Crystal Structure of CRN-4: Implications for Domain Function in
Apoptotic DNA Degradation
Yu-Yuan Hsiao,1,2 Akihisa Nakagawa,3 Zhonghao Shi,2 Shohei Mitani,4
Ding Xue,3 and Hanna S. Yuan2,5*
Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan, Republic of China1; Institute of Molecular
Biology, Academia Sinica, Taipei, Taiwan, Republic of China2; Department of Molecular, Cellular and Developmental Biology,
University of Colorado, Boulder, Colorado3; Department of Physiology, Tokyo Women’s Medical University, School of
Medicine, and CREST, JST, Tokyo 162-8666, Japan4; and Graduate Institute of Biochemistry and Molecular Biology,
National Taiwan University, Taipei, Taiwan, Republic of China5
Received 26 June 2008/Returned for modification 4 September 2008/Accepted 23 October 2008

Cell death related nuclease 4 (CRN-4) is one of the apoptotic nucleases involved in DNA degradation in Caenorhabditis elegans. To understand how CRN-4 is involved in apoptotic DNA fragmentation, we analyzed CRN-4's biochemical properties, in vivo cell functions, and the crystal structures of CRN-4 in apo-form, Mn2+ -bound active form, and Er3+bound inactive form. CRN-4 is a dimeric nuclease with the optimal enzyme activity in cleaving double-stranded DNA in apoptotic salt conditions. Both mutational studies and the structures of the Mn2+ -bound CRN-4 revealed the geometry of the functional nuclease active site in the N-terminal DEDDh domain. The C-terminal domain, termed the Zn-domain, contains basic surface residues ideal for nucleic acid recognition and is involved in DNA binding, as confirmed by deletion assays. Cell death analysis in C. elegans further demonstrated that both the nuclease active site and the Zn-domain are required for crn-4's function in apoptosis. Combining all of the data, we suggest a structural model where chromosomal DNA is bound at the Zn -domain and cleaved at the DEDDh nuclease domain in CRN-4 when the cell is undergoing apoptosis.

mcb2009
Electrostatic surface potential and DNA-binding sites of CRN-4. (A) The front and top views of the electrostatic surface potential of CRN-4 show that the surface of Zn-domain is highly basic. The color scale was set from 5 kT/e (red) to 5 kT/e (blue), as calculated by Pymol(9). (B) The top view of the ribbon model of CRN-4 shows the five arginine residues—Arg216, Arg248, Arg253, Arg261, and Arg264—in the Zn-domain (in salmon) exposing on the surface. (C) CRN-4 digested a double-stranded 49-mer DNA more efficiently than the C-terminal Zn-domain deletion mutant. The DNA was digested at 23°C for 180 min because the deletion mutant was less stable. (D) The Zn-domain deletion mutant bound to double-stranded 31-mer DNA with an 8-fold reduced affinity compared to the wild-type CRN-4, as assayed by gel shift experiments. The three arrowheads mark the protein-DNA complexes.