Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 112 (48), 14817-22

Architecture of the Human XPC DNA Repair and Stem Cell Coactivator Complex

Affiliations

Architecture of the Human XPC DNA Repair and Stem Cell Coactivator Complex

Elisa T Zhang et al. Proc Natl Acad Sci U S A.

Abstract

The Xeroderma pigmentosum complementation group C (XPC) complex is a versatile factor involved in both nucleotide excision repair and transcriptional coactivation as a critical component of the NANOG, OCT4, and SOX2 pluripotency gene regulatory network. Here we present the structure of the human holo-XPC complex determined by single-particle electron microscopy to reveal a flexible, ear-shaped structure that undergoes localized loss of order upon DNA binding. We also determined the structure of the complete yeast homolog Rad4 holo-complex to find a similar overall architecture to the human complex, consistent with their shared DNA repair functions. Localized differences between these structures reflect an intriguing phylogenetic divergence in transcriptional capabilities that we present here. Having positioned the constituent subunits by tagging and deletion, we propose a model of key interaction interfaces that reveals the structural basis for this difference in functional conservation. Together, our findings establish a framework for understanding the structure-function relationships of the XPC complex in the interplay between transcription and DNA repair.

Keywords: DNA repair; biochemistry; stem cells; structure; transcription.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
3D reconstruction of the human XPC complex and localization of subunits. (A) Schematic representation of the subunits and domains of the human XPC complex. Transglutaminase homology domain (TGD), β-hairpin domains 1–3 (BH), ubiquitin-like domain (UbL), ubiquitin-associated domains 1 and 2 (UBA), EF-hand domains (EF), and protein- and DNA-binding domains (BD) are indicated accordingly. (B) Front and back views of the XPC complex reconstructed in EMAN2 (49). Estimated dimensions are indicated. (C) Positive (yellow) and negative (purple) 3D difference densities at 4σ between the complex containing MBP-CETN2 and untagged complex. (D) Positive (pink) 3D difference density at 5σ between the full complex and the XPC-RAD23B subcomplex, indicating the likely position for the CETN2 subunit. No negative difference density was observed at this threshold. (E) Docking of the yeast Rad4/Rad23 [Protein Data Bank (PDB) ID 2QSF; cyan/green] into the model with the human CETN2 and XPC interaction peptide (PDB ID 2GGM; pink/cyan) by Situs (31) in a manner consistent with the difference density data in A and B. Shown are predicted approximate positions of the UbL, UBA1, and UBA2 domains of RAD23B based on positional information of the Rad23Rad4-BD (dark green) N and C termini in the crystal structure.
Fig. S1.
Fig. S1.
XPC complex sample preparation and generation of ab initio model by random conical tilt. (A) Purification and sample preparation strategy for EM for the XPC complex expressed recombinantly in Sf9 insect cells. (B) SDS/PAGE and Coomassie staining of the purified fractions of the XPC complex (Left) and the complex cross-linked with glutaraldehyde (Right). (C) Migration of the XPC complex at ∼275 kDa in size on a Superose 6 gel filtration column, as calculated by fitting to the Kav values (Kav = (VeVo)/(VtVo), where Ve = elution volume of the protein, Vo = column void volume, and Vt = total bed volume) of the molecular weight standards ferritin (440 kDa), aldoloase (158 kDa), conalbumin (75 kDa), and ovalbumin (44 kDa). (D) Representative tilt pair of images used for RCT. (Scale bar: 200 nm.) (E) Two-dimensional class averages of the RCT data set of 2,384 particles. The class average corresponding to the initial model selected for subsequent analysis is indicated by the green box. (F) Front and back views of the top RCT initial volume #1 selected for subsequent refinement. (G) Comparisons between its 3D reprojections and 2D class averages from the entire RCT data set.
