Crystal Structure Of Restriction Endonuclease Kpn2i Of Ccgg-Family

Background: Restriction endonucleases belong to prokaryotic restriction-modification systems, that protect host cells from invading DNA. Type II restriction endonucleases recognize short 4-8 bp sequences in the target DNA and cut both DNA strands producing double strand breaks. Type II restriction endonuclease Kpn2I cleaves 5'-T/CCGGA DNA sequence ("/" marks the cleavage position). Analysis of protein sequences suggested that Kpn2I belongs to the CCGG-family, which contains ten enzymes that recognize diverse nucleotides outside the conserved 5'-CCGG core and share similar motifs for the 5'-CCGG recognition and cleavage.Methods: We solved a crystal structure of Kpn2I in a DNA-free form at 2.88 A resolution. From the crystal structure we predicted active center and DNA recognition residues and tested them by mutational analysis. We estimated oligomeric state of Kpn2I by SEC-MALS and performed plasmid DNA cleavage assay to elucidate DNA cleavage mechanism.Results: Structure comparison confirmed that Kpn2I shares a conserved active site and structural determinants for the 5'-CCGG tetranucleotide recognition with other restriction endonucleases of the CCGG-family. Guided by structural similarity between Kpn2I and the CCGG-family restriction endonucleases PfoI and AgeI, Kpn2I residues involved in the outer base pair recognition were proposed.Conclusions: Kpn2I is an orthodox Type IIP restriction endonuclease, which acts as a dimer. Kpn2I shares structural similarity to the CCGG-family restriction endonucleases PfoI, AgeI and PspGI.General significance: The Kpn2I structure concluded the studies of the CCGG-family, covering detailed structural and biochemical characterization of eleven restriction enzymes and their complexes with DNA.