PDBsum entry 2pie

Ligase

PDB id: 2pie

Contents

Protein chain

132 a.a. *

Ligands

GLU-LEU-LYS-TPO-GLU-ARG-TYR

Waters ×190

* Residue conservation analysis

PDB id:

2pie

Name:

Ligase

Title:

Crystal structure of the fha domain of rnf8 in complex with its optimal phosphopeptide

Structure:

E3 ubiquitin-protein ligase rnf8. Chain: a. Fragment: n terminal fha domain of rnf8. Synonym: ring finger protein 8. Engineered: yes. Phosphopeptide. Chain: f. Engineered: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: rnf8, kiaa0646. Expressed in: escherichia coli. Expression_system_taxid: 562. Synthetic: yes. Other_details: synthetic phosphopeptide

Resolution:

1.35Å R-factor: 0.182 R-free: 0.198

Authors:

R.A.Grant,M.B.Yaffe

Key ref:

M.S.Huen et al. (2007). RNF8 transduces the DNA-damage signal via histone ubiquitylation and checkpoint protein assembly. Cell, 131, 901-914. PubMed id: 18001825 DOI: 10.1016/j.cell.2007.09.041

Date:

13-Apr-07 Release date: 11-Dec-07

Protein chain

O76064  (RNF8_HUMAN) -  E3 ubiquitin-protein ligase RNF8 from Homo sapiens

Seq: 485 a.a.

Struc: 132 a.a.*

* PDB and UniProt seqs differ at 4 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.2.3.2.27 - RING-type E3 ubiquitin transferase.
• Reaction: S-ubiquitinyl-[E2 ubiquitin-conjugating enzyme]-L-cysteine + [acceptor protein]-L-lysine = [E2 ubiquitin-conjugating enzyme]-L-cysteine + N₆- ubiquitinyl-[acceptor protein]-L-lysine

DOI no: 10.1016/j.cell.2007.09.041

Cell 131:901-914 (2007)

PubMed id: 18001825

RNF8 transduces the DNA-damage signal via histone ubiquitylation and checkpoint protein assembly.

M.S.Huen, R.Grant, I.Manke, K.Minn, X.Yu, M.B.Yaffe, J.Chen.

ABSTRACT

DNA-damage signaling utilizes a multitude of posttranslational modifiers as molecular switches to regulate cell-cycle checkpoints, DNA repair, cellular senescence, and apoptosis. Here we show that RNF8, a FHA/RING domain-containing protein, plays a critical role in the early DNA-damage response. We have solved the X-ray crystal structure of the FHA domain structure at 1.35 A. We have shown that RNF8 facilitates the accumulation of checkpoint mediator proteins BRCA1 and 53BP1 to the damaged chromatin, on one hand through the phospho-dependent FHA domain-mediated binding of RNF8 to MDC1, on the other hand via its role in ubiquitylating H2AX and possibly other substrates at damage sites. Moreover, RNF8-depleted cells displayed a defective G2/M checkpoint and increased IR sensitivity. Together, our study implicates RNF8 as a novel DNA-damage-responsive protein that integrates protein phosphorylation and ubiquitylation signaling and plays a critical role in the cellular response to genotoxic stress.

Selected figure(s)

Figure 2.
Figure 2. Structural Basis for Phosphorylation-Dependent Binding by RNF8 FHA Domain
(A) Amino acid selectivity values for the RNF8 FHA domain determined using the phosphothreonine-oriented degenerate peptide library MAXXXX-pT-XXXXAKKK, where X indicates all amino acids except Cys. Values ≥ 1.4 indicate moderate selection; values ≥ 2.0 indicate strong selection.
(B) Cartoon representation of the RNF8 FHA domain bound to the optimal phosphopeptide ELKpTERY.
(C) stereo view of the phosphopeptide-binding surface.
(D) Closeup of the phosphate-binding pocket, with 2Fo-Fc density map contoured at 2σ. A bound water molecule is evident in the upper center.
(E–G) Molecular interaction surfaces of the RNF8:phosphopeptide complex, the MDC1 tandem BRCT domain:γ-H[2]AX phosphopeptide complex, and the Rad53 FHA1:LEVpTEAD phosphopeptide complex. Peptide surfaces are contoured in salmon, protein surfaces are contoured in lime. In the RNF8 FHA domain (E), selection for Tyr over Phe in the +3 position likely results from a water-mediated contact between the Tyr hydroxyl and the backbone nitrogen of Leu57. In the Rad53 FHA1 structure (G), an Arg residue from the FHA domain occupies the equivalent position as the peptide +3 Tyr in the RNF8 structure (dashed line).
(H) Divergence in the phospho-amino acid +3 binding surfaces of the FHA domains of RNF8 and Rad53. The Cα traces of the FHA domains of the RNF8 FHA domain:phosphopeptide complex and the Rad53 FHA1 domain:phosphopeptide complex were optimally aligned. The phosphopeptide +3 interacting region is shown in cartoon representation, with the RNF8 FHA domain shaded blue and its bound phosphopeptide shaded cyan, while the Rad53 FHA1 domain is shaded yellow and its bound phosphopeptide is shaded green. The +3 Tyr residue in the RNF8 optimal phosphopeptide and the +3 Asp in the Rad53 FHA1 optimal phosphopeptide are shown in stick representation. Note that the +3 Tyr binding site in RNF8 is occluded in the Rad53 FHA domain by an Arg residue that mediates selection for Asp in the +3 position.

Figure 6.
Figure 6. RNF8 Is Required for H2AX Ubiquitylation following DNA Damage
(A) H2AX is ubiquitylated in vivo. 293T cells were transiently transfected with plasmids encoding myc-tagged ubiquitin with or without plasmids encoding SBP-Flag-H2AX. Immunoprecipitation and immunoblotting were carried out using indicated antibodies. Black arrow indicates doubly ubiquitylated species of H2AX, while gray arrow indicates monoubiquitinated H2AX. Multiple-ubiquitinated H2AX species are also pointed out.
(B) HeLa cells stably expressing HA-tagged H2AX were transfected with control siRNA or RNF8 siRNA were treated with 10 Gy or left untreated. Cells were harvested 1 hr post-irradiation. Cell lysates were prepared, separated by SDS-PAGE, and blotted with indicated antibodies.
(C) HeLa cells transfected with control siRNA or RNF8 siRNA were treated as described in (B) and immunoblotting experiments were carried out using indicated antibodies.
(D) IR-induced H2AX ubiquitylation in H2AX^+/+ and H2AX^−/− MEFs. Cell lysates prepared from WT or H2AX^−/− cells before and after irradiation were immunoblotted with anti-H2AX and anti-pH2AX antibodies.
(E) IR-induced H2AX ubiquitylation requires H2AX phosphorylation. H2AX-deficient MEFs stably expressing HA-tagged H2AX or S139A mutant of H2AX were treated with 0 Gy or 10 Gy and immunoblotting was performed using indicated antibodies.
(F) IR-induced H2AX ubiquitylation requires RNF8 FHA and RING domains. Experiments were carried out as that described in Figure 4C. Immunoblotting experiments were conducted with antibodies as indicated. Arrow indicates ubiquitylated species of H2AX that only appear after radiation in cells expressing WT RNF8.

The above figures are reprinted from an Open Access publication published by Cell Press: Cell (2007, 131, 901-914) copyright 2007.

Figures were selected by an automated process.