Dynamic conformational switching underlies TFIIH function in transcription and DNA repair and impacts genetic diseases

Abstract

Transcription factor IIH (TFIIH) is a protein assembly essential for transcription initiation and nucleotide excision repair (NER). Yet, understanding of the conformational switching underpinning these diverse TFIIH functions remains fragmentary. TFIIH mechanisms critically depend on two translocase subunits, XPB and XPD. To unravel their functions and regulation, we build cryo-EM based TFIIH models in transcription- and NER-competent states. Using simulations and graph-theoretical analysis methods, we reveal TFIIH's global motions, define TFIIH partitioning into dynamic communities and show how TFIIH reshapes itself and self-regulates depending on functional context. Our study uncovers an internal regulatory mechanism that switches XPB and XPD activities making them mutually exclusive between NER and transcription initiation. By sequentially coordinating the XPB and XPD DNA-unwinding activities, the switch ensures precise DNA incision in NER. Mapping TFIIH disease mutations onto network models reveals clustering into distinct mechanistic classes, affecting translocase functions, protein interactions and interface dynamics.

Fig. 1. Cryo-EM based models of TFIIH in apo state, holo-PIC and NER-TFIIH reveal how this multifaceted assembly reorganizes with changed functional context.

Anterior and posterior view of TFIIH in a the holo-PIC assembly; b apo-TFIIH; and c the NER lesion scanning complex. XPD anchor (light green), BSD2 (light blue) and ATP-cap (gold) elements of p62 positioned on the XPD surface in d PIC-TFIIH; e apo-TFIIH; and f NER-TFIIH. The TFIIH subunits are shown in cartoon representation colored as follows: XPD dark red, p62 blue, p44 orange, p34 green, p52 purple, p8 dark cyan, XPB pink; MAT1 and XPA are shown in tan and DNA is cyan.

Fig. 2. Chain-of-replicas path optimization methods reveal the DNA translocation mechanisms of XPB and XPD.

Global motions (gray arrows) of the XPB domains in a top and b side orientation. RecA1 is shown in red, RecA2 in dark cyan, NTE in purple, DRD in tan, dsDNA in cyan. XPB latch is indicated by violet dashed outline; opening/closure of XPB ATPase domain cleft is indicated by a black double arrow. Global motions (gray arrows) of the XPD domains in c top and d side orientation. The Arch domain is shown in magenta, RecA1 in light blue, RecA2 in gold, Fe–S in green, NTE-CTE in gray, ssDNA in cyan. Opening/closure of the Arch and Fe–S domains is indicated by a black double arrow.

Fig. 3. TFIIH dramatically alters its flexibility and dynamics depending on functional context.

Computed B-factors mapped onto the structural models of a PIC-TFIIH, b apo-TFIIH and c NER-TFIIH. B-factor values are colored from low (blue) to high (red). Close-up views of the rigid (blue) versus flexible (red) structural elements at the XPB–XPD interface in d PIC-TFIIH, e apo-TFIIH and f NER-TFIIH. Black dashed outline highlights an unexpected rigid anchor region formed by p44/XPB and part of XPD in the TFIIH-NER complex. The interface of XPB and XPD is highlighted by a red dashed line. The TFIIH lever arm (p8, p52, p34) is indicated by a green dashed outline.

Fig. 4. Community networks underlying changes in TFIIH dynamics in transcription versus NER.

a Consensus communities are identified from difference contact network analysis and mapped to the structure of apo-TFIIH. Communities are color-coded and labeled. Net change in contact probabilities across TFIIH communities during b the PIC to apo transition and c the apo to NER transition. The radius of each community vertex denotes the size (number of residues) of the community. The gray and black edges between communities indicate overall gain(+)/loss(-) of dynamic contacts.

Fig. 5. Network analysis reveals conformational switching at TFIIH community interfaces.

Residues experiencing the largest gain/loss of contact probability during the PIC to apo and apo to NER transitions are mapped onto the TFIIH structure and shown as green or purple dots, respectively. Dynamic communities of XPB, XPD and adjacent subunits are colored as in Fig. 4. TFIIH lever arm communities are shown in gray. dCNA subgraphs for these communities are shown as insets, with gray or black edges indicating gain(+)/loss(-) of dynamic contacts Contact changes are shown for the following transitions: a PIC to apo; b apo to NER. c community identities in the consensus network are indicated; labels denote the principal domains/structural elements belonging to a community. d–f, Close-up views of interfaces with the largest contacts gain (green)/loss (purple) during the PIC to apo transition: d the XPB–XPD interface; e XPD-p44 and XPD-p62 interfaces; f the XPB ATPase domains and the collar region. Close-up views of interfaces with the largest contacts gain (green)/loss (purple) during the apo to NER transition: g the XPB–XPD interface; h the XPD-p44 and XPD-p62 interfaces; i the XPB ATPase domains and the collar region.

Fig. 6. Mapping of the global motions of the NER-TFIIH complex onto the dCNA communities shows that translocation dynamics is enabled for XPD and suppressed for XPB during lesion scanning.

a The first principal mode from PCA analysis of the NER-TFIIH complex; gray arrows indicate the directionality of the motions of the Cα atoms of the assembly; b Zoomed view showing the motion of the XPB communities; c Zoomed view showing the motion of XPD communities. Communities are colored the same as in Fig. 4.

Fig. 7. Human disease mutations mapped onto TFIIH show distinct patterns within protein-protein and community interfaces.

a TTD, XP, XP/CS and XP/TTD point mutations mapped onto XPD, XPB, and p8 subunits do not co-localize by disease on primary sequence. b Map of human disease mutations (spheres) onto the PIC-TFIIH structure shown in cartoon representation and colored by community. c Map of human disease mutations (spheres) onto the NER-TFIIH structure shown in cartoon representation and colored by community. d Zoomed view of mutations within XPD. e Zoomed view of mutations at the XPB–XPD interface. f Zoomed view of mutations lining the path of ssDNA in XPD.

Fig. 8. Conformational switching mechanism enabling sequential coordination of XPB and XPD activities in NER.

The schematic represents key steps in the NER pathway—XPC lesion recognition, NER bubble extension, XPD-mediated lesion scanning, PInC assembly and DNA incision by XPG and XPF/ERCC1, gap filling synthesis, and DNA restoration. Protein participants in NER are shown as cartoons, color-coded, and labeled. The DNA lesion position is shown as a red star. Red dashed arrows indicate the direction in which ssDNA is displaced during the different stages of NER and the rotation of the DNA duplex by XPB. White dotted arrow indicates the displacement of the XPB ATPase domains. Black dotted arrow indicates the opening/closing of the XPD Arch and Fe–S domains during ssDNA translocation. A red cross indicates the blocking of a bulky lesion inside XPD and damage verification. Two red arrows indicate the incision points on DNA by XPF/ERCC1 and XPG.