10.1021/ja307974e.s001
Megan
Brunjes Brophy
Megan
Brunjes
Brophy
Joshua A. Hayden
Joshua A.
Hayden
Elizabeth M. Nolan
Elizabeth M.
Nolan
Calcium Ion Gradients
Modulate the Zinc Affinity and
Antibacterial Activity of Human Calprotectin
American Chemical Society
2016
Kd 2 values
ZP
Zn
pM
CP
Zinc competition titrations
Kd 1
3Asp
EPR spectroscopic signatures
4 sites
Calcium Ion Gradients Modulate
extracellular space
2016-02-20 07:45:23
Journal contribution
https://acs.figshare.com/articles/journal_contribution/Calcium_Ion_Gradients_Modulate_the_Zinc_Affinity_and_Antibacterial_Activity_of_Human_Calprotectin/2474128
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<sub>3</sub>Asp and His<sub>4</sub> 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<sub>3</sub>Asp and His<sub>4</sub> 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 <i>K</i><sub>d1</sub> = 133 ± 58 pM and <i>K</i><sub>d2</sub> = 185 ± 219 nM for CP in the absence of Ca(II). These values
decrease to <i>K</i><sub>d1</sub> ≤ 10 pM and <i>K</i><sub>d2</sub> ≤ 240 pM in the presence of excess
Ca(II). The <i>K</i><sub>d1</sub> and <i>K</i><sub>d2</sub> values are assigned to the His<sub>3</sub>Asp and His<sub>4</sub> sites, respectively. <i>In vitro</i> 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.