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

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 ^{mu t-1} (K -206, -209), P ^{mut -2} (K -206, -207) and P ^{mu t-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 ^{mu t-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-1 in 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.
Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-021-02659-1.
Competing Interests: Conflicts of interestAll authors consent to this submission and declare no conflicts of interest.
(© King Abdulaziz City for Science and Technology 2021.)