Crystal structure of the human Polϵ B-subunit in complex with the C-terminal domain of the catalytic subunit

Abstract

The eukaryotic B-family DNA polymerases include four members: Polα, Polδ, Polϵ, and Polζ, which share common architectural features, such as the exonuclease/polymerase and C-terminal domains (CTDs) of catalytic subunits bound to indispensable B-subunits, which serve as scaffolds that mediate interactions with other components of the replication machinery. Crystal structures for the B-subunits of Polα and Polδ/Polζ have been reported: the former within the primosome and separately with CTD and the latter with the N-terminal domain of the C-subunit. Here we present the crystal structure of the human Polϵ B-subunit (p59) in complex with CTD of the catalytic subunit (p261_C). The structure revealed a well defined electron density for p261_C and the phosphodiesterase and oligonucleotide/oligosaccharide-binding domains of p59. However, electron density was missing for the p59 N-terminal domain and for the linker connecting it to the phosphodiesterase domain. Similar to Polα, p261_C of Polϵ contains a three-helix bundle in the middle and zinc-binding modules on each side. Intersubunit interactions involving 11 hydrogen bonds and numerous hydrophobic contacts account for stable complex formation with a buried surface area of 3094 Ų Comparative structural analysis of p59-p261_C with the corresponding Polα complex revealed significant differences between the B-subunits and CTDs, as well as their interaction interfaces. The B-subunit of Polδ/Polζ also substantially differs from B-subunits of either Polα or Polϵ. This work provides a structural basis to explain biochemical and genetic data on the importance of B-subunit integrity in replisome function in vivo.

Keywords: B-subunit; DNA polymerase epsilon; DNA replication; Dpb2; crystal structure; human; protein complex; zinc; zinc-binding module.

Figure 1.

Overall structure of Polϵ p59–p261_C. A, schematic representation of the domain organization. The dark blue lines in the schematics of p261_C present the relative positions of the zinc-coordinating residues in two zinc-binding modules: Zn1 (Cys²¹⁵⁸, Cys²¹⁶¹, Cys²¹⁸⁷, and Cys²¹⁹⁰) and Zn2 (Cys²²²¹, Cys²²²⁴, Cys²²³⁶, and Cys²²³⁸). B, stereo view of p59–p261_C. p261_C is colored light pink; the PDE domain (excluding region 84–121) and OB domain are colored cyan and green, respectively. The N-terminal portion of the PDE domain (residues 84–121) is colored red. Zinc atoms are depicted as dark blue spheres.

Figure 2.

Model of the p59–p261_C structure. The model comprised of the crystal structure of p59–p261_C and the NMR structure of NTD (Protein Data Bank code 2v6z) (39). The dashed line indicates the linker between NTD and PDE. The domains are colored as in Fig. 1.

Figure 3.

Close-up stereo view of the hydrophilic contacts between p261_C and p59. The secondary structure elements are shown with 60% transparency. The domains and carbons are colored according to Fig. 1. The side chains or main chains of the residues involved in intersubunit interactions are shown as sticks. The nitrogens, oxygens, and sulfurs are colored blue, red, and yellow, respectively.

Figure 4.

Close-up stereo view of the hydrophobic contacts between p261_C and p59. The presentation details are described in the Fig. 3 legend.

Figure 5.

Side-by-side comparison of the Polϵ p59–p261_C, Polα p70–p180_C, and Polδ/Polζ p50–p66_N crystal structures. Red arrows indicate the structural elements contributing to significant differences between the three structures. The coloring of molecules is similar to Fig. 1, except the p66_N domain of Polδ/Polζ C-subunit is colored purple. Two different orientations are shown for each molecule. The molecules are displayed using Protein Data Bank entries 5vbn, 5exr, and 3e0j.

Figure 6.

Structure-based sequence alignments of p261_C with p180_C (A) and p125_C with p353_C (B). Blue- and green-shaded boxes depict the α-helices and β-strands, respectively. The gray-shaded boxes indicate the residues that are disordered in the crystal structures of p261_C and p180_C. The secondary structure elements are from the crystal structures of p261_C and p180_C (A) and from Phyre (57) predictions for p125_C and p353_C (B). The conserved residues are highlighted in red. Magenta-shaded boxes depict the zinc-coordinating cysteines. Yellow bars indicate the sequences comprising the zinc-binding modules Zn1 and Zn2. The red bar indicates the sequences implicated in iron–sulfur cluster binding.

Figure 7.