Fig. S2.
Fig. S2.
3D reconstruction of the apo human XPC complex and subunit labeling. (A) Representative image (Left) with DoG-picker (53) selected particles indicated in red (Right) of uranyl-formate stained sample taken at 80,000× magnification. (Scale bar: 200 nm.) (B) Representative 2D class averages of ∼46,000 total particles. (C) Fourier shell correlation (FSC) curve indicating the estimated resolution to be 24.7 Å using the FSC = 0.5 criterion (60). (D) Euler angular distribution plot. (E) Comparison of 3D reprojections of the refined model to their corresponding reference-free 2D class averages. (F) SDS/PAGE and Coomassie staining of purified XPC complex containing MBP-CETN2 (M-C) (Left) and the complex cross-linked with glutaraldehyde (Right). (G) SDS/PAGE and Coomassie staining of the purified and cross-linked XPC-RAD23B subcomplex.
Fig. S3.
Fig. S3.
Model validation. (A) Front (Upper) and back (Lower) views of cross-linked (pink), uncross-linked (yellow), and cryo-EM models generated in RELION (32) using ∼20,000–30,000 particles each. (B) Negative stain 3D reprojections and cryo-EM reference-free 2D class averages corresponding to A. (C, Left) Positive (blue) and negative (purple) 3D difference maps at 3σ and 2σ between the cross-linked and native complexes. (C, Right) Corresponding 2D class averages of cross-linked (Left) and native (Right) particles. (D) Free-hand test of the XPC complex using 93 particles taken at a 0° and a 30° tilt.
Fig. 2.
Fig. 2.
The XPC complex adopts highly flexible conformations. (A) Three models of the XPC complex generated in RELION with Situs-based docking (31) of the yeast Rad4/Rad23 (PDB ID 2QSF; cyan/green) and the human CETN2/XPCCETN2-BD (PDB ID 2GGM; pink/cyan) crystal structures. (B) Comparison of 3D reprojections of the models to their corresponding reference-free 2D class averages.
Fig. 3.
Fig. 3.
DNA binding by XPC is accompanied by large but localized conformational changes distal from the sites of presumed DNA contact. (A) Front, side, and back views of the XPC complex bound to a mismatch bubble substrate generated in RELION (32) (yellow) shown with the apo structure (mesh gray), with the 3D [apo] − [DNA-bound] difference density at 3σ (cyan), and with the [DNA-bound] − [apo] difference density (brown). Also shown is the Situs-based docking (31) of the yeast Rad4/Rad23 apo (PDB ID 2QSF; purple/orange) structure with the DNA-bound (PDB ID 2QSH; green/yellow) structure aligned to the apo via the Rad23 subunit. (B) Comparison of 3D reprojections of the model to their corresponding reference-free 2D class averages.
Fig. S4.
Fig. S4.
Purification of DNA-bound XPC complexes and alternate DNA-bound structural models. (A) Schematic of purification strategy of DNA-bound complexes. (B) SDS/PAGE and Coomassie staining of XPC-RAD23B-CETN2 copurifying with indicated DNA substrates (Upper); gel cropped for clarity. Native PAGE and SYBR Gold staining of DNA substrates eluted during sample preparation (Lower). IN = input before pull-down. (C) Alternative docking of the Rad4/Rad23 crystal structure (21) (PDB ID 2QSF) to better illustrate the proposed overlap between the primary difference density and the observed positional shift of Rad4 in the crystal structure. Shown are front and top-side views of the XPC complex bound to a mismatch bubble substrate generated in RELION (mesh yellow) with the apo structure (solid gray) and the [apo] – [DNA-bound] 3D difference density at 4σ (for clarity) in light blue. No [DNA-bound] - [apo] difference density was observed at this threshold. Here, the N- and C-terminal portions of the TGD domain (residues 99–295 and 298–433, respectively) are split to better reflect the human loop insertion (Fig. 1A and Fig. S5). Rad4TGD2-Rad23 structures were docked manually and are shown with their Rad23 domains aligned to one another. Rad4TGD1 was docked in the context of the full crystal structure using Situs (31) and Chimera (61). Rad4TGD1 is shown in blue, Rad4-TGD2apo is in purple, Rad23apo is in orange, Rad4-TGD2DNA-bound is in green, and Rad23DNA-bound is in solid yellow. (D) Front and side views of the ssDNA-bound XPC complex (mesh pink) shown with the mismatch-bound structure (solid yellow) with the [mismatch] – [ssDNA] difference density at 3σ shown in blue. No difference density is observed at 4σ. (E) Alignment of the apo (purple) and DNA-bound (green) Rad4 crystal structures by their TGD domain. Rad23 was omitted for clarity. Shown also is an enlarged view with distances in Ångstroms between indicated residues.
