 | Acta Crystallographica Section F Acta Crystallographica Section F STRUCTURAL BIOLOGY COMMUNICATIONS |
journal menu
research communications
| STRUCTURAL BIOLOGY COMMUNICATIONS |
accessStructural analysis of the flexibility of the Ubl2 domain within the papain-like protease of SARS-CoV-2
aEuropean Synchrotron Radiation Facility (ESRF), 71 Avenue des Martyrs, 38000 Grenoble, France, and bNuvisan (Germany), Muellerstrasse 178, 13353 Berlin, Germany
*Correspondence e-mail: [email protected]
The papain-like protease (PLpro) of SARS-CoV-2 is part of the multi-domain nonstructural protein 3 (NSP3) and consists of two domains: a ubiquitin-like domain 2 (Ubl2) and a protease domain. PLpro plays a crucial role in the replication cycle of SARS-CoV-2, facilitating host immune-system evasion and the formation of double-membrane vesicles where replication occurs. While the function of the Ubl2 domain is still not clear, it is critical for the stability and the functional efficiency of PLpro. Despite its predicted inherent flexibility, nearly all SARS-CoV-1 and SARS-CoV-2 PLpro crystal structures deposited in the Protein Data Bank show the Ubl2 domain in a highly similar, closed conformation against the Here, we present a of PLpro exhibiting Ubl2 in two distinct conformations: the well characterized closed state, where Ubl2 is positioned near the PLpro domain, and an as yet uncharacterized open state, where Ubl2 is displaced by 4 Å from the PLpro core. This conformational variability in our structure appears to be related to the occupancy of a zinc ion within the zinc-finger domain. These results provide new insights into the flexibility of Ubl2, suggesting potential avenues for targeting and harnessing this dynamic behaviour for drug discovery.
Keywords: SARS-CoV-2; papain-like proteases; Ubl2 domain; COVID-19; zinc binding; flexibility; NSP3.
1. Introduction
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for the COVID-19 pandemic, has infected over 770 million people and caused seven million deaths from its first occurrence in 2019 until March 2026 (World Health Organization, 2026
). While emerging variants challenge the effectiveness of existing vaccines and antiviral drugs such as Paxlovid, remdesivir and molnupiravir, they also indicate an urgent need for the discovery of new effective therapeutics, particularly for combinatorial treatments (Chan et al., 2024). Addressing this challenge will require both de novo antiviral drug discovery and systematic drug-repurposing strategies (Du et al., 2026; Oktavianawati et al., 2023). However, the development of novel therapies relies on a thorough understanding of the viral assembly and life cycle, as well as the exploration of several viral functional proteins as potential therapeutic targets.
The life cycle of SARS-CoV-2 within the host cell starts with its genome translation into two polyproteins, pp1a and pp1ab, which are subsequently cleaved by two viral proteases, the main protease (Mpro) and the papain-like protease (PLpro), into 16 nonstructural proteins (NSP1–NSP16). NSP3 is the largest of these, with a molecular mass of approximately 200 kDa. As NSP3 is one of the few (poly)proteins which become functional at the start of the virus life cycle and as it is highly conserved in all coronaviruses (Lei et al., 2018), NSP3 has been spotlighted as a key viral protein (Dable-Tupas & Derecho, 2022; Chan et al., 2024). The intricate complexity of NSP3, which consists of approximately 15 different domains, gives the protein its versatile functions, including evasion of the host immune system and involvement in different steps of the viral replication cycle such as polyprotein maturation, double-membrane vesicle (DMV) formation and pore creation in the DMVs (Lei et al., 2018; Wolff, Limpens et al., 2020; Wolff, Melia et al., 2020; Huang et al., 2024). However, this complexity also poses challenges in obtaining high-resolution structural information for the development of therapeutics. Because the protease activity of the PLpro domain of NSP3 is of central importance for many of its critical functions above and because the PLpro domain is highly amenable for structural and fragment-binding studies compared with full-length NSP3, the PLpro domain has emerged as a potential therapeutic target.
PLpro and the adjacent ubiquitin-like domain 2 (Ubl2) together are often considered as a single protein (von Soosten et al., 2022). The Ubl2–PLpro protomer (referred to as PLpro in this paper) is a 35 kDa papain-like cysteine protease that includes Ubl2 at its N-terminus followed by its catalytic core (Supplementary Fig. S1), which itself consists of three subdomains (Osipiuk et al., 2021): an α-helical `thumb', a β-stranded `finger' and a β-stranded `palm'. The fingers subdomain contains a zinc-binding site coordinated by four conserved cysteine residues (Cys189, Cys192, Cys224 and Cys226; Gao et al., 2021) distributed within the loops between the β-strands of the finger domain (Supplementary Fig. S1). Mutational studies of SARS-CoV-1 have shown that the zinc-finger loop residues are essential for proper folding and solubility of the enzyme and for maintaining the integrity of the active site (Barretto et al., 2005). The active centre is located in the palm domain and comprises a catalytic triad formed by the amino acids Cys111, His272 and Asp286 at the interface between the thumb and palm subdomains (Gao et al., 2021). Asp286 promotes the deprotonation of Cys111 by His272, rendering it a more potent nucleophile (Osipiuk et al., 2021). Cys111 then binds the substrate via a covalent thioether linkage, followed by substrate (peptide) cleavage (Rut et al., 2020). PLpro recognizes the target tetrapeptide motif LXGG↓XX by blocking loop 2, BL2, situated in the palm domain between β-strands 11 and 12 (residues 267–271; Gao et al., 2021).
