Calcium Ion Gradients Modulate the Zinc Affinity and Antibacterial Activity of Human Calprotectin

Calprotectin (CP) is an antimicrobial protein produced and released by neutrophils that inhibits the growth of pathogenic microorganisms by sequestering essential metal nutrients in the extracellular space. In this work, spectroscopic and thermodynamic metal-binding studies are presented to delineate the zinc-binding properties of CP. Unique optical absorption and EPR spectroscopic signatures for the interfacial His₃Asp and His₄ sites of human calprotectin are identified by using Co­(II) as a spectroscopic probe. Zinc competition titrations employing chromophoric Zn­(II) indicators provide a 2:1 Zn­(II):CP stoichiometry, confirm that the His₃Asp and His₄ sites of CP coordinate Zn­(II), and reveal that the Zn­(II) affinity of both sites is calcium-dependent. The calcium-insensitive Zn­(II) competitor ZP4 affords dissociation constants of K_d1 = 133 ± 58 pM and K_d2 = 185 ± 219 nM for CP in the absence of Ca­(II). These values decrease to K_d1 ≤ 10 pM and K_d2 ≤ 240 pM in the presence of excess Ca­(II). The K_d1 and K_d2 values are assigned to the His₃Asp and His₄ sites, respectively. In vitro antibacterial activity assays indicate that the metal-binding sites and Ca­(II)-replete conditions are required for CP to inhibit the growth of both Gram-negative and -positive bacteria. Taken together, these data provide a working model whereby calprotectin responds to physiological Ca­(II) gradients to become a potent Zn­(II) chelator in the extracellular space.