Calcium Ion Gradients Modulate the Zinc Affinity and Antibacterial Activity of Human Calprotectin
Author(s)Brophy, Megan Brunjes; Hayden, Joshua A.; Nolan, Elizabeth M.
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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 His[subscript 3]Asp and His[subscript 4] 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[subscript d1] = 133 ± 58 pM and K[subscript d2] = 185 ± 219 nM for CP in the absence of Ca(II). These values decrease to K[subscript d1] ≤ 10 pM and K[subscript d2] ≤ 240 pM in the presence of excess Ca(II). The K[subscript d1] and K[subscript d2] values are assigned to the His[subscript 3]Asp and His[subscript 4] 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.
DepartmentMassachusetts Institute of Technology. Department of Chemistry
Journal of the American Chemical Society
American Chemical Society
Brophy, Megan Brunjes, Joshua A. Hayden, and Elizabeth M. Nolan. “Calcium Ion Gradients Modulate the Zinc Affinity and Antibacterial Activity of Human Calprotectin.” Journal of the American Chemical Society 134, no. 43 (October 31, 2012): 18089-18100.
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