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Manganese Binding Properties of Human Calprotectin under Conditions of High and Low Calcium: X-ray Crystallographic and Advanced Electron Paramagnetic Resonance Spectroscopic Analysis
- Gagnon, Derek
M., Brophy, Megan Brunjes, Bowman, Sarah E. J., Stich, Troy A., Drennan, Catherine
L., Britt, R. David, Nolan, Elizabeth M.
- Journal of the American Chemical Society 2015 v.137 no.8 pp. 3004-3016
- X-ray diffraction, antimicrobial proteins, binding capacity, binding sites, calcium, electron paramagnetic resonance spectroscopy, histidine, humans, ions, ligands, manganese, nitrogen, sodium, spectral analysis, stable isotopes
- The antimicrobial protein calprotectin (CP), a hetero-oligomer of the S100 family members S100A8 and S100A9, is the only identified mammalian Mn(II)-sequestering protein. Human CP uses Ca(II) ions to tune its Mn(II) affinity at a biologically unprecedented hexahistidine site that forms at the S100A8/S100A9 interface, and the molecular basis for this phenomenon requires elucidation. Herein, we investigate the remarkable Mn(II) coordination chemistry of human CP using X-ray crystallography as well as continuous-wave (CW) and pulse electron paramagnetic resonance (EPR) spectroscopies. An X-ray crystallographic structure of Mn(II)-CP containing one Mn(II), two Ca(II), and two Na(I) ions per CP heterodimer is reported. The CW EPR spectrum of Ca(II)- and Mn(II)-bound CP prepared with a 10:0.9:1 Ca(II):Mn(II):CP ratio is characterized by an unusually low zero-field splitting of 485 MHz (E/D = 0.30) for the S = 5/2 Mn(II) ion, consistent with the high symmetry of the His₆ binding site observed crystallographically. Results from electron spin–echo envelope modulation and electron–nuclear double resonance experiments reveal that the six Mn(II)-coordinating histidine residues of Ca(II)- and Mn(II)-bound CP are spectroscopically equivalent. The observed ¹⁵N (I = 1/2) hyperfine couplings (A) arise from two distinct classes of nitrogen atoms: the coordinating ε-nitrogen of the imidazole ring of each histidine ligand (A = [3.45, 3.71, 5.91] MHz) and the distal δ-nitrogen (A = [0.11, 0.18, 0.42] MHz). In the absence of Ca(II), the binding affinity of CP for Mn(II) drops by two to three orders of magnitude and coincides with Mn(II) binding at the His₆ site as well as other sites. This study demonstrates the role of Ca(II) in enabling high-affinity and specific binding of Mn(II) to the His₆ site of human calprotectin.