Structural and dynamical insights into the PH domain of p62 in human TFIIH

Abstract

TFIIH is a crucial transcription and DNA repair factor consisting of the seven-subunit core. The core subunit p62 contains a pleckstrin homology domain (PH-D), which is essential for locating TFIIH at transcription initiation and DNA damage sites, and two BSD (BTF2-like transcription factors, synapse-associated proteins and DOS2-like proteins) domains. A recent cryo-electron microscopy (cryo-EM) structure of human TFIIH visualized most parts of core, except for the PH-D. Here, by nuclear magnetic resonance spectroscopy we have established the solution structure of human p62 PH-D connected to the BSD1 domain by a highly flexible linker, suggesting the flexibility of PH-D in TFIIH. Based on this dynamic character, the PH-D was modeled in the cryo-EM structure to obtain the whole human TFIIH core structure, which indicates that the PH-D moves around the surface of core with a specific but limited spatial distribution; these dynamic structures were refined by molecular dynamics (MD) simulations. Furthermore, we built models, also refined by MD simulations, of TFIIH in complex with five p62-binding partners, including transcription factors TFIIEα, p53 and DP1, and nucleotide excision repair factors XPC and UVSSA. The models explain why the PH-D is crucially targeted by these factors, which use their intrinsically disordered acidic regions for TFIIH recruitment.

Figure 1.

Structure comparison between human and yeast TFIIH p62/Tfb1. (A) Domain organization of TFIIH p62/Tfb1. The figure is based on sequence alignment of the two proteins (Supplementary Figure S2). Arrow, β-strand; rectangle, α-helix. (B) Ribbon representation of cryo-EM TFIIH structures. Left, human TFIIH, apo form (PDB code 6NMI); right, yeast TFIIH, holo form (PDB code 5OQM). For clarity, only the p62/Tfb1 domain is colored.

Figure 2.

NMR signal assignment and secondary structure of the PH and BSD1 domains of human TFIIH p62. (A) p62_1–158 construct used in the NMR experiments. (B) ¹H–¹⁵N-HSQC spectrum. Residues 1–103, 104–108 and 109–158 are labeled in black, yellow and blue, respectively. (C) Chemical shift index (CSI) of ¹³Cα, ¹³Cβ and ¹³C′, and identified secondary structure elements. Arrow: β-strand; rectangle; α-helix. The secondary structure identification was performed using the CSI 2.0 (http://csi.wishartlab.com) web server (79). (D) Random coil index (RCI), calculated by using the difference between observed and reference random coil shifts of ¹³Cα, ¹³Cβ, ¹³C′, ¹⁵N and ¹H. RCI should be proportional to the flexibility of the protein backbone (80).

Figure 3.

Solution structure of the PH and BSD1 domains of human TFIIH p62. Overlay of the backbone structures (A, B) and ribbon representation (C, D) of the 20 best structures, superimposed on the PH (A, C) and BSD1 (B, D) domains. (E) Ribbon representation of a single structure. The PH-D is shown in magenta/pink, the BSD1 domain in blue/cyan and the interdomain linker in yellow.

Figure 4.

Dynamics of the linker between the PH and BSD1 domains of human TFIIH p62. (A) ¹⁵N longitudinal relaxation rate (R₁). (B) Transverse relaxation rate (R₂). (C) Ratio of R₂ to R₁. (D) ¹⁵N–{¹H} NOE. Water–amide proton exchange rate (k_ex) measured by HET_ex-BEST-TROSY (54) (E) and CLEANEX-PM (55) (F).

Figure 5.

Structural model of human TFIIH. Structural model of human TFIIH built by a combination of the cryo-EM (PDB code 6NMI) and NMR (PDB code 7BUL) structures. The 275 models (A), single representative models (B, D, F, H, J, L) and their corresponding MD simulation models (C, E, G, I, K, M) are shown.

Figure 6.

MD simulation models of the complexes of human TFIIH with p62-binding partners. (A) Overall view. Extended view of the area indicated by the square in (A): (B) complex of TFIIH and TFIIEα; (C) complex of TFIIH and p53; (D) complex of TFIIH and DP1; (E) complex of TFIIH and XPC; and (F) complex of TFIIH and UVSSA.