Crystal structures of PCNA1 and PCNA2 from Aeropyrum pernix: implications for a distorted heterotrimeric sliding clamp

Aeropyrum pernix is a hyperthermophilic archaeon that possesses three proliferating cell nuclear antigen (PCNA) isoforms (ApePCNA1, ApePCNA2 and ApePCNA3) that form a heterotrimeric sliding clamp. To gain more detailed structural insights into this heterotrimeric assembly, we determined the crystal structures of ApePCNA1 and ApePCNA2. ApePCNA1 was crystallized under a new condition, and the 1.60 Å resolution structure revealed a unique non­proline cis-peptide bond between Arg187 and Arg188, which was not deeply discussed in a previous report. The structure of ApePCNA2 was determined at 2.17 Å resolution, and it forms a typical homotrimeric ring. In the cubic crystal form, its crystal packing shows an intriguing tetrahedral assembly of four trimers. Modeling the ApePCNA1–ApePCNA2–ApePCNA3 heterotrimer based on these structures suggests that the cis-peptide in ApePCNA1 induces significant steric hindrance at the subunit interface, leading to a symmetry-broken or distorted ring conformation rather than the canonical pseudo-threefold-symmetric assembly.

1. Introduction

DNA replication is an essential process that is conserved across all living organisms (O'Donnell et al., 2013; Bell & Kaguni, 2013; Ishino et al., 2013). Efficient DNA replication requires ring-shaped sliding clamps to tether DNA polymerases and other enzymes onto DNA, increasing their processivity. In eukaryotes and archaea, the proliferating cell nuclear antigen (PCNA) homotrimer serves this role, while the β-subunit dimer and the gp45 trimer function in bacteria and phages, respectively (Kong et al., 1992; Krishna et al., 1994). DNA-metabolizing enzymes contact PCNA through the PCNA-interacting peptide (PIP) motif or its variants (Warbrick, 1998; De Biasio & Blanco, 2013). These clamps are loaded onto DNA by pentameric AAA⁺ clamp loaders (Miyata et al., 2005; Kelch et al., 2011; Gaubitz et al., 2022).

Despite the fundamental differences in cellular organization and their extreme habitats, archaeal DNA-replication systems are remarkably similar to those of eukaryotes (Ishino & Cann, 1998; Kelman & White, 2005). Certain Crenarchaeota possess multiple PCNA isoforms (Daimon et al., 2002; Dionne et al., 2003). In Saccharolobus solfataricus and Aeropyrum pernix, three PCNA isoforms assemble into a stable heterotrimeric ring (Pascal et al., 2006; Williams et al., 2006; Imamura et al., 2007; Hlinkova et al., 2008), which is considered to be evolutionarily related to the eukaryotic Rad9–Rad1–Hus1 heterotrimer for DNA repair (Doré et al., 2009).

Although detailed intersubunit interactions have been analyzed (Imamura et al., 2007), structural data for A. pernix were restricted to ApePCNA1 (Yamauchi et al., 2024). Here, we report the crystal structure of ApePCNA1 determined under a new condition and the newly solved ApePCNA2 homotrimer. The ApePCNA1 structure revealed a unique protrusion formed by the nonproline cis-peptide bond. Modeling of the ApePCNA1–ApePCNA2–ApePCNA3 heterotrimer based on these structural data suggests that the cis-peptide protrusion in ApePCNA1 may induce a distorted or symmetry-broken ring conformation, rather than the canonical threefold-symmetric ring.

2. Materials and methods

2.1. Macromolecule production

Recombinant ApePCNA1 (UniProt code Q9YFT8, APE_0162) and ApePCNA2 (Q9Y9V7, APE_2182) proteins (Table 1), without any non-native residues, were expressed and purified following the methods previously described by Daimon et al. (2002). It should be noted that ApePCNA2 was referred to as ApePCNA3 in the previous literature. These proteins were overexpressed in an Escherichia coli system. The harvested cells were disrupted, followed by heat treatment, polyethyleneimine treatment in the presence of high-concentration NaCl and ammonium sulfate precipitation to obtain crude extracts. The proteins were then purified using anion-exchange and heparin column chromatography to achieve sufficient purity for crystallization. The purified proteins were concentrated to 20 mg ml⁻¹ using Amicon Ultra-4 centrifugal filter units (Merck) for subsequent crystallization experiments.