The enzymatic protease activity of PLpro is responsible for the cleavage of the viral polyproteins pp1a and pp1ab, leading to the release of mature NSP1–NSP3 (von Soosten et al., 2022). The rest of the polyprotein cleavage to produce NSP4–NSP15 is performed by the main protease (MPro; Gao & Zhang, 2025). PLpro harbours two distinct substrate-binding sites, SUb1 and SUb2 (Supplementary Fig. S1), each with unique specificity and activity (Bosken et al., 2020). Notably, the SUb1 site primarily interacts with ubiquitin, ubiquitin-like proteins and ISG15, while the SUb2 site accommodates polyubiquitin-linked chains (von Soosten et al., 2022). The binding capability of PLpro to these proteins underscores its direct involvement in evading the host immune response, a mechanism shared with other coronaviruses such as Middle East respiratory syndrome-related coronavirus (MERS-CoV) (Bailey-Elkin et al., 2014; Clasman et al., 2020). Consequently, these two sites emerge as promising targets for drug discovery.
In spite of the importance and technical accessibility of PLpro for structural therapeutic studies, no clinically approved drugs have been developed against it to date (Chan et al., 2024). One important factor to consider is the accessibility of its drug-binding sites in situ. Although most crystal structures of PLpro deposited in the Protein Data Bank show the Ubl2 domain in a single, linearly aligned conformation, molecular-dynamics simulations indicate considerable intrinsic mobility of Ubl2 and the adjacent loops, including the BL2 and zinc-binding loops (Bosken et al., 2020; Báez-Santos et al., 2015). Consistent with this, recent in situ cryo-electron tomography of the NSP3–NSP4 pore reveals a markedly different (∼45° bent) Ubl2 orientation (Huang et al., 2024). Although this orientation is distinct from the linear arrangement seen in most isolated PLpro crystal structures, the overall picture emerging from structural and computational studies points to pronounced conformational plasticity of this domain. In our 1.95 Å resolution crystal structure of SARS-CoV-2 PLpro, two protomers in the asymmetric unit adopt different Ubl2 orientations, namely `closed' and `open'. The `closed' conformation corresponds to the canonical orientation described in most SARS-CoV PLpro structures, whereas the `open' conformation is rotated and features an ∼4 Å displacement with respect to the closed form. Here, we present a detailed analysis of this previously uncharacterized conformation of PLpro from SARS-CoV-2 in comparison to the closed state and provide an overview of all crystal structures reported so far with respect to Ubl2 conformational variability. The diversity of Ubl2 positioning observed across crystallographic and in situ structures underscores the intrinsic mobility of the domain and suggests that PLpro can access multiple conformational states depending on its molecular context.
2. Materials and methods
2.1. PLpro recombinant protein production
The SARS-CoV-2 PLpro wild-type (WT) and C111S mutant Escherichia coli codon-optimized genes inserted into a pET-28a(+) vector were synthesized by GenScript (https://www.genscript.com). The bacterial vector contains a C-terminal TEV protease cleavage site and a His6 polyhistidine tag (Supplementary Fig. S1). Plasmids were amplified using the E. coli MACH1 strain and were verified by DNA sequencing at Eurofins (https://www.eurofinsus.com/genomic-services/dna-sequencing). The final protein sequences are shown in Table 1.
| ||||||||
Protein expression was performed in E. coli BL21(DE3) cells. Cell cultures were grown at 37°C until the at 600 nm (OD600) reached 0.6. Expression was induced by adding isopropyl β-D-1-thiogalactopyranoside to a final concentration of 1 mM and ZnSO4 to 0.1 mM, followed by a temperature decrease to 18°C for ∼20 h. The cells were harvested via centrifugation at 5000g at 4°C. The cell pellets were resuspended in lysis buffer [50 mM HEPES pH 7.5, 500 mM NaCl, 5 mM β-mercaptoethanol (β-ME), 10 µM ZnCl2, 5% glycerol, 0.5% Tween, lysozyme and a complete protease-inhibitor EDTA-free tablet (Roche)] and lysed by sonication on ice. The lysate was centrifuged for 20 min at 40 000g at 4°C. The supernatant was loaded onto a 5 ml HisTrap column (GE Healthcare) for affinity-chromatography purification at 4°C. The target protein was eluted from the column with elution buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5 mM β-ME, 10 µM ZnCl2, 500 mM imidazole). TEV protease was added in a 1:20(w/w) ratio to the protein for His-tag cleavage and dialysed overnight at 4°C into 50 mM HEPES pH 7.5, 300 mM NaCl, 5 mM β-ME, 10 µM ZnCl2. A second HisTrap run was performed and the flowthrough containing the His-tag-cleaved protein was collected and dialysed again overnight at 4°C with 50 mM HEPES pH 7.5, 150 mM NaCl, 5 mM β-ME, 10 µM ZnCl2. The protein was then further purified by size-exclusion chromatography using a Superdex 75 10/300 GL column (Cytiva) at 4°C with final buffer consisting of 50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM TCEP, 10 µM ZnCl2. Protein-containing elution peaks were pooled and concentrated using a centrifugal filter unit (Amicon Ultra-4, 3.5 kDa molecular-weight cutoff) to around 10 mg ml−1. Concentrated proteins were flash-frozen using liquid nitrogen and stored at −80°C, ready to be used for crystallization or other downstream experiments.
