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Structural interpretation of site-directed mutagenesis and specificity of the catalytic subunit of protein kinase CK2 using comparative modelling

Srinivasan, N and Antonelli, Marcelo and Jacob, Germaine and Korn, Iris and Jedlicki, Ana and Dhanaraj, V and Sayed, Muhammed FR and Blundell, Tom L and Allende, Catherine C and Allende, Jorge E (1999) Structural interpretation of site-directed mutagenesis and specificity of the catalytic subunit of protein kinase CK2 using comparative modelling. In: Protein Engineering, 12 (2). pp. 119-127.

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Abstract

The catalytic subunit of protein kinase casein kinase $2 (CK2\alpha)$, which has specificity for both ATP and GTP, shows significant amino acid sequence similarity to the cyclin-dependent kinase 2 (CDK2). We constructed site-directed mutants of $CK2\alpha$ and used a three-dimensional model to investigate the basis for the dual specificity. Introduction of Phe and Gly at positions 50 and 51, in order to restore the pattern of the glycine-rich motif, did not seriously affect the specificity for ATP or GTP. We show that the dual specificity probably originates from the loop situated around the position His115 to Asp120 (HVNNTD). The insertion of a residue in this loop in $CK2\alpha$ subunits, compared with CDK2 and other kinases, might orient the backbone to interact with the base A and G; this insertion is conserved in all known $CK2\alpha$. The mutant$\Delta N118$, the design of which was based on the modelling, showed reduced affinity for GTP as predicted from the model. Other mutants were intended to probe the integrity of the catalytic loop, alter the polarity of a buried residue and explore the importance of the carboxy terminus. Introduction of Arg to replace Asn189, which is mapped on the activation loop, results in a mutant with decreased $k_{cat}$, possibly as a result of disruption of the interaction between this residue and basic residues in the vicinity. Truncation at position 331 eliminates the last 60 residues of the \alpha subunit and this mutant has a reduced catalytic efficiency compared with the wild-type. Catalytic efficiency is restored in the truncation mutant by the replacement of a potentially buried Glu at position 252 by Lys, probably owing to a higher stability resulting from the formation of a salt bridge between Lys252 and Asp208.

Item Type: Journal Article
Additional Information: Copyright of this article belongs to Oxford University Press.
Keywords: Casein kinase 2;Comparative modelling;Mutagenesis;Protein kinase;Protein phosphorylation;Structure prediction
Department/Centre: Division of Biological Sciences > Molecular Biophysics Unit
Date Deposited: 23 Oct 2007
Last Modified: 19 Sep 2010 04:40
URI: http://eprints.iisc.ernet.in/id/eprint/12160

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