Crystal structure of the Rad3/XPD regulatory domain of Ssl1/p44

Abstract

The Ssl1/p44 subunit is a core component of the yeast/mammalian general transcription factor TFIIH, which is involved in transcription and DNA repair. Ssl1/p44 binds to and stimulates the Rad3/XPD helicase activity of TFIIH. To understand the helicase stimulatory mechanism of Ssl1/p44, we determined the crystal structure of the N-terminal regulatory domain of Ssl1 from Saccharomyces cerevisiae. Ssl1 forms a von Willebrand factor A fold in which a central six-stranded β-sheet is sandwiched between three α helices on both sides. Structural and biochemical analyses of Ssl1/p44 revealed that the β4-α5 loop, which is frequently found at the interface between von Willebrand factor A family proteins and cellular counterparts, is critical for the stimulation of Rad3/XPD. Yeast genetics analyses showed that double mutation of Leu-239 and Ser-240 in the β4-α5 loop of Ssl1 leads to lethality of a yeast strain, demonstrating the importance of the Rad3-Ssl1 interactions to cell viability. Here, we provide a structural model for the Rad3/XPD-Ssl1/p44 complex and insights into how the binding of Ssl1/p44 contributes to the helicase activity of Rad3/XPD and cell viability.

Keywords: Crystal Structure; DNA Repair; Nucleotide Excision Repair; Rad3/XPD Helicase; Regulatory Domain of Ssl1/p44; TFIIH; Transcription; Yeast Genetics.

FIGURE 1.

The overall structure of the Ssl1 regulatory domain. A, residues 1–252 of the human p44 fragment (residues 50–322 of ScRad3), but not the C-terminal fragment comprising residues 252–395, interact with human XPD. Sf9 cells infected with XPD baculoviruses were lysed and incubated with either full-length p44, p44 (residues 1–252), or p44 (residues 252–395) Sf9 cell extracts. The Ab-XPD immunoprecipitated (IP) complexes were resolved by SDS-PAGE and immunoblotted. Lanes 1–3 represent full-length p44 (full-length), p44 (residues 1–252), and p44 (residues 252–395), respectively; lanes 4–6 represent the negative control, in which the Ab-XPD were incubated with various p44 proteins only. Lanes 7–9 represent the immunoprecipitated complexes containing Ab-XPD, which were incubated with XPD, and subsequently with p44 proteins. The positions of XPD, p44 full-length, p44 (residues 1–252) fragment, p44 (residues 252–395) fragment, immunoglobulin heavy chain (HC), and light chain (LC) are indicated in the right panel. B and C, in Ssl1, a central β-sheet is surrounded by the seven amphipathic helices. The β4-α5 loop is indicated as a red line.

FIGURE 2.

Sequence alignment of seven Ssl1/p44 homologs and five different VWA proteins. Shown are Ssl1 from S. cerevisiae (Sc), Schizosaccharomyces pombe (Sp), Homo sapiens (Hs), Mus musculus (Mm), Drosophila melanogaster (Dm), Caenorhabditis elegans (Ce), and Arabidopsis thaliana (At), S. pombe (Sp) Rpn10, Chaetomium thermophilum (Ct) Tfb4/p34, Catenulispora acidiphila (Ca) VWFA, H. sapiens Ku70, and H. sapiens integrin αLβ2. α helices and β strands are shown as red rectangles and blue arrows, respectively. The secondary structures of the proteins are underlined. The conserved residues are indicated by yellow boxes, and fully and partly exposed residues are indicated by open and half-filled circles, respectively. Metal ion-dependent adhesion site motif residues are indicated by blue dots. The positions of the mutations (Leu-174/Ser-175 and Glu-203) are indicated as red dots. Promals program (34) was used for multiple sequence alignments.

FIGURE 3.

Structural comparisons of Ssl1 and VWA family proteins. A, from left, Ssl1 (orange) was superimposed onto C. acidiphila VWFA (Protein Data Bank 4FX5, RMSD 2.0 Å, blue), S. pombe Rpn10 (Protein Data Bank code 2X5N, RMSD 2.5 Å, cyan), C. thermophilum Tfb4/p34 (Protein Data Bank code 4PN7, RMSD 2.2 Å, deep blue), H. sapiens Ku70 (Protein Data Bank code 1JEY, RMSD 2.6 Å, pink), or H. sapiens integrin αLβ2 (Protein Data Bank code 1T0P, RMSD 2.6 Å, green). The β4-α5 loop is indicated by dotted black circles. The structures are shown in the same orientation. B, structural superposition of Ssl1 (light orange) and VWA family proteins. The variable regions of VWFA (Protein Data Bank code 4FX5), RPN10 (Protein Data Bank code 2X5N), and Tfb4/p34 (Protein Data Bank code 4PN7) are shown in magenta, blue, and teal, respectively. C, surface representation of the Ssl1 regulatory domain. The frequencies with which specific regions of VWA family members interact with their counterparts are indicated by dark green, green, and pale green, which represent high, moderate, and low frequencies. The binding frequencies were calculated from the interface analyses of VWA family protein structures that are complexed with their binding partners (30, 33, 35–38). The β4-α5 loop is indicated as a white dotted line.

FIGURE 4.

Protein interaction, helicase activity assays, and yeast viability tests of XPD and mutant p44 proteins. A, the positions of the following residues that were mutated in this study are indicated: Glu-203 (Thr-138 of p44, blue line) and Leu-239/Ser-240 (Leu-174/Thr-175 of p44, dark green lines) at the top side of Ssl1. The β4-α5 loop is indicated as red lines. The Thr-242 residue is indicated by a dotted circle. The top figure is in the same orientation as that of Fig. 1C. B, binding of WT, T138R, or L174W/T175R p44 proteins to XPD. Equivalent residues of Ssl1 are marked in parentheses. The positions of XPD and p44 are indicated at the right side of the panel. C, the stimulation of XPD helicase activity by WT and mutant p44 proteins. Lane 2, WT; lane 3, T138R; lane 4, L174W/T175R. The positions of the substrate and product are shown at the right side of the panel. Lanes 1 and 6 show results for WT-XPD and WT-p44 only, respectively. Lane 5 contains XPD mutant (R722W) and p44. Lanes 7 (−) and 8 (Δ) show the results for nonboiled and boiled substrate, respectively. The helicase activities were quantified using ImageJ software and are shown as a bar graph (bottom panel). D, 5-fold serial dilutions of yeast strains containing the pRS316_WT Ssl1 plasmid and the pRS314_mutant or WT Ssl1 plasmid were spotted onto Trp−/Ura− and Trp−/FOA plates and photographed after growth for 2 days at 30 °C in the dark. The positions of the Ssl1 mutants are indicated at the left side of the panel. The strain that harbored the pRS314 vector only was spotted at the empty vector line.

FIGURE 5.

The predicted position of Ssl1 in the TFIIH complex. A, the x-ray structures of T. acidophilum XPD (Protein Data Bank code 2VSF) and Ssl1 were fitted into the EM density of the ScTFIIH complex. The predicted locations of the Rad3 C-terminal is depicted as blue dotted circles. The β4-α5 loop is shown as a red line. The Ssl1 N-terminal end is indicated by a red dot. B, a close-up view of the interface between Rad3 and Ssl1 in the complex model.

FIGURE 6.

Close-up views of the p44 mutants. A–D, previously studied p44 mutant residues Asp-66, Arg-165, Glu-166, Asp-178, and Gly-200 are shown in stick representation and colored in cyan (12). Water molecules are shown as red dots, and hydrogen bonds are indicated by black dotted lines.