Dataset Information

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-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.

SUBMITTER: Joshi R

PROVIDER: S-EPMC7859019 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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

Joshi Rini R Singh Prabhjot P Sharma Naresh K NK Ponnan Prija P Saluja Daman D Saluja Daman D Gambhir Jasvinder K JK Rawat Diwan S DS Parmar Virinder S VS Dwarakanath Bilkere S BS Prasad Ashok K AK Raj Hanumantharao G HG

3 Biotech 20210203 3

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 ...[more]

PMID: 33585151

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Dataset Information

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

Supplementary information

Publications

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

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