CP responds to physiological Ca(Il)-ion gradients to become a potent Zn(ll)- and Mn(Il)-chelating agent in the extracellular space. Individual chapter summaries follow. Chapter 1: Bioinorganic Chemistry of the Host Pathogen Interaction. Transition metal ions are required for all forms of life. During the course of infection, pathogenic microorganisms must acquire transition metals from the host. Three metals of interest from this standpoint are iron, zinc, and manganese. This chapter describes bacterial metal-ion homeostasis machineries, and metal-requiring processes with a focus on Zn(II) and Mn(II). This chapter then highlights the S100 family of Ca(ll)-binding proteins and discuses the Zn(Il)-, Cu(ll)-, and Mn(Il)-binding properties of S100B, S100A12, S100A7, S10OA15, and S100A8/S100A9. Finally, an overview of the scope of this thesis is presented. Chapter 2: Calcium Ion Gradients Modulate the Zinc(Il) Affinity and Antibacterial Activity of Human Calprotectin. Calprotectin (CP) is a human neutrophil protein that is produced and released by neutrophils at sites of infection, where it prevents the growth of microorganisms by sequestering bioavailable zinc(II) and manganese(II). In this chapter, we present metalbinding studies to elucidate the Zn(ll)-binding properties of CP. We report unique optical absorption and EPR spectroscopic signatures for the interfacial His 3Asp and His 4 sites of human CP by using Co(II) as a spectroscopic probe. Zinc competition titrations employing colorimetric and fluorimetric Zn(II) sensors establish that CP coordinates two Zn(II) ions / CP heterodimer. The Ca(ll)-insensitive Zn(ll) sensor ZP4 is used to determine the Kd of CP for Zn(II) in Ca(Il)-deplete and Ca(Il)-replete conditions. These competition titrations afford apparent Kdsitel = 133 58 pM and Kdsite2 = 185 219 nM in the absence of Ca(II). In the presence of excess Ca(Il) these values decrease to Kd,sitel 5 10 pM and Kd,site2 : 240 pM. In vitro antibacterial assays indicate that the metal-binding sites and Ca(ll)-replete conditions are required to inhibit the growth of Gram-negative and Gram-positive bacteria. We propose a model in which Ca(II) ion gradients modulate the antibacterial activity and Zn(Il)-binding properties of human CP. Chapter 3: High-Affinity Manganese Coordination by Human Calprotectin Is Calcium- Dependent and Requires the Histidine-Rich Site at the Dimer Interface. In this chapter, we report that the His 4 motif at the S10OA8/S100A9 dimer interface of CP is required for high-affinity Mn(II) coordination. We identify a low-temperature EPR spectroscopic signal for this site that is consistent with high-spin Mn(II) in an octahedral coordination sphere. This site could be simulated with zero-field splitting parameters D = 270 MHz and EID = 0.30 (E = 81 MHz). This analysis, combined with studies of mutant proteins, suggests that (A8)Hisl7, (A8)His27, (A9)His9l, (A9)His95 and two as-yet unidentified ligands coordinate Mn(ll) at site 2. These studies support a model in which CP responds to Ca(ll) ion gradients to become a potent metal-ion chelator in the extracellular space. Chapter 4: Contributions of the C-terminal Tail of S100A9 to High-Affinity Manganese Binding by Human Calprotectin. This chapter examines the role of the S100A9 C-terminal tail to high-affinity Mn(ll) coordination by human CP. We present a 16-member mutant family with mutations in the S100A9 C-terminal tail (residues 96-114), which houses three histidine and four acidic residues, to evaluate its contribution to Mn(ll) sequestration. These studies confirm that two His residues at positions 103 and 105 complete the octahedral coordination sphere of CP in solution. Appendix 1: Sequence Alignments of Transition-Metal Binding S100 Proteins. Sequence alignments of S100A7, S100A8, S100A9, S100A12, S100A15, and S100B proteins from multiple organisms are presented. Appendix 2: Characterization of CP Mutant Proteins by Circular Dichroism and Analytical Size Exclusion Chromatography. Additional characterization of CP and mutant proteins employed in Chapters 2-4 is presented. Appendix 3: Structures of Sensors Used In this Work. The structures of Zincon, MagFura-2, Zinpyr-1, and Zinpyr-4 are presented. Appendix 4: Manganese Binding Properties of Human Calprotectin under Conditions of High and Low Calcium. This appendix represents a collaborative work with the Drennan Lab (MIT) and Britt Lab (UC Davis) to study the Mn(Il)-CP complex in low- and high-Ca(II) conditions. We report a crystal structure of Mn(Il)-, Ca(Il)-, and Na(l)-bound CP with Mn(II) exclusively coordinated to the His6 motif. Electron spin-echo envelope modulation and electron-nuclear double resonance experiments demonstrate that the six coordinating histidine residues are spectroscopically equivalent. The observed 15N ( = %/h)y perfine couplings (A) arise from two distinct classes of nitrogen atoms: the coordinating E-nitrogen of the imidazole ring of each histidine (A = [3.45, 3.71, 5.91] MHz) and the distal 6-nitrogen (A = [0.11, 0.18, 0.42] MHz). In the absence of Ca(II), the affinity of CP for Mn(II) drops by two to three orders of magnitude, and Mn(II) coordinates to the His6 site as well as other sites on the protein. by Megan Brunjes Brophy. Ph. D. in Biological Chemistry 2015-09-17T19:14:44Z 2015-09-17T19:14:44Z 2015 2015 Thesis http://hdl.handle.net/1721.1/98823 921148805 eng M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission. http://dspace.mit.edu/handle/1721.1/7582 345 pages application/pdf Massachusetts Institute of Technology |
| spellingShingle | Chemistry. Brophy, Megan Brunjes Bioinorganic Chemistry of the Human Host-Defense Protein Calprotectin |
| title | Bioinorganic Chemistry of the Human Host-Defense Protein Calprotectin |
| title_full | Bioinorganic Chemistry of the Human Host-Defense Protein Calprotectin |
| title_fullStr | Bioinorganic Chemistry of the Human Host-Defense Protein Calprotectin |
| title_full_unstemmed | Bioinorganic Chemistry of the Human Host-Defense Protein Calprotectin |
| title_short | Bioinorganic Chemistry of the Human Host-Defense Protein Calprotectin |
| title_sort | bioinorganic chemistry of the human host defense protein calprotectin |
| topic | Chemistry. |
| url | http://hdl.handle.net/1721.1/98823 |
| work_keys_str_mv | AT brophymeganbrunjes bioinorganicchemistryofthehumanhostdefenseproteincalprotectin |