Crystal structure of human XLF/Cernunnos reveals unexpected differences from XRCC4 with implications for NHEJ.
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 A 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.
Li
Yi
Y
Department of Biochemistry, University of Cambridge, Cambridge, UK. jessica@cryst.bioc.cam.ac.uk
Chirgadze
Dimitri Y
DY
Bolanos-Garcia
Victor M
VM
Sibanda
Bancinyane L
BL
Davies
Owen R
OR
Ahnesorg
Peter
P
Jackson
Stephen P
SP
Blundell
Tom L
TL
D000595:Amino Acid Sequence; D000818:Animals; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D004260:DNA Repair / Q000502:physiology*; D045643:DNA Repair Enzymes / Q000737:chemistry* / Q000502:physiology; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000502:physiology; D019281:Dimerization; D019143:Evolution, Molecular; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D051381:Rats; D013499:Surface Properties; D029867:Xenopus Proteins / Q000737:chemistry; D029961:Zebrafish Proteins / Q000737:chemistry
D004268:DNA-Binding Proteins; C508551:NHEJ1 protein, human; C101467:XRCC4 protein, human; D029867:Xenopus Proteins; D029961:Zebrafish Proteins; D045643:DNA Repair Enzymes