The intricate network between the p34 and p44 subunits is central to the activity of the transcription/DNA repair factor TFIIH

Abstract

The general transcription factor IIH (TFIIH) is a multi-protein complex and its 10 subunits are engaged in an intricate protein-protein interaction network critical for the regulation of its transcription and DNA repair activities that are so far little understood on a molecular level. In this study, we focused on the p44 and the p34 subunits, which are central for the structural integrity of core-TFIIH. We solved crystal structures of a complex formed by the p34 N-terminal vWA and p44 C-terminal zinc binding domains from Chaetomium thermophilum and from Homo sapiens. Intriguingly, our functional analyses clearly revealed the presence of a second interface located in the C-terminal zinc binding region of p34, which can rescue a disrupted interaction between the p34 vWA and the p44 RING domain. In addition, we demonstrate that the C-terminal zinc binding domain of p34 assumes a central role with respect to the stability and function of TFIIH. Our data reveal a redundant interaction network within core-TFIIH, which may serve to minimize the susceptibility to mutational impairment. This provides first insights why so far no mutations in the p34 or p44 TFIIH-core subunits have been identified that would lead to the hallmark nucleotide excision repair syndromes xeroderma pigmentosum or trichothiodystrophy.

Figure 1.

Organization of TFIIH and characterization of a minimal p34/p44 complex. (A) Composition of TFIIH and domain organization of the p34 and p44 TFIIH subunits: the Homo sapiens (hs)/Chaetomium thermophilum (ct) nomenclature of each subunit is indicated. The different structural domains of the p34/p44 subunits are represented in blue, gray and green shades, respectively. The interaction between the two subunits as observed in the structures is indicated for each protein and framed by a black diamond. (B) Structure based alignment of H. sapiens (hs) and C. thermophilum (ct) p34 and p44. Secondary structure elements are indicated below the sequence, with arrows representing β-strands and coils for α-helices. Red (largely buried) and orange circles (partially buried) indicate interface residues as defined by PDBsum. Residues that were mutated are boxed and numbered. Conserved residues are color coded (light purple for moderate conservation and dark purple for highly conserved residues (threshold 30% and 50% identify computed from a multiple sequence alignment of the human and ct sequences with orthologs from Danio rerio, Caenorhabditis elegans, Drosophila melanogaster, Saccharomyces cerevisiae and Arabidopsis thaliana). The highly conserved Zn knuckles of the p44 subunits are shown.

Figure 2.

Overall structure of the p34/p44 complex. (A and B) Ribbon representation of the ct and hs p34/p44 complexes. The topology of the p44 RING domain is shown on the upper left side of panel A. The kinked loop between helix α1 and strand β2 is shown in red. (C) Overall superposition of the human (in magenta and yellow for p34 and p44, respectively) and fungal (in blue and green for p34 and p44, respectively) p34/p44 complex structures. Close-up view of the interactions between the two proteins. The blue spheres represent the Zn ions. The anomalous electron density map from the human complex calculated using the phases of the p34 molecule is contoured at 3σ. (D) Schematic diagram of the p34 vWA/p44 RING interface. The fungal interface was analyzed with PDBsum: interacting residues are colored by residue type (blue positive (H,K,R); red negative (D,E); green neutral (S,T,N,Q); gray aliphatic (A,V,L,I,M); magenta aromatic (F,Y,W); orange (P,G); yellow (C) cysteine) and interactions are indicated by colored lines (blue and red lines for potential H-bonds and salt bridges, dashed orange lines for non-bonded contacts). Structurally similar residues (identified from a geometry-based alignment of the human and fungal interfaces) are represented using the same color code.

Figure 3.

Mutational analysis of the p34/p44 interface. (A) Detailed view of the contact area for the fungal and the human complex: selected side chains of interface residues are shown. Residues 76–85, 131–151 and 213–231 of p34ct (blue) and 398–407 and 484–506 of p44ct (green) of the fungal complex are represented in the left panel. Residues 65–74, 131–151 and 172–188 of p34hs (magenta) and 343–351 and 368–506 of p44hs (yellow) are represented in the right panel. (B) SEC of p34ct(1–277) with p44ct(368–534) wild-type or variants. p34ct(1–277) (red) and p44ct(368–534) (blue) were analyzed separately and after mixing them in a 1:1 stoichiometry (green) prior to SEC. (C) Quantification of the interaction between wild-type or variant p34ct(1–277) and p44ct(368–534) by ITC. The thermodynamic parameters for the association of the wild-type protein domains are: K_D = 11 nM, n = 0.71, ΔH = −25 kcal/mol and ΔS = −45 cal/mol/degree. (D) Core-TFIIH wild-type or variant were co-expressed in insect cells and purified using immobilized metal affinity chromatography (IMAC) followed by immuno-precipitation using an anti-p44 antibody. Equal amounts of purified rIIH6 were analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by western blot (WB) using antibodies directed against XPB, p62, p52, p44 and p34. (E) Basal transcription activity. Increasing amounts of purified wild-type and variant rIIH6 (20, 40 and 80 ng) were added to an in vitro reconstituted transcription system lacking core-TFIIH. Transcripts were analyzed by electrophoresis followed by autoradiography. The length of the corresponding transcript is indicated on the right. (F) In vitro dual-incision assay. Increasing amounts of immune purified wild-type and variant rIIH6 (60 and 160 ng) were added to an incision/excision assay using recombinant NER factors, and the reaction was analyzed by electrophoresis followed by autoradiography. Quantification of the basal transcription and of the double incision assays are presented in Supplementary Figure S5C.

Figure 4.

Association between p34 and p44 also involves the C-terminal domain of p34. (A) SEC of the p34ct full length protein with p44ct(368–534) wild-type or p44ct(368–534)-F490E and p34ct-A151E with p44ct(368–534) wild-type. p34ct full length (red) and p44ct(368–534) (blue) were analyzed separately and mixed in a 1:1 stoichiometry (green) prior to SEC. (B) HDX-MS differential heatmap of p34hs versus p34hs/p44hs. Hydrogen–Deuterium eXchange coupled to mass spectrometry experiments, in which the deuterium exchange labeling of p34hs in the presence and absence of p44hs was compared. Data could be measured only for the highlighted areas as depicted in the sequence coverage presented in Supplementary Figure S6B. (C) Densitometry analysis of a Coomassie stained gel of rIIH6/p34wt and rIIH6/p34(1–233) purified using IMAC followed by immuno-precipitation using an anti-p44 antibody. The gel as well as a western blot analysis of the complexes are presented in the supplementary data. (D) Extracts from SF9 cells co-infected by viruses expressing core-TFIIH wild-type (rIIH6/p34 wt) or mutant (rIIH6/p34(1–233)) were incubated with XPD plus CAK, immuno-precipitated using an antibody directed against cdk7 and immobilized proteins were analyzed using specific antibodies. Heavy (HC) and light (LC) chains of the anti-cdk7 antibody are indicated. (E) and(F) Complexes purified in (C) were analyzed for their transcription and NER activities as described in Figure 3.

Figure 5.

Cartoon representation depicting the summary of our observations with the same coloring scheme as used in Figure 1 showing core-TFIIH with XPD. Left panel: wild-type active TFIIH. Middle panel: TFIIH mutated within the minimal p34/p44 interface (structurally characterized in this study) maintaining the active complex. Right panel: TFIIH with the C4 domain of p34 missing. This p34 variant still interacts with p44 but other vital interactions are missing leading to an inactive TFIIH.