Novel protein fold discovered in the PabI family of restriction enzymes.
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 beta-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 Mg(2+). 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.
Miyazono
Ken-ichi
K
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, Japan.
Watanabe
Miki
M
Kosinski
Jan
J
Ishikawa
Ken
K
Kamo
Masayuki
M
Sawasaki
Tatsuya
T
Nagata
Koji
K
Bujnicki
Janusz M
JM
Endo
Yaeta
Y
Tanokura
Masaru
M
Kobayashi
Ichizo
I
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D014176:Protein Biosynthesis; D017510:Protein Folding; D017433:Protein Structure, Secondary; D043903:Pyrococcus abyssi / Q000201:enzymology; D016415:Sequence Alignment
D019843:Archaeal Proteins; D004268:DNA-Binding Proteins; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific