Glutamate carboxypeptidase II (GCPII) is a transmembrane dimeric metalloprotease harboring two zinc ions indispensable for its enzymatic activity in the active site. In addition, a calcium ion of an unknown function is observed at the dimer interface in the distance of 20 Å from the active site. The present work is aimed at elucidating the structural and functional role of the Ca2+ ion in vitro.
To this end, we designed, expressed and purified a panel of GCPII variants with mutated amino acids coordinating Ca2+ and/or positioned at the homodimer interface. Size-exclusion chromatography reveales that a significant portion of the protein is either aggregated or eluted in fractions corresponding to the monomeric species that have not been described in the literature. Activity measurements showed no or very limited activity of the mutant enzymes. At the same time circular-dichroism spectra didn’t reveal any significant structural changes in comparison with wtGCPII. Based on these findings we assume that Ca2+ is crucial for enzyme dimerization that is in turn required for GCPII enzymatic activity.