Table 1
Macromolecule-production information

Source organism	Aeropyrum pernix
Expression vector	pET-21a
Expression host	Escherichia coli BL21 (DE3)
Complete amino-acid sequence of the construct produced
ApePCNA1 (Q9YFT8, APE_0162)	MSSEATLDSEFTDYKAMFRYEAKVFKELVDSVSKILDEGLFIITGEGLRLRGMDPARVALVDIEIPSSSFFDFYMAGDVERVELGVNMETLKGVVARAKKGDQLEVRVREDKVLFIVESVVLRRYLLPNLEVIVDVPEDISLEFDATATVIADVVKKTLRDVELVGDIVEFDAGEDYLSIRSVGPERRRVETRLTRESPALIDLEVKEPATSRYDVGYLKRMLGVAKIAESIELSFSTDKPLKMVFKSPDGSRVTYLLAPSTG
ApePCNA2 (Q9Y9V7, APE_2182)	MFRLVYTASSKFKYIAQTLAKINDEGVFEFSLDGLRAWIMSPDKTSLAILEMPSLSFEEYMVEEEMRVVLRTDELNKISKRATRNDDIIFQWNAEEQALEVELRDRKLGFSRKFLVPATSVGAEEMRRLKLEPTVSFTILTDDLKAMIQDVKVVGDFAEFEASEGQVVVRSQAEEKEYEWVMKPGDVLLSLEVEEDAKSIYSRQVLEIATKPVGAAESVKVSFASDYPMKIEYTFPNGERMELYMAPSLAG

2.2. Crystallization

Crystallization of ApePCNA1 and ApePCNA2 was performed at 298 K using the hanging-drop vapor-diffusion method (Table 2). We attempted crystallization based on the conditions reported for Pyrococcus furiosus PCNA (Matsumiya et al., 2002), which shares 34% and 29% sequence identity with ApePCNA1 and ApePCNA2, respectively. Single crystals suitable for X-ray diffraction experiments were successfully obtained without extensive screening. Specifically, 1 µl protein solution was mixed with an equal volume of reservoir solution consisting of 0.1 M citric acid pH 4.5 and 1.8 M ammonium sulfate. The initial drops were equilibrated against 500 µl of the same reservoir solution. Polyhedral crystals with a maximum dimension of approximately 0.2 mm were grown within a week.

Table 2
Crystallization

Method	Hanging-drop vapor diffusion
Plate type	VDX plate
Temperature (K)	293
Protein concentration (mg ml^−1)	20
Buffer composition of protein solution	50 mM Tris–HCl pH 8.0, 0.1 mM EDTA, 0.5 mM dithiothreitol, 10% glycerol, 250 mM (ApePCNA1) or 600 mM (ApePCNA2) NaCl
Composition of reservoir solution	0.1 M citric acid pH 4.5, 1.8 M ammonium sulfate
Volume and ratio of drop	1 µl protein solution and 1 µl reservoir solution
Volume of reservoir (µl)	500

2.3. Data collection and processing

X-ray diffraction data (Table 3) were collected on beamline BL38B1 at the SPring-8 synchrotron-radiation facility, Harima, Japan. Prior to data collection, crystals were briefly soaked in a cryoprotectant solution consisting of the reservoir solution supplemented with a final concentration of 20% glycerol. The crystals were then directly mounted and flash-cooled in a stream of cold nitrogen gas at 100 K. Diffraction images were recorded on a Rigaku Jupiter 210 CCD detector. For each dataset, a total of 180 images were collected with an oscillation angle of 1.0° per frame. The ApePCNA1 crystal belonged to the tetragonal space group P4₃2₁2, with unit-cell parameters a = b = 68.93, c = 120.165 Å, and diffracted X-rays to a resolution of 1.60 Å. The crystal contained one molecule in the asymmetric unit, which led to a Matthews coefficient (V_M) of 2.43 ų Da⁻¹ and a solvent content of 49.4%. The ApePCNA2 crystal belonged to the cubic space group P2₁3 and diffracted to 2.17 Å resolution. The unit-cell parameters for ApePCNA2 were a = b = c = 169.79 Å. The asymmetric unit contained four copies of the protein, with a V_M of 2.16 ų Da⁻¹ and a solvent content of 43.2%. Data processing was performed using XDS (Kabsch, 2010) and AIMLESS (Evans & Murshudov, 2013).

