Structural integrity of the beta beta alpha-Metal finger motif is required for DNA binding and stable protein-DNA complex formation in R.KpnI.
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 beta beta alpha-Me finger motif. Structurally, this motif consists of a twisted beta-hairpin followed by an alpha-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 alpha-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 alpha-helix in intersubunit interactions and/or stabilization of the active site. We show here that mutations of residues in the alpha-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 beta beta alpha-Me finger motif. The results of mutational analysis suggest that the alpha-helix of the beta beta alpha-Me finger of R.KpnI plays an important role for the stability of the protein-DNA complex.
Vasu
Kommireddy
K
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India.
Saravanan
Matheshwaran
M
Bujnicki
Janusz M
JM
Nagaraja
Valakunja
V
D020816:Amino Acid Motifs; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019281:Dimerization; D008345:Manganese / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D011489:Protein Denaturation; D020836:Protein Structure, Quaternary; D013696:Temperature
D008345:Manganese; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; C029312:GGTACC-specific type II deoxyribonucleases