Site-directed mutagenesis in the P-domain of calreticulin transacylase identifies Lys-207 as the active site residue

Abstract

In silico-docking studies from previous work have suggested that Lys-206 and lys-207 of calreticulin (CR) play a pivotal key role in its well-established transacetylation activity. To experimentally validate this prediction, we introduced three mutations at lysine residues of P-domain of CR: K → A, P^mut−1 (K -206, -209), P^mut−2 (K -206, -207) and P^mut−3 (K -207, -209) and analyzed their immunoreactivity and acetylation potential. The clones of wild-type P-domain (P^wt) and three mutated P-domain (P^mut−1, P^mut−2 and P^mut−3) were expressed in pTrcHis C vector and the recombinant P^wt, P^mut−1, P^mut−2 and P^mut−3 proteins were purified by Ni–NTA affinity chromatography. Screening of the transacylase activity (TAase) by the Glutathione S Transferase (GST) assay revealed that the TAase activity was associated with the P^wt and P^mut−1 while P^mut−2 and P^mut−3 did not show any activity. The immune-reactivity to anti-lysine antibody was also retained only by the P^mut−1in which the Lys-207 was intact. Retention of the TAase activity and immunoreactivity of P^mut−1 with mutations introduced at Lys-206, Lys-209, while its loss with a mutation at Lys-207 residue indicated that lysine-207 of P-domain constitutes the active site residue controlling TAase activity.