Differences between the Polϵ p261_C and Polα p180_C structures. The molecules were aligned by superposition of the conservative helices α₁ and α₂. p261_C and p180_C are colored light pink and gray, respectively.

Figure 8.

Comparison of CTD docking on the B-subunit between p59–p261_C and p70–p180_C. Both CTD–B-subunit complexes were aligned by superposition of the B-subunits; p70 is omitted for clarity. The p59–p261_C domains are colored according to Fig. 1, and p180_C is colored gray.

Figure 9.

Structure-based sequence alignments of the B-subunits (A) and CTDs (B) of human and yeast Polϵ. The α-helices and β-strands are depicted by green- and blue-shaded boxes, respectively. The gray-shaded boxes indicate the residues that are disordered in the crystal structure of p59–p261_C. The conserved residues are shown in red. The blue, pink, and black circles indicate amino acids involved in intersubunit hydrophobic, hydrophilic, and both types of interactions, respectively. The Dpb2 residues listed in Table 2 are underlined. The fold prediction for the yeast Polϵ subunits was performed using Phyre (57).

Figure 10.

Modeling of the amino acid changes mapped to the intersubunit interface. A, modeling of p59 Cys³³³ and p261_C Val²¹⁹⁷ in place of asparagine and serine, respectively. Only one conformation of Cys³³³ is allowed to avoid steric hindrance with main-chain atoms. In both allowed conformations of Val²¹⁹⁷ (only one is shown for clarity), there is a clash with Cys³³³. B, modeling of p59 Ser⁴⁶⁰ in place of proline. The p59–p261_C domains are colored according to Fig. 1. Carbons of the modeled residues are colored purple. A blue dashed line depicts the distance between atoms. Zn1 and Zn2 labels show the position of corresponding zinc-binding modules.

Figure 11.

Modeling of the amino acid changes inducing steric hindrance. A, close-up view of a hydrophobic pocket with modeled proline in place of Val¹⁸³. Val¹⁸³ makes two main-chain–to–main-chain hydrogen bonds with Ala²²⁶ (not shown). Leu¹⁷¹, Val¹⁸³, Leu¹⁹⁷, Ala²²⁶, and Phe²³⁵ of p59 correspond to Leu²⁷⁴, Leu²⁸⁴, Leu²⁹⁸, Val³²⁸, and Phe³³⁹ of Dpb2, respectively. B, close-up view of a hydrophobic pocket with modeled tryptophan in place of Leu¹⁸⁴. In the shown conformation, tryptophan clashes with the side chains of Pro²⁴³ and Leu¹³⁷. Tyr¹³⁴, Leu¹³⁷, Thr¹⁴¹, Leu¹⁶⁵, Leu¹⁸⁴, Phe²²³, Leu²²⁵, and Pro²⁴³ of p59 correspond to Tyr²²², Thr²²⁵, Val²²⁹, Ile²⁶⁸, Leu²⁸⁵, Met³²⁵, Leu³²⁷, and Pro³⁴⁷ of Dpb2, respectively. p59 and p261_C are colored according to Fig. 1. Carbons of the modeled residues are colored purple. The residues are shown as sticks. The secondary structure elements are shown with 20% transparency. Blue dashed lines depict the closest distance between the residues. Proline and tryptophan rotamers, shown in A and B, respectively, correspond to minimal steric hindrance.

Figure 12.

Analysis of the role of Pro⁵¹⁴ and the extreme C-terminal region on p59 folding. A, close-up view of a hydrophobic pocket containing Leu⁵²⁴ and Phe⁵²⁷. The carbons of p59 residues corresponding to the deleted region in the Dpb2-200 mutant are colored purple. Lys³⁰³, Ile³⁰⁶, Met³⁰⁷, Phe⁵⁰², Phe⁵⁰⁷, and Phe⁵⁰⁹ of p59 correspond to Ala⁴¹⁶, Lys⁴¹⁹, Ile⁴²⁰, Phe⁶⁶⁴, Ala⁶⁷⁰, and Tyr⁶⁷² of Dpb2, respectively. B, close-up view of a hydrophobic pocket containing Pro⁵¹⁴. Ala²⁸⁵, Phe²⁸⁷, Tyr⁴⁰⁷, and Phe⁵¹² of p59 correspond to His³⁹⁷, Phe³⁹⁹, Tyr⁵³⁴, and Tyr⁶⁷⁵ of Dpb2, respectively. The p59 domains are colored according to Fig. 1. Side chains of the residues are shown as sticks.

Figure 13.

Photomicrograph of the p59–p261_C crystal.