Manganese Binding Properties of Human Calprotectin under Conditions of High and Low Calcium: X‑ray Crystallographic and Advanced Electron Paramagnetic Resonance Spectroscopic Analysis

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 (<i>E/D</i> = 0.30) for the <i>S</i> = 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>I</i> = 1/2) hyperfine couplings (<i>A</i>) arise from two distinct classes of nitrogen atoms: the coordinating ε-nitrogen of the imidazole ring of each histidine ligand (<i>A</i> = [3.45, 3.71, 5.91] MHz) and the distal δ-nitrogen (<i>A</i> = [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