2.2. Crystallization
The purified protein was used in crystallization trials at a concentration of 10 mg ml−1. Initial intergrown crystals were obtained for the PLproC111S mutant using Grid Screen Salt (Hampton Research) in a condition consisting of 0.1 M bicine pH 9, 1.6 M ammonium sulfate in sitting drops set up by the EMBL Grenoble HTX crystallization facility (Dimasi et al., 2007; Márquez & Cipriani, 2014). Single crystals of the PLproC111S mutant were obtained by refining the starting condition to 0.1 M bicine pH 8, 1.45 M ammonium sulfate, 10% glycerol in 24-well plates and performing micro-seeding (Table 2). Seeds were made from crystals of the PLproC111S mutant using the Hampton Research seed bead kit following the manufacturer's protocol. Seed stock was prepared by crushing crystals in one crystallization drop and transferring this solution to a reaction tube with one seed bead and 50 µl reservoir solution. The tube was vortexed for 3 min, stopping every 30 s to cool the solution on ice for 20 s. New drops were set up with a dilution series of the seed stock ranging from a 50-fold to 1500-fold dilution in reservoir buffer. PLproWT crystals were obtained by cross-seeding with previously obtained PLproC111S crystals in a hanging-drop vapour-diffusion setup. The reservoir volume was 500 µl with a drop size of 2.5 µl (1 µl protein solution, 1 µl reservoir solution and 0.5 µl seed solution).
| ||||||||||||||||||
2.3. X-ray diffraction data collection and processing
Crystals originally grown without any glycerol in the crystallization buffer were screened for a suitable cryoprotectant. However, all cryoprotectants tested reduced the diffraction quality and the diffraction limit of the crystals. This was ultimately overcome by crystallizing the protein in the presence of 10% glycerol in the crystallization buffer, followed by directly flash-cooling the crystals in liquid nitrogen.
Diffraction data from three different crystal types were collected: PLproWT and PLproC111S, both of which were harvested two months after the drops were set up, and PLproWT_`aged', which was harvested eight months after crystallization setup. X-ray diffraction data were collected on beamline ID30B (McCarthy et al., 2018) at the European Synchrotron Radiation Facility (ESRF), Grenoble, France, using an EIGER2 X 9M detector at 100 K at a wavelength of 0.873 Å for PLproWT and PLproC111S and 1.282 Å for PLproWT_`aged' (Table 3). Data processing was performed using autoPROC and STARANISO (Vonrhein et al., 2011, 2024).
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
2.4. Structure solution and refinement
The structure-solution and are listed in Table 4. All structures were solved by molecular replacement using Phenix (Liebschner et al., 2019). The search model for the C111S mutant structure was the PLpro structure from SARS-CoV-2 (PDB entry 7d7k; Zhao et al., 2021). The WT structures were solved using the C111S mutant structure reported here as a search model. Model refinement was performed using Coot and phenix.refine (Emsley et al., 2010; Liebschner et al., 2019). Experimental phases using the anomalous scattering contribution of zinc at an energy of 9.67 keV of the PLproWT_'aged' crystal were calculated with SFALL, merged with the MTZ file using CAD and the anomalous difference Fourier map was subsequently generated with fast Fourier transform (all as part of the CCP4 suite; Agarwal, 1978; Immirzi, 1966; Potterton et al., 2003; Agirre et al., 2023; Ten Eyck, 1973; Read & Schierbeek, 1988).
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
2.5. SAXS data collection, processing and modelling
SEC-SAXS measurements were carried out on BM29 at the ESRF (Tully et al., 2023); in brief, 100 µl of 10 mg ml−1 PLproC111S was loaded onto a Superdex 75 10/300 column (Cytiva) pre-equilibrated with five column volumes of running buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM TCEP, 10 µM ZnCl2). SAXS data were collected using X-rays of wavelength 0.9919 Å (12.5 keV) and a sample-to-detector distance of 2.81 m, corresponding to a q-range of 0.007–0.5 Å−1. 1400 frames were collected at 2 s per frame. Data were initially processed using the BM29 automated software pipeline FreeSAS (Kieffer et al., 2022) with additional post-processing using ScatterIV (Tully et al., 2021). Flexibility was measured using BilboMD (Pelikan et al., 2009), in which molecular-dynamics (MD) simulations are used to explore conformational space and provide an ensemble of molecular models from which a SAXS curve is calculated and compared with the experimental curve. Residues 57–60 of PLproC111S were allowed to be flexible between two rigid bodies.
3. Results and discussion
3.1. Crystallization and X-ray diffraction of PLpro WT and the active-site mutant C111S
Both PLpro protein constructs, PLproWT and PLproC111S, were expressed as a C-terminal His-tag fusion protein. The His-tag was subsequently cleaved by TEV protease prior to crystallization. During expression of the protein, 100 mM ZnSO4 was added to the cell culture and 10 mM ZnCl2 was added to each purification buffer, which led to an increase in protein yields. Both PLproWT and PLproC111S proteins eluted as monomers in (SEC; Fig. 1a). However, analytical ultracentrifugation (AUC) also showed a small population of <2% with a molecular weight of 70.2 ± 2.5 kDa, corresponding to a PLpro dimer (71.4 kDa; Supplementary Fig. S2). In spite of the similar behaviour of both constructs in SEC and AUC, only the C111S mutant crystallized readily. However, the initial crystals only diffracted to approximately 4 Å resolution. We were able to optimize the mutant crystals by micro-seeding using crystals obtained from an initial crystallization screen. Crystallization of the WT protein was achieved through cross-seeding, starting from a seed stock prepared from the optimized mutant crystals. While these crystals diffracted to below 3 Å resolution when directly flash-cooled in liquid nitrogen, they showed considerably degraded diffraction quality upon the addition of cryoprotectants. This limitation was overcome by incorporating 10% glycerol in the crystallization buffer, followed by direct flash-cooling of the obtained crystals in liquid nitrogen. Under these conditions, the crystals consistently diffracted to better than 2 Å resolution and the structures of PLproWT and PLproC111S were resolved in the orthorhombic P21212 with very similar unit-cell dimensions (Table 3). Each structure contains two molecules in the asymmetric unit, with Matthews coefficients of 2.66 and 2.58 Å3 Da−1 for PLproWT and PLproC111S, respectively, indicating solvent contents of 54% and 52% in the crystals.
