Novel protein fold discovered in the PabI family of restriction enzymes

• Published in: Nucleic acids research Vol. 35; no. 6; pp. 1908 - 1918
• Main Authors: Miyazono, Ken-ichi, Watanabe, Miki, Kosinski, Jan, Ishikawa, Ken, Kamo, Masayuki, Sawasaki, Tatsuya, Nagata, Koji, Bujnicki, Janusz M, Endo, Yaeta, Tanokura, Masaru, Kobayashi, Ichizo
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01-03-2007; Oxford Publishing Limited (England)
• Subjects: Amino Acid Sequence; Archaeal Proteins - chemistry; Archaeal Proteins - genetics; Archaeal Proteins - metabolism; Binding Sites; Crystallography, X-Ray; Deoxyribonucleases, Type II Site-Specific - chemistry; Deoxyribonucleases, Type II Site-Specific - genetics; Deoxyribonucleases, Type II Site-Specific - metabolism; DNA - chemistry; DNA Mutational Analysis; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Models, Molecular; Molecular Sequence Data; Protein Binding; Protein Biosynthesis; Protein Folding; Protein Structure, Secondary; Pyrococcus abyssi; Pyrococcus abyssi - enzymology; Sequence Alignment; Structural Biology
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkm091

Although structures of many DNA-binding proteins have been solved, they fall into a limited number of folds. Here, we describe an approach that led to the finding of a novel DNA-binding fold. Based on the behavior of Type II restriction-modification gene complexes as mobile elements, our earlier work identified a restriction enzyme, R.PabI, and its cognate modification enzyme in Pyrococcus abyssi through comparison of closely related genomes. While the modification methyltransferase was easily recognized, R.PabI was predicted to have a novel 3D structure. We expressed cytotoxic R.PabI in a wheat-germ-based cell-free translation system and determined its crystal structure. R.PabI turned out to adopt a novel protein fold. Homodimeric R.PabI has a curved anti-parallel β-sheet that forms a 'half pipe'. Mutational and in silico DNA-binding analyses have assigned it as the double-strand DNA-binding site. Unlike most restriction enzymes analyzed, R.PabI is able to cleave DNA in the absence of Mg²⁺. These results demonstrate the value of genome comparison and the wheat-germ-based system in finding a novel DNA-binding motif in mobile DNases and, in general, a novel protein fold in horizontally transferred genes.