Structure-based functional identification of Helicobacter pylori HP0268 as a nuclease with both DNA nicking and RNase activities.
HP0268 is a conserved, uncharacterized protein from Helicobacter pylori. Here, we determined the solution structure of HP0268 using three-dimensional nuclear magnetic resonance (NMR) spectroscopy, revealing that this protein is structurally most similar to a small MutS-related (SMR) domain that exhibits nicking endonuclease activity. We also demonstrated for the first time that HP0268 is a nicking endonuclease and a purine-specific ribonuclease through gel electrophoresis and fluorescence spectroscopy. The nuclease activities for DNA and RNA were maximally increased by Mn(2+) and Mg(2+) ions, respectively, and decreased by Cu(2+) ions. Using NMR chemical shift perturbations, the metal and nucleotide binding sites of HP0268 were determined to be spatially divided but close to each other. The lysine residues (Lys7, Lys11 and Lys43) are clustered and form the nucleotide binding site. Moreover, site-directed mutagenesis was used to define the catalytic active site of HP0268, revealing that this site contains two acidic residues, Asp50 and Glu54, in the metal binding site. The nucleotide binding and active sites are not conserved in the structural homologues of HP0268. This study will contribute to improving our understanding of the structure and functionality of a wide spectrum of nucleases.
Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742, Korea.
Ki-Young
KY
Lee
Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742, Korea.
Kyu-Yeon
KY
Lee
Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742, Korea.
Ji-Hun
JH
Kim
Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742, Korea.
In-Gyun
IG
Lee
Department of Biotechnology, College of Biomedical and Health Science, Konkuk University, Chungju, Chungbuk 380-701, Korea.
Sung-Hee
SH
Lee
Department of Biotechnology, College of Biomedical and Health Science, Konkuk University, Chungju, Chungbuk 380-701, Korea.
Dae-Won
DW
Sim
Department of Biotechnology, College of Biomedical and Health Science, Konkuk University, Chungju, Chungbuk 380-701, Korea.
Hyung-Sik
HS
Won
Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742, Korea lbj@nmr.snu.ac.kr.
Bong-Jin
BJ
Lee
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D020134:Catalytic Domain; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000378:metabolism; D016480:Helicobacter pylori / Q000201:enzymology*; D008670:Metals / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism; D011687:Purines / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry* / Q000378:metabolism
D001426:Bacterial Proteins; D008670:Metals; D009711:Nucleotides; D011687:Purines; D004706:Endodeoxyribonucleases; D012260:Ribonucleases