Table 3
Data collection and processing

Values in parentheses are for the outer shell.

Crystal	ApePCNA1	ApePCNA2
X-ray source	BL38B1, SPring-8	BL38B1, SPring-8
Wavelength (Å)	1.00000	1.00000
Temperature (K)	100	100
Detector	Rigaku Jupiter 210	Rigaku Jupiter 210
Space group	P43212	P213
a, b, c (Å)	68.93, 68.93, 120.17	169.79, 169.79, 169.79
α, β, γ (°)	90, 90, 90	90, 90, 90
Mosaicity (°)	0.30	0.12
Resolution range (Å)	45.3–1.60 (1.63–1.60)	47.1–2.17 (2.25–2.17)
Total No. of reflections	412127 (31722)	1614594 (122163)
No. of unique reflections	38871 (3729)	86020 (8530)
Completeness (%)	99.7 (97.7)	99.9 (100.0)
Multiplicity	10.6 (8.5)	18.8 (14.3)
〈I/σ(I)〉	20.3 (4.1)	24.82 (4.23)
Rmeas	0.076 (0.389)	0.098 (0.686)
Rp.i.m.	0.023 (0.131)	0.022 (0.180)
CC1/2	0.998 (0.936)	0.999 (0.933)
Overall B factor from Wilson plot (Å^2)	16.49	33.23

2.4. Structure solution and refinement

The crystal structures of both ApePCNA1 and ApePCNA2 were determined by the molecular-replacement method. Calculations were performed using Phaser within the Phenix package (Liebschner et al., 2019). A polyalanine model based on the P. furiosus PCNA monomer (PDB entry 1ge8) was used as a search probe. Following the correct placement of the probe in the crystal lattice, initial models including side chains were generated using phenix.autobuild. The structures were further improved through cycles of crystallographic refinement using phenix.refine and manual model rebuilding with Coot (Emsley et al., 2010) until the refinement converged (Table 4). Most of the ApePCNA1 structure could be built into the electron-density map, excluding some residues at both termini (Met1–Ser3, Ser9–Asp13 and Gly263). In the case of ApePCNA2, all amino-acid residues in the four molecules of the asymmetric unit were visible in the electron-density map.

Table 4
Structure refinement

Crystal	ApePCNA1	ApePCNA2
Resolution range (Å)	45.3–1.60	47.1–2.17
Final Rcryst	0.198	0.205
Final Rfree	0.218	0.237
No. of non-H atoms
Protein	2005	8197
Ligand	0	60 (sulfate ions)
Water	89	168
R.m.s. deviations
Bond lengths (Å)	0.007	0.005
Angles (°)	0.88	0.73
Average B factors (Å^2)
Protein	22.86	39.34
Ligand	—	30.84
Water	25.62	35.15
Ramachandran plot
Most favored (%)	99.2	97.6
Allowed (%)	0.80	2.41
PDB code	24ku	24jd

3. Results and discussion

3.1. Molecular arrangement in the crystals

Despite very similar crystallization conditions and morphologies, ApePCNA1 and ApePCNA2 crystallized in different space groups. The structure of ApePCNA1 was determined at 1.60 Å resolution as a monomer in the same space group and with similar unit-cell parameters as the previously reported structure (PDB entry 6aig; Yamauchi et al., 2024), with a root-mean-square deviation (r.m.s.d) of 0.37 Å for 252 C^α atoms. Notably, we identified a nonproline cis-peptide bond between Arg187 and Arg188 in the C-terminal domain (Fig. 1b), a feature that despite being present in PDB entry 6aig was not deeply discussed in the previous study. As discussed below, this unique local conformation likely contributes to the specific assembly characteristics of the A. pernix heterotrimer by potentially hindering homotrimerization (Imamura et al., 2007). For ApePCNA2, four monomers in the asymmetric unit form a noncrystallographic symmetry (NCS)-related trimer (chains A–C) and a crystallographic trimer (chain D), which together assemble into a tetrahedral cluster (Fig. 2).

Figure 1 The nonproline cis-peptide between Arg187 and Arg188 of ApePCNA1. (a) Overall structure of the ApePCNA1 monomer shown as a cartoon model, colored in a rainbow gradient from the N-terminus (blue) to the C-terminus (red). (b) Stereo pair of a close-up view showing the Fo − Fc omit electron-density map (contoured at 2.5σ) for the unique nonproline cis-peptide bond identified between Arg187 and Arg188. All structural figures were prepared using PyMOL (Schrödinger).

