Abstract
Zinc and caspase-6 have independently been implicated in several neurodegenerative disorders. Depletion of zinc intracellularly leads to apoptosis by an unknown mechanism. Zinc inhibits cysteine proteases, including the apoptotic caspases, leading to the hypothesis that zinc-mediated inhibition of caspase-6 might contribute to its regulation in a neurodegenerative context. Using inductively coupled plasma optical emission spectroscopy, we observed that caspase-6 binds one zinc per monomer, under the same conditions where the zinc leads to complete loss of enzymatic activity. To understand the molecular details of zinc binding and inhibition, we performed an anomalous diffraction experiment above the zinc edge. The anomalous difference maps showed strong 5σ peaks, indicating the presence of one zinc/monomer bound at an exosite distal from the active site. Zinc was not observed bound to the active site. The zinc in the exosite was liganded by Lys-36, Glu-244, and His-287 with a water molecule serving as the fourth ligand, forming a distorted tetrahedral ligation sphere. This exosite appears to be unique to caspase-6, as the residues involved in zinc binding were not conserved across the caspase family. Our data suggest that binding of zinc at the exosite is the primary route of inhibition, potentially locking caspase-6 into the inactive helical conformation.
Highlights
Caspase-6 is a critical factor in neurodegeneration, which is regulated by zinc binding
The anomalous difference maps showed strong 5 peaks, indicating the presence of one zinc/monomer bound at an exosite distal from the active site
The zinc in the exosite was liganded by Lys-36, Glu-244, and His-287 with a water molecule serving as the fourth ligand, forming a distorted tetrahedral ligation sphere. This exosite appears to be unique to caspase-6, as the residues involved in zinc binding were not conserved across the caspase family
Summary
Caspase-6 is a critical factor in neurodegeneration, which is regulated by zinc binding. One zinc binds at the active site, and the second zinc binds at an exosite composed of residues Cys230, His-224, and Cys-272 [36] Together, these studies suggest that when the zinc concentration in the cell is lowered, zincmediated inhibition of caspases is relieved, and an increase in caspase activation leads to apoptotic cell death. In Alzheimer disease patients, zinc concentrations in post-mortem brain tissue correlated with the severity of the case, whereas the concentrations of other metals did not differ with severity or relative to control tissues [40] These tissues with zinc accumulation are the same tissues in which caspase-6 activation precedes and leads to the formation of neurofibrillary tangles but does not lead to cell death [7, 42,43,44]. We explore the ability of divalent metals to inactivate caspase-6 and uncover the structural mechanism by which caspase-6 metal binding results in inhibition
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