Structural and functional studies on CHFR autoubiquitination

The antephase checkpoint plays an important role in delaying eukaryotic cell division in the presence of numerous stress conditions. Checkpoint with Fork-Head Associated (FHA) and RING domain (CHFR) is an E3 ubiquitin ligase and an integral component of the antephase checkpoint. Responsible for delaying mitotic entry in the presence of adverse conditions, downregulation of CHFR is frequently observed in numerous cancer cell lines and tumours. Previous studies have demonstrated N-terminal FHA-domain and C-terminal cysteine rich domain deficient CHFR proteins (CRD; ΔFHA-ΔCRD-CHFR) can form polyubiquitin chains in vitro. However, the oligomeric state of the full-length (FL-CHFR) and N-terminal FHA-domain deletion mutant (ΔFHA-CHFR) proteins remains unexplored. This study has demonstrated that the FL-CHFR protein is dimeric in solution, with ΔFHA-CHFR proteins retaining both the capacity to dimerize in solution and form polyubiquitin chains. With di-, tetra- and octomeric CRD species identified, both the FHA domain and the CRD therefore mediate dimerization of FL-CHFR. SEC in-line with Small Angle X-ray Scattering (SEC-SAXS) has verified segment-swapped dimerization of the FHA domain (13-180) in solution, previously observed within a published crystal structure (PDB: 1LGP, 13-125). A screen of 34 E2s has identified 5 previously unreported ubiquitin conjugating enzymes (UbcH5C, UbcH5D, UbcH6, UbcH8, Ubc1) responsible for CHFR mediated polyubiquitin chain formation in vitro, corroborating with phylogenetic analysis. A RING domain homology model was generated using X-ray structures of RING homologues, featuring an alpha helix, three antiparallel beta strands and two zinc metal ions; with molecular dynamic simulations revealing considerable Root Mean Square Fluctuations (RMSFs) within the N and C-terminal loop regions. By modelling key interactions within the RING: Ubc13 (:Mms2) ~ ubiquitin complex, site directed FL-CHFR (I306A, P340A, W332A, R345A, H322C/ Y362A, R335A, R343A, E300A, H322C, Y362A) and ubiquitin (E34A, Q40A, R72A, G35A) mutants have identified essential interactions responsible for CHFR-mediated polyubiquitin chain formation in vitro.