Structural and functional evidence that Rad4 competes with Rad2 for binding to the Tfb1 subunit of TFIIH in NER

Abstract

XPC/Rad4 (human/yeast) recruits transcription faction IIH (TFIIH) to the nucleotide excision repair (NER) complex through interactions with its p62/Tfb1 and XPB/Ssl2 subunits. TFIIH then recruits XPG/Rad2 through interactions with similar subunits and the two repair factors appear to be mutually exclusive within the NER complex. Here, we show that Rad4 binds the PH domain of the Tfb1 (Tfb1PH) with high affinity. Structural characterization of a Rad4-Tfb1PH complex demonstrates that the Rad4-binding interface is formed using a motif similar to one used by Rad2 to bind Tfb1PH. In vivo studies in yeast demonstrate that the N-terminal Tfb1-binding motif and C-terminal TFIIH-binding motif of Rad4 are both crucial for survival following exposure to UV irradiation. Together, these results support the hypothesis that XPG/Rad2 displaces XPC/Rad4 from the repair complex in part through interactions with the Tfb1/p62 subunit of TFIIH. The Rad4-Tfb1PH structure also provides detailed information regarding, not only the interplay of TFIIH recruitment to the NER, but also links the role of TFIIH in NER and transcription.

Figure 1.

The N-terminal region of Rad4 contains a high-affinity Tfb1PH-binding site. (a) Identification of an amino acid segment located between residues 86 and 106 of Rad4 that aligns with the Tfb1PH-binding motif found in Rad2 and TFIIEαCTD. A similar motif is also located between residues 124 and 144 of XPC. In these alignments, the residues of TFIIEαCTD and Rad2_642–690 that form the binding interface with p62PH/Tfb1PH are underlined and crucial hydrophobic residues are shaded in gray. (b) Comparison of the dissociation constant (K_d) values for the binding of Rad4_76–115 and its mutants (F95P and V98P) to Tfb1PH. (c) Thermogram of the Tfb1PH titration with successive additions of Rad4_76–115. Experiments are performed at 25°C, in 20 mM NaPO₄ pH 7.5 buffer, and the results fit to a single-binding site model with 1:1 stoichiometry.

Figure 2.

Rad4_76–115 and Rad2_359–383 share a common binding site on Tfb1PH. (a and b) Ribbon model of the 3D structure of Tfb1PH (blue; PDB code 1Y5O). The amino acids of Tfb1PH showing a significant chemical shift change {Δδ(ppm) > 0.15; Δδ = [(0.17ΔN_H)² + (ΔH_N)²]^1/2} upon formation of a complex with either Rad4_76–115 (a) or Rad2_359–383 (b) are highlighted in orange and brown, respectively. (c) Overlay of a selected region from the ¹H –¹⁵N HSQC spectra of ¹⁵N-labeled Rad4_76–115 (0.5 mM) in the free form (green) and in the presence of unlabeled Tfb1PH (0.4 mM; blue). (d) Same overlay as in (c), but after the addition of unlabeled Rad2_359–383 (1.5 mM; black). Rad4_76–115 signals that undergo significant changes in ¹H and ¹⁵N chemical shifts upon formation of the complex with Tfb1PH (c), and return towards their original position following the addition of Rad2_359–383 (d) are indicated by arrows.

Figure 3.

Rad34 contains a Tfb1PH-binding motif. (a) Identification of an amino acid segment located between residues 41 and 63 from Rad34 that aligns with the Tfb1PH-binding motif from Rad4 and XPC. The two crucial hydrophobic residues in the motif are shaded in gray. (b) Comparison of the dissociation constant (K_d) values for the binding of Rad4_76–115 and Rad34_41–63 with Tfb1PH. No binding is observed with the W54S mutant of Rad34_41–63 under the experimental conditions indicating a K_d > 10 µM. (c) Ribbon model of the 3D structure of Tfb1PH (blue). The amino acids of ¹⁵N-labeled Tfb1PH showing a significant chemical shift change {Δδ(ppm) > 0.15; Δδ = [(0.17ΔN_H)² + (ΔH_N)²]^1/2} upon formation of a complex with Rad34_41–63 are highlighted in magenta.

Figure 4.

The two TFIIH-binding regions of Rad4 are crucial for survival following UV irradiation. (a) The survival of RAD4 (blue), rad4-PP (black) and rad4-AAA yeast were determined following increasing doses of UV irradiation. (b) The survival of RAD4 (blue), rad4 (red), rad4-PP (black), rad4-AAA (orange) and rad4-PPAAA (aqua) yeast were determined following increasing doses of UV irradiation. In both (a) and (b), the y-axis represents the percentage of surviving cells (normalized to the number of viable cells not exposed to UV light) and the x-axis shows the energy levels of the UV irradiation applied (J/m²). The results are the mean ± SEM of three independent experiments.

Figure 5.

NMR structure of the Rad4_76–115–Tfb1PH complex. (a) Stereo view of the 20 lowest-energy structures of the complex between Tfb1PH (blue) and Rad4_76–115 (orange; PDB code 2M14). The 3D structures were superimposed using the backbone atoms C′, C^α and N of residues 4–65 and 85–112 of Tfb1PH and residues 90–104 of Rad4_76–115. (b) Ribbon representation of Tfb1PH (blue) and backbone trace of the region of Rad4_76–115 (orange) interacting in the first binding pocket. In this pocket, Phe95 of Rad4 forms a cation–π interaction with Arg61 of Tfb1 and van der Waals interactions with Met59. (c) Ribbon representation of Tfb1PH (blue) and backbone trace of the region of Rad4_76–115 (orange) interacting in the second binding pocket. On one side of the pocket Val98 of Rad4 interacts with Leu48, Ala50, Lys101 and Gln105 of Tfb1. On the other side of the pocket Thr99 of Rad4 interacts with Gln105, Ile108 and Lys112 of Tfb1.

Figure 6.

The interfaces of Rad2–Tfb1PH and Rad4–Tfb1PH complexes are very similar. The 3D structures of Tfb1PH are shown as molecular surfaces (blue) and Rad4_76–115 (a; orange), Rad2_642–690 (b; yellow) are shown as ribbons. Selected residues in the Tfb1PH-binding modif of Rad4_76–115 and Rad2_642–690 are also shown to demonstrate the similarity between their binding interfaces.