Structural integrity of the Beta Beta Alpha-Metal finger motif is required for DNA binding and stable protein–DNA complex formation in R.KpnI

Vasu, Kommireddy and Saravanan, Matheshwaran and Bujnicki, Janusz M and Nagaraja, Valakunja (2008) Structural integrity of the Beta Beta Alpha-Metal finger motif is required for DNA binding and stable protein–DNA complex formation in R.KpnI. In: Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics, 1784 (2). pp. 269-275.

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Abstract

Restriction endonuclease (REase) R.KpnI from Klebsiella pneumoniae is a homodimeric enzyme, which recognizes palindromic sequence GGTAC|C and cleaves generating 4 base 3′ end overhangs. R.KpnI belongs to the HNH superfamily of nucleases, which are characterized by the presence of the ββα-Me finger motif. Structurally, this motif consists of a twisted β-hairpin followed by an α-helix, and serves as a scaffold for side chains of residues involved in co-ordination of a divalent metal ion that is required for catalysis. Homology modeling studies of R.KpnI suggested a crossover structure for the α-helix, which could possibly form dimeric interface and/or structural scaffold for the active site. We have evaluated the role of the residues present in this α-helix in intersubunit interactions and/or stabilization of the active site. We show here that mutations of residues in the α-helix lead to a loss of the enzyme activity, but not dimerization ability. Intrinsic fluorescence and circular dichroism studies revealed that the loss of function phenotype was due to the structural perturbation of the ββα-Me finger motif. The results of mutational analysis suggest that the α-helix of the ββα-Me finger of R.KpnI plays an important role for the stability of the protein–DNA complex.

Item Type:	Journal Article
Publication:	Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics
Publisher:	Elsevier
Additional Information:	Copyright of this article belongs to Elsevier.
Keywords:	Restriction endonuclease; R.KpnI; ββα-Me finger; HNH Motiff; Site directed mutagenesis; Protein DNA complex.
Department/Centre:	Division of Biological Sciences > Microbiology & Cell Biology
Date Deposited:	08 Mar 2008
Last Modified:	19 Sep 2010 04:43
URI:	http://eprints.iisc.ac.in/id/eprint/13341

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