![[Figure 1]](nq5003fig1thm.gif) | Figure 1 (a) SEC elution purification profile of PLproWT showing a single peak corresponding to the monomeric size and SDS–PAGE confirming the purity of the target protein. (b) Ribbon-diagram representation of the asymmetric unit containing two protomers. Protomer A is represented in orange and protomer B in blue. The thumb, finger, palm and Ubl2 domains are marked. (c) Cartoon-diagram representation of differences between protomer A and B [using the same colour scheme as shown in (b)] of the asymmetric unit, showing the differences in two different insets for the zinc-finger and the Ubl2 domain (from left to right). (d) Electron density of the protomer A zinc finger showing the coordination of the ion by the four cysteine residues and an inset showing the density around the zinc ion present at a contour level of 10 r.m.s.d.. (e) Interactions between the protomer A zinc-finger and protomer B Ubl2 domain. Amino acids involved in hydrogen bonds are labelled and the hydrogen bonds are marked as black dashed lines. |
Unless specified, all descriptions given here are for the PLproWT crystals. The two protomers in the are organized in an almost dimeric arrangement (Fig. 1b). For protomer A, residues 3–322 were modelled into the electron density, while protomer B showed clearly interpretable electron density for residues 1–315 of the total 322 residues. Globally, the two protomers show almost identical structures (r.m.s.d. of 0.36 Å for all Cα atoms in common). However, local differences are visible, especially in the two functionally important regions known for their dynamic nature: the Ubl2 domain and the zinc-binding loop (Frieman et al., 2009; Bosken et al., 2020; Báez-Santos et al., 2015).
3.2. Open and closed conformations of the Ubl2 domain
The Ubl2 domain is a ubiquitous domain associated with all coronavirus PLpro proteins and has been shown to be essential for the stability and catalytic efficiency of PLpro in SARS-CoV-1, SARS-CoV-2 and murine hepatitis virus (Frieman et al., 2009; Mielech et al., 2015; Arya et al., 2025). It is a small domain comprising approximately 60 amino acids attached to the ridge helix (residues 61–68) of the thumb domain of PLpro. In both the PLproWT and PLproC111S structures reported here, we observe distinct conformations of the Ubl2 domain in the two protomers. The Ubl2 domain of protomer B has undergone a rotation and is in a different position relative to the `ridge' helix (Asp62–His73) compared with protomer A (Fig. 1c, Ubl2 inset). This rotation places the Ubl2 domain of chain B spatially further away from the PLpro domain, defining what we refer as the `open' conformation. A maximum displacement of 3.9 Å is observed between the two protomers at around residue Ala39 (Cα), highlighting a notable structural rearrangement that distinguishes protomer B from protomer A. This positioning of the Ubl2 domain in protomer B cannot be attributed to crystal packing as the Ubl2 domain does not engage in lattice interactions, except for the N-terminal tip, which contacts a short loop (residues 293–297) of a symmetry-related molecule (Supplementary Fig. S3). Moreover, the Ubl2 residues of this monomer show higher B-factor values than those of protomer A (Fig. 2 and Supplementary Fig. S4). To investigate flexibility in the solution state, SAXS measurements were taken. The SAXS results from PLproC111S show a monomeric globular protein with a (Rg) of 24.98 Å and a maximum dimension (Dmax) of 98 Å (Supplementary Fig. S5). To explore any flexibility, the BilboMD software (Pelikan et al., 2009) was used to create an ensemble of molecular models that conformationally sample the molecular space. An initial fit to the monomeric PLproC111S structure was taken (χ2 = 2.33), the linker region between the Ubl2 domain was then allowed freedom to move and the resulting ensemble of models generated indicate that the Ubl2 domain does have movement about the linker region (Supplementary Fig. S6); a three-state model gave the best fit (χ2 = 1.40). Thus, this supports the proposal that localized flexibility may arise from the Ubl2 domain about a relatively rigid PLPro core domain. Importantly, the SAXS data are well described by monomeric models, supporting that the observed flexibility in the Ubl2 domain reflects intrinsic conformational variability rather than intermolecular interactions as in the crystal.