Figure 2 Crystal packing and tetrahedral assembly of ApePCNA2. Among the four molecules in the asymmetric unit, three molecules (chains A, B and C; shown as green cartoons) form a homotrimer via noncrystallographic threefold symmetry. Two additional trimers generated by the crystallo­graphic symmetry are shown as yellow and gray surface models. The remaining molecule (chain D; dark blue cartoon) forms another homotrimer with its symmetry mates (light blue cartoons). Consequently, four homotrimers are assembled into a tetrahedral cluster within the crystal lattice.

3.2. Overall structure of the ApePCNA1 and ApePCNA2 subunits

Both ApePCNA1 and ApePCNA2 adopt the canonical PCNA fold, which is characterized by a pseudo-sixfold-symmetric architecture (Figs. 1 and 2). Although the inter-domain connecting loop (IDCL) is often disordered in unliganded PCNAs, it was clearly defined in both structures, likely stabilized by crystal packing.

ApePCNA1 and ApePCNA2 share 28.7% sequence identity (Daimon et al., 2002) and exhibit high structural similarity, with an r.m.s.d. of 1.41 Å for 231 C^α atoms (Fig. 3). Similarly, the AlphaFold3-predicted ApePCNA3 model aligns closely with ApePCNA2 (r.m.s.d. of 1.37 Å for 239 C^α atoms). This structural conservation across the three subunits supports the feasibility of the heterotrimer model described below.

Figure 3 Structural comparison between ApePCNA1 and ApePCNA2. (a) Structure-based sequence alignment. Asterisks and colons indicate identical and highly similar residues, respectively. (b) Stereoview of the superposed structures. ApePCNA1 and ApePCNA2 are represented as C^α traces in sky blue and green, respectively.

3.3. Implications for A. pernix PCNA heterotrimer assembly

Previous biochemical analysis suggested that the A. pernix heterotrimer assembles in the order ApePCNA1–ApePCNA3–ApePCNA2, as ApePCNA3 interacts stably with both ApePCNA1 and ApePCNA2, whereas the interaction between ApePCNA1 and ApePCNA2 is weak (Imamura et al., 2007). We constructed a heterotrimer model using our ApePCNA2 homotrimer as a template, incorporating the experimental ApePCNA1 structure and an AlphaFold3-predicted model (Abramson et al., 2024) of ApePCNA3, with the S. solfataricus PCNA heterotrimer (PDB entry 2ix2) as a reference (Fig. 4a).

Figure 4 Proposed structural model of the A. pernix PCNA heterotrimer. (a) Model of the ApePCNA1–ApePCNA2–ApePCNA3 heterotrimer constructed using the ApePCNA2 homotrimer as a template. Subunits are replaced with ApePCNA1 (cyan) and an ApePCNA3 model (orange). The cis-peptide is shown as yellow sticks. (b) Stereo pair of a close-up view of the clashing region between ApePCNA1 and ApePCNA2, viewed from the direction indicated by the arrow in (a). Arg188 causes a steric clash with ApePCNA2. (c) Schematic diagram of the predicted ApePCNA heterotrimer. The threefold rotational symmetry is predicted to be broken by the cis-peptide protrusion (yellow triangle) of ApePCNA1.

This modeling revealed significant steric hindrance at the interface between ApePCNA1 and ApePCNA2 (Fig. 4b), caused by the protruding segment of ApePCNA1 containing the Arg187-Arg188 cis-peptide bond. This structural distortion is likely attributed to a unique sequence that forms the cis-peptide, rather than an insertion, at the subunit interface of ApePCNA1: a region that, despite its critical role in ring assembly, is surprisingly not conserved among PCNA homologs (Daimon et al., 2002). AlphaFold3 failed to accurately predict this cis conformation (data not shown), highlighting the importance of experimental validation for capturing such rare nonproline cis-peptides. Since nonproline cis-peptide bonds are typically rigid (Jabs et al., 1999), this segment likely maintains its conformation in solution. Consequently, the A. pernix PCNA heterotrimer may adopt a distorted or symmetry-broken ring architecture instead of the canonical pseudo-threefold-symmetric closed ring (Fig. 4c). Experimental determination of the full heterotrimer structure remains a high priority to verify this model.