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| Subject Categories: Cell Cycle | Structural Biology |
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| The EMBO Journal
(2008) 27, 290–300, doi:10.1038/sj.emboj.7601942 Published online 29 November 2007 |
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| Crystal structure of human XLF/Cernunnos reveals unexpected differences from XRCC4 with implications for NHEJ EMBO Open |
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| Yi Li1, Dimitri Y Chirgadze1, Victor M Bolanos-Garcia1, Bancinyane L Sibanda1, Owen R Davies1, Peter Ahnesorg2, 3, Stephen P Jackson2, 3 and Tom L Blundell1 |
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1 Department of Biochemistry, University of Cambridge, Cambridge, UK 2 The Wellcome Trust and Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, UK 3 Department of Zoology, University of Cambridge, Cambridge, UK To whom correspondence should be addressed Yi Li, Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, UK. Tel.: +44 1223 333628 629; Fax: +44 1223 333345; E-mail: jessica@cryst.bioc.cam.ac.uk Tom L Blundell, Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, UK. Tel.: +44 1223 333628 629; Fax: +44 1223 333345; E-mail: tom@cryst.bioc.cam.ac.uk Received 27 July 2007; Accepted 2 November 2007; Published online 29 November 2007. |
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| Abstract |
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| The recently characterised 299-residue human XLF/Cernunnos protein plays a crucial role in DNA repair by non-homologous end joining (NHEJ) and interacts with the XRCC4–DNA Ligase IV complex. Here, we report the crystal structure of the XLF (1–233) homodimer at 2.3 Å resolution, confirming the predicted structural similarity to XRCC4. The XLF coiled-coil, however, is shorter than that of XRCC4 and undergoes an unexpected reverse in direction giving rise to a short distorted four helical bundle and a C-terminal helical structure wedged between the coiled-coil and head domain. The existence of a dimer as the major species is confirmed by size-exclusion chromatography, analytical ultracentrifugation, small-angle X-ray scattering and other biophysical methods. We show that the XLF structure is not easily compatible with a proposed XRCC4:XLF heterodimer. However, we demonstrate interactions between dimers of XLF and XRCC4 by surface plasmon resonance and analyse these in terms of surface properties, amino-acid conservation and mutations in immunodeficient patients. Our data are most consistent with head-to-head interactions in a 2:2:1 XRCC4:XLF:Ligase IV complex. |
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| Keywords: coiled-coil, homodimer, non-homologous end-joining (NHEJ), structure, XRCC4 |
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