![[Figure 2]](nq5003fig2thm.gif){kind=small} | Figure 2 Representation of B factors in a rainbow colour spectrum (ranging from 10 to 150 Å2) of all three structures for protomer A and protomer B. Higher B factors can be observed for protomer B zinc-finger regions, especially for the PLproWT_`aged' structure that has no zinc bound. |
The conformation observed in protomer A, which we refer to as `closed', has been commonly observed, with slight variations, in several SARS-CoV-1 and SARS-CoV-2 PLpro structures deposited in the Protein Data Bank (PDB entry 2fe8, Ratia et al., 2006; PDB entry 7nfv, Srinivasan et al., 2022). Both Ubl2 conformations seem to exhibit hydrogen-bond interactions with PLpro. In protomer A (the closed state), hydrogen bonds are observed between His17 and Glu67 (3.1 Å) and between Asp37 and Lys91 (2.8 Å). In contrast, the open conformation in protomer B maintains the hydrogen bond between His17 and Glu67 (2.8 Å) but lacks the interaction between Asp37 and Lys91. Instead, a new hydrogen bond forms between Gly38 and Lys91 (3.3 Å). In SARS-CoV-1 (for example PDB entry 5y3e), equivalent hydrogen bonds occur between His17 and Glu67 (2.8 Å) and Asp37 and Lys91 (2.8 Å), as observed in protomer A (Lin et al., 2018). The presence of only two hydrogen bonds between the Ubl2 and catalytic domains of PLpro may be considered weaker but is perhaps necessary to accommodate the conformational flexibility of the Ubl2 domain (Bosken et al., 2020; Báez-Santos et al., 2015). Nevertheless, in both conformations residues involved in SUb2, which is located proximal to the Ubl2 domain, appear to be structurally conserved, suggesting that the open conformation is unlikely to impact substrate binding at this site.
The `closed' conformation is prevalent across all known SARS-CoV-2 PLpro apo structures (13 in total; PDB entries 6w9c, 6wrh, 6wzu, 6xg3, 7d47, 7d6h, 7d7k, 7cjd, 7nfv, 7ybg, 8fwn, 8fwo and 8vec), whether crystallized as monomers or as dimers. However, to our knowledge, the `open' conformation observed in our structure has not been reported in any of these structures. Interestingly, one SARS-CoV-2 PLpro structure (PDB entry 7d7k; Zhao et al., 2021), crystallized in space group P6522 with two molecules in the asymmetric unit, reveals one protomer in the `closed' conformation and the other in an intermediate state positioned between the `open' conformation observed in our protomer B and the `closed' conformation (Supplementary Fig. S7). The observation of these conformations across different space groups and different protomer–protomer packings reduces the likelihood that the observation is linked to crystal-packing artefacts and confirms the existing notion of flexibility within the Ubl2 domain (Bosken et al., 2020).
3.3. Flexibility of the zinc-binding loop
Next, we analysed the flexibility of the zinc-binding loop and its correlation to zinc occupancy in both protomers. Interestingly, protomer A of PLproWT has a fully occupied zinc ion coordinated by four conserved cysteine residues, Cys189, Cys192, Cys224 and Cys226 (Fig. 1d and inset). The electron density for the zinc ion is clearly visible at a contour level exceeding 12 r.m.s.d., with a corresponding B factor of 28 Å2. The surrounding loop residues in protomer A exhibit defined electron density and low B factors (for example, the B factor of Cys189 is 29 Å2; Table 5). In contrast, protomer B shows only weak electron density for the zinc ion (disappearing at contour levels of >3 r.m.s.d.) with a significantly higher B factor of 110 Å2. The associated zinc-binding loop residues in protomer B also display elevated B factors (see Table 5). A similar situation is observed in the PLproC111S structure, where the B factors of these residues in protomers A and B remain comparable to those of PLproWT. In our hands, the removal of bound zinc by EDTA in the purification buffer resulted in protein precipitation, while addition of zinc to the cell culture and the purification buffer increased the protein yield (data not shown). These observations support the notion that zinc binding to PLpro is essential for stabilizing the flexibility of the zinc-binding loop and thereby maintaining the structural integrity of the protein (Jiang et al., 2022).
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
We extended this analysis to explore whether zinc-ion occupancy and zinc-binding loop stability contribute to the conformational variability observed in the Ubl2 domain in the In our structures, both protomers are arranged such that the N-terminal region of one protomer is positioned near the C-terminal region of the other, although not symmetrically, forming a configuration reminiscent of a `yin–yang' pattern. This arrangement positions the zinc-binding loop of protomer A to interact with the Ubl2 domain of protomer B (Fig. 1e). Specifically, there are hydrogen bonds from the main-chain O atom of Thr191 (in the zinc-binding loop) in protomer A to the OG1 atom of Thr26 and the NE2 atom of Gln29 in protomer B. Additionally, the main-chain O atom of Lys190 (in the zinc-binding loop) in protomer A forms a hydrogen bond to the NE2 atom of Gln29 in protomer B. In contrast, the zinc-binding loop of protomer B does not form any interactions with the Ubl2 domain of protomer A. This suggests that the interaction with a stable zinc-binding loop could lock the Ubl2 domain in this open conformation, however in a specific environment such as our crystal packing. This raised the question: would removal of the zinc ion from protomer A weaken its interaction with the Ubl2 domain of protomer B, thereby inducing it to adopt a `closed' conformation? To answer this question, we examined PLproWT crystals that were 7–8 months old, based on the fact that zinc binding to proteins is pH-dependent (Kluska et al., 2018). Over time, we noticed that the pH of our crystallization drops shifted from basic to acidic (as measured with pH paper). To assess the presence of zinc within these `aged' crystals, we performed X-ray anomalous scattering experiments slightly above the zinc absorption edge (9.7 keV), which showed no measurable anomalous signal at the zinc-binding site. Diffraction data collected from several PLproWT_'aged' crystals showed weak electron density at the zinc-binding site and ill-defined electron density for zinc-binding-loop residues, with elevated B factors for both protomers (Fig. 2 and Supplementary Figs. S4, S8 and S9). As anticipated, the Ubl2 domain of protomer B was no longer in an `open' conformation in this structure, but adopted an `intermediate' conformation between the open and the closed states (Supplementary Fig. S8). Interestingly, the crystals remained intact despite a minor reduction in the resolution obtained, implying that they can tolerate the conformational variability of Ubl2.
