Contributions of the S100A9 C-Terminal Tail to High-Affinity Mn(II) Chelation by the Host-Defense Protein Human Calprotectin

Abstract

Human calprotectin (CP) is an antimicrobial protein that coordinates Mn(II) with high affinity in a Ca(II)-dependent manner at an unusual histidine-rich site (site 2) formed at the S100A8/S100A9 dimer interface. We present a 16-member CP mutant family where mutations in the S100A9 C-terminal tail (residues 96–114) are employed to evaluate the contributions of this region, which houses three histidines and four acidic residues, to Mn(II) coordination at site 2. The results from analytical size-exclusion chromatography, Mn(II) competition titrations, and electron paramagnetic resonance spectroscopy establish that the C-terminal tail is essential for high-affinity Mn(II) coordination by native CP in solution. The studies indicate that His103 and His105 (HXH motif) of the tail complete the Mn(II) coordination sphere in solution, affording an unprecedented biological His6 site. These solution studies are in agreement with a Mn(II)-CP crystal structure reported recently (PNAS 2013, 110, 3841). Remarkably high-affinity Mn(II) binding is retained when either H103 or H105 are mutated to Ala, when the HXH motif is shifted from positions 103–105 to 104–106, and when the human tail is substituted by the C-terminal tail of murine S100A9. Nevertheless, antibacterial activity assays employing human CP mutants reveal that the native disposition of His residues is important for conferring growth inhibition against Escherichia coli and Staphylococcus aureus. Within the S100 family, the S100A8/S100A9 heterooligomer is essential for providing high-affinity Mn(II) binding; the S100A7ox, S100A9(C3S), S100A12, and S100B homodimers do not exhibit such Mn(II)-binding capacity.

Nickel Sequestration by the Host-Defense Protein Human Calprotectin

“…The bond distances and angles indicate distorted octahedral geometry (Tables S3–S4). These results are consistent with previous studies of Mn(II), 41-43, 45 Fe(II), 31, 44 and Zn(II) 39, 46 bound at this site. The two Mn(II)- and Ca(II)-bound CP structures comprise the Mn(II)-His 6 site, 41, 42 and structural alignments of the metal-bound His 6 motifs indicate little difference between Mn(II) and Ni(II) coordination at this site (Figure S2).…”

“…Site 1 has high affinity for Zn(II) 39 and has been observed to chelate Mn(II) 42,43 and Fe(II), 44 albeit with relatively low affinity. 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.…”

Zinc and Manganese Chelation by Neutrophil S100A8/A9 (Calprotectin) Limits Extracellular Aspergillus fumigatus Hyphal Growth and Corneal Infection

“…These data indicate that Zn chelation is essential for anti-fungal activity, whereas Mn has only a minor contributing role. This is in contrast to bacteria, in which Mn binding has a significant role in the anti-microbial activity of CP (12, 43). CP-mediated Mn chelation has been shown to inhibit superoxide dismutase activity and reduce the virulence of Staphylococcus aureus (44).…”

Biophysical Examination of the Calcium-Modulated Nickel-Binding Properties of Human Calprotectin Reveals Conformational Change in the EF-Hand Domains and His3Asp Site

“…Similar behavior was observed for Mn(II) binding to the tail variants, where the presence of the His residues in the S100A9 C-terminal tail are important for complete transformation to the heterotetrameric species. 11 …”