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 His(3)Asp and His(4) 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(3)Asp and His(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(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 His(3)Asp and His(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.
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Megan Brunjes
MB
Brophy
Joshua A
JA
Hayden
Elizabeth M
EM
Nolan
D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology; D002118:Calcium / Q000737:chemistry*; D006090:Gram-Negative Bacteria / Q000187:drug effects*; D006094:Gram-Positive Bacteria / Q000187:drug effects*; D006801:Humans; D007477:Ions / Q000737:chemistry; D039841:Leukocyte L1 Antigen Complex / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D013816:Thermodynamics; D015032:Zinc / Q000737:chemistry* / Q000494:pharmacology*
D000900:Anti-Bacterial Agents; D007477:Ions; D039841:Leukocyte L1 Antigen Complex; D015032:Zinc; D002118:Calcium