While this is the case within the asymmetric unit arrangement, a detailed analysis of B factors within each protomer reveals a trend; elevated B factors of the zinc-binding loop residues correlate with higher B factors in the Ubl2 domain of the same monomer. This is also confirmed across several existing structures (Supplementary Figs. S4 and S10). The potential allosteric link between zinc-binding loop flexibility and Ubl2 conformational variability, along with the possibility that zinc ion binding regulates this variability, warrant further investigation.
3.4. Intrinsic conformational variability of Ubl2 in PLpro
To decipher whether the open form could be one of the physiologically relevant states of PLpro, we expanded our search to PLpro structures from other coronaviruses. Our analyses showed that several MERS-CoV PLpro structures (PDB entries 5v69, 4rna, 4pt5, 4p16, 4rez and 4r3d) have a conformation similar to our `open' conformation (Supplementary Figs. S1 and S11), highlighting the possibility that the `open' conformation represents an intrinsic state of PLpro and may be relevant to consider in future inhibitor studies. This is the first time that this conformation has been characterized from crystallographic analyses for SARS-CoV-1 and SARS-CoV-2. Further supporting the physiological relevance of Ubl2 flexibility, a recent cryo-EM structure of the NSP3–NSP4 pore complex manifested an altogether different conformation for Ubl2, differing from the reported open or closed states, which exhibited unanticipated interactions with C-terminal domains of NSP3 (Y1-CoVG; Huang et al., 2024). While the crystal and cryo-EM structures provide valuable snapshots of Ubl2 conformational variability, they do not capture its full dynamic behaviour. Additional approaches such as molecular-dynamics simulations could further explore the conformational landscape of the Ubl2 domain, particularly in relation to ligand and substrate binding and its potential allosteric coupling to the zinc-binding site.
Together, these findings show the essential nature of Ubl2 flexibility in the structural and functional integrity of the papain-like protease of SARS-CoV-2. We believe that this intrinsic flexibility of the Ubl2 domain of PLpro, which may open up a pocket for potential inhibitors, could be exploited in hit- and lead-finding campaigns to identify starting points for antiviral drug development.
4. Related literature
The following reference is cited in the supporting information for this article: Robert et al. (2025).
Supporting information
Supplementary Figures. DOI: https://doi.org/10.1107/S2053230X26003699/nq5003sup1.pdf
Acknowledgements
We acknowledge the European Synchrotron Radiation Facility (ESRF) for the provision of beamtime on ID30B and BM29 through the in-house research programme. This work used the platforms of the Grenoble Instruct-ERIC center (ISBG; UAR 3518 CNRS–CEA–UGA–EMBL) within the Grenoble Partnership for Structural Biology (PSB), supported by FRISBI (ANR-10-INBS-0005-02) and GRAL, financed within the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH–EUR–GS (ANR-17-EURE-0003). We thank Samira Acajjaoui and Melissa Saidi for training and assistance in the biochemistry laboratory. We thank Aline Le Roy for assistance and access to the analytical ultracentrifugation (AUC) platform.
Conflict of interest
MS and RCH are employees of Nuvisan ICB GmbH.
Data availability
The atomic coordinates and structure-factor amplitudes of the reported structures have been deposited in the Protein Data Bank under accession codes 9hhg for PLproWT, 9hhh for PLproC111S and 9hhi for PLproWT_'aged'.
Funding information
EK acknowledges InnovaXN (European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 847439) for supporting a three-year PhD fellowship (InnovaXN_8_2020). Open access publication funding provided by COUPERIN CY26.
References
Agarwal, R. C. (1978). Acta Cryst. A34, 791–809. CrossRef CAS IUCr Journals Web of Science Google Scholar
