High-Affinity Manganese Coordination by Human Calprotectin Is Calcium-Dependent and Requires the Histidine-Rich Site Formed at the Dimer Interface

Abstract

Calprotectin (CP) is a transition metal-chelating antimicrobial protein of the calcium-binding S100 family that is produced and released by neutrophils. It inhibits the growth of various pathogenic microorganisms by sequestering the transition metal ions manganese and zinc. In this work, we investigate the manganese-binding properties of calprotectin. We demonstrate that the unusual His4 motif (site 2) formed at the S100A8/S100A9 dimer interface is the site of high-affinity Mn(II) coordination. We identify a low-temperature Mn(II) spectroscopic signal for this site consistent with an octahedral Mn(II) coordination sphere with simulated zero-field splitting parameters D = 270 MHz and E/D = 0.33 (E = 81 MHz). This analysis, combined with studies of mutant proteins, suggests that four histidine residues (H17 and H27 of S100A8; H91 and H95 of S100A9) coordinate Mn(II) in addition to two as-yet unidentified ligands. The His3Asp motif (site 1), which is also formed at the S100A8/S100A9 dimer interface, does not provide a high-affinity Mn(II) binding site. Calcium binding to the EF-hand domains of CP increases the Mn(II) affinity of the His4 site from the low-micromolar to the mid-nanomolar range. Metal-ion selectivity studies demonstrate that CP prefers to coordinate Zn(II) over Mn(II). Nevertheless, the specificity of Mn(II) for the His4 site provides CP with the propensity to form mixed Zn:Mn:CP complexes where one Zn(II) ion occupies site 1 and one Mn(II) ion occupies site 2. These studies support the notion that CP responds to physiological calcium ion gradients to become a high-affinity transition metal ion chelator in the extracellular space where it inhibits microbial growth.

Production and Release of Antimicrobial and Immune Defense Proteins by Mammary Epithelial Cells following Streptococcus uberis Infection of Sheep

“…Alone or in association as calprotectin (54), S100A8 and S100A9 exert strong proinflammatory and chemotactic activities (60) by promoting leukocyte recruitment (61,62). In addition, the calprotectin heterodimer exerts direct antimicrobial functions by sequestering zinc and manganese (59,(63)(64)(65)(66). The concerted increase in abundance of the two monomers S100A9 and S100A8 observed in MFGs upon infection with S. uberis (Table 3 and Fig.…”

Nutritional Immunity: S100 Proteins at the Host-Pathogen Interface

“…An x-ray crystal structure, as well as spectroscopic and mutagenesis studies, revealed that the Zn 2ϩ binding in Site I involves His-17 and His-27 from S100A8 and His-91 and His-95 from S100A9, whereas chelation of Mn 2ϩ involves the same four residues along with two additional histidine residues from the C-terminal tail of the S100A9 subunit (Fig. 1A), which enable binding in the requisite octahedral geometry (12,21,22 (Fig. 1A) (12,20,21), but lacks appropriately positioned additional ligands to enable high affinity binding of Mn 2ϩ .…”

Nickel Sequestration by the Host-Defense Protein Human Calprotectin

“…Site 2 comprises a unique hexahistidine metal-binding motif that coordinates Mn(II) 32,41-43, 45 Fe(II), 31,44 and Zn(II) 39,46 with high affinity. Our metal-substitution studies demonstrated that site 2 exhibits thermodynamic preference for these divalent cations (i.e., K d,Zn < K d,Fe < K d,Mn ) 31,43 consistent with the Irving-Williams series. 47 Moreover, our prior work revealed that CP treatment of bacterial growth medium also reduces the concentrations of nickel.…”