Structural Basis of Poly(ADP-ribose) Recognition by the Multizinc Binding Domain of Checkpoint with Forkhead-associated and RING Domains (CHFR)

Jasmeen Oberoi,Mark W Richards,Simon Crumpler,Nathan Brown,Julian Blagg,Richard Bayliss

doi:10.1074/jbc.m110.159855

Jasmeen Oberoi, Mark W Richards + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m110.159855

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Abstract

Cellular stress in early mitosis activates the antephase checkpoint, resulting in the decondensation of chromosomes and delayed mitotic progression. Checkpoint with forkhead-associated and RING domains (CHFR) is central to this checkpoint, and its activity is ablated in many tumors and cancer cell lines through promoter hypermethylation or mutation. The interaction between the PAR-binding zinc finger (PBZ) of CHFR and poly(ADP-ribose) (PAR) is crucial for a functional antephase checkpoint. We determined the crystal structure of the cysteine-rich region of human CHFR (amino acids 425-664) to 1.9 Å resolution, which revealed a multizinc binding domain of elaborate topology within which the PBZ is embedded. The PBZ of CHFR closely resembles the analogous motifs from aprataxin-like factor and CG1218-PA, which lie within unstructured regions of their respective proteins. Based on co-crystal structures of CHFR bound to several different PAR-like ligands (adenosine 5'-diphosphoribose, adenosine monophosphate, and P(1)P(2)-diadenosine 5'-pyrophosphate), we made a model of the CHFR-PAR interaction, which we validated using site-specific mutagenesis and surface plasmon resonance. The PBZ motif of CHFR recognizes two adenine-containing subunits of PAR and the phosphate backbone that connects them. More generally, PBZ motifs may recognize different numbers of PAR subunits as required to carry out their functions.

Highlights

The second PBZ motif of APLF, in which Ade site 1 is blocked by a proline at position 1A, has a similar affinity as the Y636A point mutant of CHFR in which this site is disrupted (KD of 8 and 2.5 ␮M, respectively)
These data are consistent with the model in which the single PBZ motif of CHFR and the first PBZ motif of APLF bind two subunits of PAR and the second PBZ motif of APLF binds a single subunit
Chemical shift data on the interaction between the first PBZ motif of APLF and mADPR show a large shift for Met380, and a single NOE was detected between the adenine ring and Phe396

Summary

EXPERIMENTAL PROCEDURES

Protein Expression, and Purification—CHFR cysteine-rich domain constructs 407– 664 (CHFR-C1) and 394 – 664 (CHFR-C2) were cloned into the pETM6T1 vector (derived from pET44 (Novagen)) with an N-terminal, tobacco etch virus-cleavable His6-NusA tag for expression in Escherichia coli BL21-CodonPlus (DE3)-RIL cells (Stratagene). Cells were lysed in a buffer containing 200 mM NaCl, 50 mM Tris, pH 8.0, 5% (v/v) glycerol, 10 mM 2-mercaptoethanol, and EDTA-free protease inhibitor tablet. The protein was further purified using a Superdex 200 16/60 gel filtration column as described for the CHFR cysteine-rich domain constructs. For the co-crystallization of CHFR 407– 664 with adenosine 5Ј-diphosphoribose (mADPR) and AMP, the protein was incubated on ice with 5 mM mADPR (Sigma) and 10 mM AMP (Sigma), respectively, for 1 h, before adding 1 ␮l of this mixture to 1 ␮l of well buffer. 100-␮l samples of 5 ␮M CHFR were prepared on ice in 50 mM NaCl, 25 mM Tris 8.5, 2% (v/v) glycerol, and 5ϫ Sypro Orange. CHFR proteins were dialyzed against running buffer and injected at 40 ␮l/min for 375 s over pairs of blank and PARcontaining flow cells. The final structure was energy-minimized as described in the supplemental material

RESULTS

Molprobity score

DISCUSSION