Agirre, J., Atanasova, M., Bagdonas, H., Ballard, C. B., Baslé, A., Beilsten-Edmands, J., Borges, R. J., Brown, D. G., Burgos-Mármol, J. J., Berrisford, J. M., Bond, P. S., Caballero, I., Catapano, L., Chojnowski, G., Cook, A. G., Cowtan, K. D., Croll, T. I., Debreczeni, J. É., Devenish, N. E., Dodson, E. J., Drevon, T. R., Emsley, P., Evans, G., Evans, P. R., Fando, M., Foadi, J., Fuentes-Montero, L., Garman, E. F., Gerstel, M., Gildea, R. J., Hatti, K., Hekkelman, M. L., Heuser, P., Hoh, S. W., Hough, M. A., Jenkins, H. T., Jiménez, E., Joosten, R. P., Keegan, R. M., Keep, N., Krissinel, E. B., Kolenko, P., Kovalevskiy, O., Lamzin, V. S., Lawson, D. M., Lebedev, A. A., Leslie, A. G. W., Lohkamp, B., Long, F., Malý, M., McCoy, A. J., McNicholas, S. J., Medina, A., Millán, C., Murray, J. W., Murshudov, G. N., Nicholls, R. A., Noble, M. E. M., Oeffner, R., Pannu, N. S., Parkhurst, J. M., Pearce, N., Pereira, J., Perrakis, A., Powell, H. R., Read, R. J., Rigden, D. J., Rochira, W., Sammito, M., Sánchez Rodríguez, F., Sheldrick, G. M., Shelley, K. L., Simkovic, F., Simpkin, A. J., Skubak, P., Sobolev, E., Steiner, R. A., Stevenson, K., Tews, I., Thomas, J. M. H., Thorn, A., Valls, J. T., Uski, V., Usón, I., Vagin, A., Velankar, S., Vollmar, M., Walden, H., Waterman, D., Wilson, K. S., Winn, M. D., Winter, G., Wojdyr, M. & Yamashita, K. (2023). Acta Cryst. D79, 449–461. Web of Science CrossRef IUCr Journals Google Scholar
Arya, R., Ganesh, J., Prashar, V. & Kumar, M. (2025). Biol. Direct, 20, 102. CrossRef PubMed Google Scholar
Báez-Santos, Y. M., St John, S. E. & Mesecar, A. D. (2015). Antiviral Res. 115, 21–38. Web of Science PubMed Google Scholar
Bailey-Elkin, B. A., Knaap, R. C. M., Johnson, G. G., Dalebout, T. J., Ninaber, D. K., van Kasteren, P. B., Bredenbeek, P. J., Snijder, E. J., Kikkert, M. & Mark, B. L. (2014). J. Biol. Chem. 289, 34667–34682. PubMed Google Scholar
Barretto, N., Jukneliene, D., Ratia, K., Chen, Z., Mesecar, A. D. & Baker, S. C. (2005). J. Virol. 79, 15189–15198. Web of Science CrossRef PubMed CAS Google Scholar
Bosken, Y. K., Cholko, T., Lou, Y.-C., Wu, K.-P. & Chang, C. A. (2020). Front. Mol. Biosci. 7, 174. CrossRef PubMed Google Scholar
Chan, J. F.-W., Yuan, S., Chu, H., Sridhar, S. & Yuen, K.-Y. (2024). Nat. Rev. Microbiol. 22, 391–407. CrossRef CAS PubMed Google Scholar
Clasman, J. R., Everett, R. K., Srinivasan, K. & Mesecar, A. D. (2020). Antiviral Res. 174, 104661. CrossRef PubMed Google Scholar
Dable-Tupas, G. & Derecho, C. M. P. (2022). Coronavirus Drug Discovery, edited by C. Egbuna, pp. 181–203. Amsterdam: Elsevier. Google Scholar
Dimasi, N., Flot, D., Dupeux, F. & Márquez, J. A. (2007). Acta Cryst. F63, 204–208. Web of Science CrossRef CAS IUCr Journals Google Scholar
Du, S., Hu, X., Li, P., Xu, S., Kim, M., Liu, X. & Zhan, P. (2026). Sig. Transduct. Target. Ther. 11, 69. CrossRef Google Scholar
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. (2010). Acta Cryst. D66, 486–501. Web of Science CrossRef CAS IUCr Journals Google Scholar
Frieman, M., Ratia, K., Johnston, R. E., Mesecar, A. D. & Baric, R. S. (2009). J. Virol. 83, 6689–6705. Web of Science CrossRef PubMed CAS Google Scholar
Gao, X., Qin, B., Chen, P., Zhu, K., Hou, P., Wojdyla, J. A., Wang, M. & Cui, S. (2021). Acta Pharm. Sin. B, 11, 237–245. CrossRef PubMed Google Scholar
Gao, Y. & Zhang, J. (2025). Acta Pharm. Sin. B, 15, 4497–4510. CrossRef CAS PubMed Google Scholar
Huang, Y., Wang, T., Zhong, L., Zhang, W., Zhang, Y., Yu, X., Yuan, S. & Ni, T. (2024). Nature, 633, 224–231. CrossRef CAS PubMed Google Scholar
Immirzi, A. (1966). Crystallographic Computing Techniques, edited by F. R. Ahmed, p. 399. Copenhagen: Munksgaard. Google Scholar
Jiang, H., Yang, P. & Zhang, J. (2022). Front. Chem. 10, 822785. CrossRef PubMed Google Scholar
Kieffer, J., Brennich, M., Florial, J.-B., Oscarsson, M., De Maria Antolinos, A., Tully, M. & Pernot, P. (2022). J. Synchrotron Rad. 29, 1318–1328. Web of Science CrossRef IUCr Journals Google Scholar
Kluska, K., Adamczyk, J. & Krężel, A. (2018). Coord. Chem. Rev. 367, 18–64. CrossRef CAS Google Scholar
Lei, J., Kusov, Y. & Hilgenfeld, R. (2018). Antiviral Res. 149, 58–74. Web of Science CrossRef CAS PubMed Google Scholar
Liebschner, D., Afonine, P. V., Baker, M. L., Bunkóczi, G., Chen, V. B., Croll, T. I., Hintze, B., Hung, L.-W., Jain, S., McCoy, A. J., Moriarty, N. W., Oeffner, R. D., Poon, B. K., Prisant, M. G., Read, R. J., Richardson, J. S., Richardson, D. C., Sammito, M. D., Sobolev, O. V., Stockwell, D. H., Terwilliger, T. C., Urzhumtsev, A. G., Videau, L. L., Williams, C. J. & Adams, P. D. (2019). Acta Cryst. D75, 861–877. Web of Science CrossRef IUCr Journals Google Scholar
