Probing the roles of active site residues in the 3'-5' exonuclease of the Werner syndrome protein.
Werner syndrome is a premature aging disease caused by mutations in the WS gene and a deficiency in the function of Werner protein (WRN). The lack of WRN results in a cellular phenotype of genomic instability. WRN belongs to the RecQ DNA helicase family, but unlike other RecQ family members it possesses a functional exonuclease domain. We determined the crystal structure of mWRNexo (residues 31-238) bound to Zn(2+) and the sulfate ion. Compared with the structure of human WRNexo (hWRNexo), notable conformational changes were observed in several active site residues in an H5-H6 loop and in helices H6 and H7 of mWRNexo, presumably because of the presence of sulfate, which mimics the phosphate of substrate DNA. In particular, the side chains of Lys(185) and Tyr(206) were reoriented toward the Zn(2+) ion, whereas the side chain of Arg(190) pointed away from the active site center. Mutational analysis of these conserved residues abolished WRN exonuclease activity, suggesting that these residues play a critical role in the WRNexo activity. Based on substrate modeling and mutational analyses, we propose a mechanism by which WRNexo becomes activated upon substrate DNA binding. We also describe the low resolution trimeric structure of mouse WRNexoL (mWRNexoL, residues 31-330), as elucidated by small angle x-ray scattering (SAXS) analyses.
Choi
Jung Min
JM
National Creative Research Center for Structural Biology and Department of Life Science, Pohang University of Science and Technology, Hyo-ja dong, San31, Pohang, KyungBook 790-784, South Korea.
Kang
Sung Yun
SY
National Creative Research Center for Structural Biology and Department of Life Science, Pohang University of Science and Technology, Hyo-ja dong, San31, Pohang, KyungBook 790-784, South Korea.
Bae
Won Jin
WJ
National Creative Research Center for Structural Biology and Department of Life Science, Pohang University of Science and Technology, Hyo-ja dong, San31, Pohang, KyungBook 790-784, South Korea.
Jin
Kyeong Sik
KS
Department of Chemistry, National Research Laboratory for Polymer Synthesis & Physics, Pohang Accelerator Laboratory, Center for Integrated Molecular Systems, Polymer Research Institute, and BK School of Molecular Science, Pohang University of Science and Technology, Pohang, KyungBook 790-784, South Korea.
Ree
Moonhor
M
Department of Chemistry, National Research Laboratory for Polymer Synthesis & Physics, Pohang Accelerator Laboratory, Center for Integrated Molecular Systems, Polymer Research Institute, and BK School of Molecular Science, Pohang University of Science and Technology, Pohang, KyungBook 790-784, South Korea.
Cho
Yunje
Y
National Creative Research Center for Structural Biology and Department of Life Science, Pohang University of Science and Technology, Hyo-ja dong, San31, Pohang, KyungBook 790-784, South Korea. Electronic address: yunje@postech.ac.kr.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics / Q000502:physiology*; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D018360:Crystallography, X-Ray; D004247:DNA / Q000378:metabolism; D005092:Exonucleases / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D011897:Random Allocation; D053484:RecQ Helicases / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014898:Werner Syndrome / Q000201:enzymology*; D000071657:Werner Syndrome Helicase
D000596:Amino Acids; D004247:DNA; D005092:Exonucleases; D053484:RecQ Helicases; D000071657:Werner Syndrome Helicase; C499253:Wrn protein, mouse