Structural Insights into Apoptotic DNA Degradation by CED-3 Protease Suppressor-6 (CPS-6) from Caenorhabditis elegans

• Published in: The Journal of biological chemistry 2012-03, Vol.287 (10), p.7110-7120
• Main Authors: Lin, Jason L.J., Nakagawa, Akihisa, Lin, Chia Liang, Hsiao, Yu-Yuan, Yang, Wei-Zen, Wang, Yi-Ting, Doudeva, Lyudmila G., Skeen-Gaar, Riley Robert, Xue, Ding, Yuan, Hanna S.
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; Amino Acid Substitution; Animals; Apoptosis; Apoptosis - physiology; Caenorhabditis elegans - enzymology; Caenorhabditis elegans - genetics; Caenorhabditis elegans Proteins - chemistry; Caenorhabditis elegans Proteins - genetics; Caenorhabditis elegans Proteins - metabolism; Crystal Structure; Crystallography, X-Ray; DNA Enzymes; DNA, Helminth - chemistry; DNA, Helminth - genetics; DNA, Helminth - metabolism; DNA-Protein Interaction; DNase; Endodeoxyribonucleases - chemistry; Endodeoxyribonucleases - genetics; Endodeoxyribonucleases - metabolism; Hydrolysis; Mitochondrial Proteins - chemistry; Mitochondrial Proteins - genetics; Mitochondrial Proteins - metabolism; Models, Molecular; Mutation, Missense; Nucleic Acid Enzymology; Protein Structure and Folding; Structure-Activity Relationship
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M111.316075

Endonuclease G (EndoG) is a mitochondrial protein that traverses to the nucleus and participates in chromosomal DNA degradation during apoptosis in yeast, worms, flies, and mammals. However, it remains unclear how EndoG binds and digests DNA. Here we show that the Caenorhabditis elegans CPS-6, a homolog of EndoG, is a homodimeric Mg2+-dependent nuclease, binding preferentially to G-tract DNA in the optimum low salt buffer at pH 7. The crystal structure of CPS-6 was determined at 1.8 Å resolution, revealing a mixed αβ topology with the two ββα-metal finger nuclease motifs located distantly at the two sides of the dimeric enzyme. A structural model of the CPS-6-DNA complex suggested a positively charged DNA-binding groove near the Mg2+-bound active site. Mutations of four aromatic and basic residues: Phe122, Arg146, Arg156, and Phe166, in the protein-DNA interface significantly reduced the DNA binding and cleavage activity of CPS-6, confirming that these residues are critical for CPS-6-DNA interactions. In vivo transformation rescue experiments further showed that the reduced DNase activity of CPS-6 mutants was positively correlated with its diminished cell killing activity in C. elegans. Taken together, these biochemical, structural, mutagenesis, and in vivo data reveal a molecular basis of how CPS-6 binds and hydrolyzes DNA to promote cell death. CPS-6 (EndoG) degrades chromosomal DNA during apoptosis. The crystal structure of C. elegans CPS-6 was determined, and the DNA binding and cleavage mechanisms by CPS-6 were revealed. The DNase activity of CPS-6 is positively correlated with its pro-cell death activity. This study improves our general understanding of DNA hydrolysis by ββα-metal finger nucleases and the process of apoptotic DNA fragmentation.