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Site-directed mutagenesis in the P-domain of calreticulin transacylase identifies Lys-207 as the active site residue. 2021

Rini Joshi, and Prabhjot Singh, and Naresh K Sharma, and Prija Ponnan, and Daman Saluja, and Jasvinder K Gambhir, and Diwan S Rawat, and Virinder S Parmar, and Bilkere S Dwarakanath, and Ashok K Prasad, and Hanumantharao G Raj
Department of Biochemistry, V.P. Chest Institute, Delhi, India.

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 (K -206, -209), P (K -206, -207) and P (K -207, -209) and analyzed their immunoreactivity and acetylation potential. The clones of wild-type P-domain (P ) and three mutated P-domain (P , P and P ) were expressed in pTrcHis C vector and the recombinant P , P , P and P 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 and P while P and P did not show any activity. The immune-reactivity to anti-lysine antibody was also retained only by the P in which the Lys-207 was intact. Retention of the TAase activity and immunoreactivity of P 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. BACKGROUND The online version contains supplementary material available at 10.1007/s13205-021-02659-1.

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