Fig. S5.
Fig. S5.
Conservation analysis via comparison of the human XPC complex and the yeast Rad4 complex. (A) Schematic representation of the subunits and domains of the human XPC complex and the yeast Rad4 complex. Transglutaminase homology domain (TGD), β-hairpin domains 1–3 (BH), ubiquitin-like domain (UbL), ubiquitin-associated domains 1 and 2 (UBA), EF-hand domains (EF), and protein- and DNA-binding domains (BD) are indicated accordingly. (B) Sequence alignment between human XPC and yeast Rad4. Predicted and observed α-helices (pink bars) and β-strands (green bars) are indicated. XPA-binding residues (blue), OCT4-binding residues (25) (red), SOX2-binding residues (25) (orange), and TFIIH-binding residues (15, 16, 44, 45) (violet) were designated based on sequence alignment between human XPC and yeast Rad4 and are indicated by color as noted. Regions of contact with Rad23 in the Rad4/Rad23 crystal structure (PDB ID 2QSF) are indicated with coral bars. Yellow boxes indicate DNA-binding residues in the DNA-bound Rad4/Rad23 crystal structure (PDB ID 2QSH) and light purple/aqua bars indicate these designated regions of contact with DNA. Secondary structure prediction was performed using JPred4 (62). (C) Sequence alignment between human CETN2 and yeast Rad33. Predicted and observed α-helices (pink bars) and β-strands (green bars) are indicated. XPC-binding residues are indicated in purple. Secondary structure prediction was performed using JPred4 (62).
Fig. 4.
Fig. 4.
Comparative studies of the human and yeast XPC/Rad4 complexes reveal divergence in function but not structure. (A) Purification and cross-linking of the homologous yeast Rad4-Rad23-Rad33 complex. (B) Three-dimensional model of the yeast Rad4 complex as solved by EMAN2 (49). (C) Three-dimensional difference density at 3σ between the human (mesh pink) and the yeast (solid gray) complexes. Positive difference density, or [human]-[yeast], is shown in cyan; negative difference density or [yeast] − [human] is shown in purple. (D) Comparisons of 3D reprojections of the yeast Rad4 complex with 2D class averages. (E) Titrations over a fourfold concentration range of yeast, mouse, and human XPC homolog complexes in in vitro transcription reactions of a NANOG promoter template engineered with four extra copies of the oct-sox composite binding element (bottom), performed in the presence of OCT4 and SOX2 protein. Transcripts are indicated with arrowheads. (F) Coimmunoprecipitation of human and yeast XPC/Rad4 complexes with HA-tagged SOX2 or RFP.
Fig. 5.
Fig. 5.
Model of key interaction interfaces on the XPC complex. (A) Model of predicted interaction surfaces based on docking and sequence homology with the yeast Rad4/Rad23 crystal structure (PDB ID 2QSF). Indicated are residues involved in binding to XPA (14) (blue; yeast residues 101–296), OCT4 (25) (red; yeast residues 298–392), SOX2 (25) (orange; yeast residues 392–609), and TFIIH (15, 16, 44) (violet; human residues 847–863 and yeast residues 76–115 and 610–631). (B) Top back view of the XPC complex showing an area of positive difference density between the human and yeast structures (yellow) that coincides with the predicted OCT4- (red) and SOX2-binding domains (orange) but not the DNA-binding residues (dark blue residues and circles; BD3-5). The XPA binding domain has been omitted in the enlarged view for clarity.

Similar articles

See all similar articles

Cited by 6 articles

See all "Cited by" articles

Publication types

MeSH terms

LinkOut - more resources

Feedback