Lin, M.-H., Moses, D. C., Hsieh, C.-H., Cheng, S.-C., Chen, Y.-H., Sun, C.-Y. & Chou, C.-Y. (2018). Antiviral Res. 150, 155–163. CrossRef CAS PubMed Google Scholar
Márquez, J. A. & Cipriani, F. (2014). Structural Genomics: General Applications, edited by Y. W. Chen, pp. 197–203. Totowa: Humana Press. Google Scholar
McCarthy, A. A., Barrett, R., Beteva, A., Caserotto, H., Dobias, F., Felisaz, F., Giraud, T., Guijarro, M., Janocha, R., Khadrouche, A., Lentini, M., Leonard, G. A., Lopez Marrero, M., Malbet-Monaco, S., McSweeney, S., Nurizzo, D., Papp, G., Rossi, C., Sinoir, J., Sorez, C., Surr, J., Svensson, O., Zander, U., Cipriani, F., Theveneau, P. & Mueller-Dieckmann, C. (2018). J. Synchrotron Rad. 25, 1249–1260. Web of Science CrossRef CAS IUCr Journals Google Scholar
Mielech, A. M., Deng, X., Chen, Y., Kindler, E., Wheeler, D. L., Mesecar, A. D., Thiel, V., Perlman, S. & Baker, S. C. (2015). J. Virol. 89, 4907–4917. Web of Science CrossRef CAS PubMed Google Scholar
Oktavianawati, I., Santoso, M., Bakar, M. F. A., Kim, Y.-U. & Fatmawati, S. (2023). Appl. Biol. Chem. 66, 89. CrossRef Google Scholar
Osipiuk, J., Azizi, S.-A., Dvorkin, S., Endres, M., Jedrzejczak, R., Jones, K. A., Kang, S., Kathayat, R. S., Kim, Y., Lisnyak, V. G., Maki, S. L., Nicolaescu, V., Taylor, C. A., Tesar, C., Zhang, Y.-A., Zhou, Z., Randall, G., Michalska, K., Snyder, S. A., Dickinson, B. C. & Joachimiak, A. (2021). Nat. Commun. 12, 743. Web of Science CrossRef PubMed Google Scholar
Pelikan, M., Hura, G. L. & Hammel, M. (2009). Gen. Physiol. Biophys. 28, 174–189. Web of Science CrossRef PubMed CAS Google Scholar
Potterton, E., Briggs, P., Turkenburg, M. & Dodson, E. (2003). Acta Cryst. D59, 1131–1137. Web of Science CrossRef CAS IUCr Journals Google Scholar
Ratia, K., Saikatendu, K. S., Santarsiero, B. D., Barretto, N., Baker, S. C., Stevens, R. C. & Mesecar, A. D. (2006). Proc. Natl Acad. Sci. USA, 103, 5717–5722. Web of Science CrossRef PubMed CAS Google Scholar
Read, R. J. & Schierbeek, A. J. (1988). J. Appl. Cryst. 21, 490–495. CrossRef CAS Web of Science IUCr Journals Google Scholar
Robert, X., Guillon, C. & Gouet, P. (2025). Nucleic Acids Res. 53, W277–W282. CrossRef PubMed Google Scholar
Rut, W., Lv, Z., Zmudzinski, M., Patchett, S., Nayak, D., Snipas, S. J., El Oualid, F., Huang, T. T., Bekes, M., Drag, M. & Olsen, S. K. (2020). Sci. Adv. 6, eabd4596. CrossRef PubMed Google Scholar
Srinivasan, V., Brognaro, H., Prabhu, P. R., de Souza, E. E., Günther, S., Reinke, P. Y. A., Lane, T. J., Ginn, H., Han, H., Ewert, W., Sprenger, J., Koua, F. H. M., Falke, S., Werner, N., Andaleeb, H., Ullah, N., Franca, B. A., Wang, M., Barra, A. L. C., Perbandt, M., Schwinzer, M., Schmidt, C., Brings, L., Lorenzen, K., Schubert, R., Machado, R. R. G., Candido, E. D., Oliveira, D. B. L., Durigon, E. L., Niebling, S., Garcia, A. S., Yefanov, O., Lieske, J., Gelisio, L., Domaracky, M., Middendorf, P., Groessler, M., Trost, F., Galchenkova, M., Mashhour, A. R., Saouane, S., Hakanpää, J., Wolf, M., Alai, M. G., Turk, D., Pearson, A. R., Chapman, H. N., Hinrichs, W., Wrenger, C., Meents, A. & Betzel, C. (2022). Commun. Biol. 5, 805. CrossRef PubMed Google Scholar
Ten Eyck, L. F. (1973). Acta Cryst. A29, 183–191. CrossRef CAS IUCr Journals Web of Science Google Scholar
Tully, M. D., Kieffer, J., Brennich, M. E., Cohen Aberdam, R., Florial, J. B., Hutin, S., Oscarsson, M., Beteva, A., Popov, A., Moussaoui, D., Theveneau, P., Papp, G., Gigmes, J., Cipriani, F., McCarthy, A., Zubieta, C., Mueller-Dieckmann, C., Leonard, G. & Pernot, P. (2023). J. Synchrotron Rad. 30, 258–266. Web of Science CrossRef IUCr Journals Google Scholar
Tully, M. D., Tarbouriech, N., Rambo, R. P. & Hutin, S. (2021). J. Vis. Exp., e61578. Google Scholar
Vonrhein, C., Flensburg, C., Keller, P., Fogh, R., Sharff, A., Tickle, I. J. & Bricogne, G. (2024). Acta Cryst. D80, 148–158. Web of Science CrossRef IUCr Journals Google Scholar
Vonrhein, C., Flensburg, C., Keller, P., Sharff, A., Smart, O., Paciorek, W., Womack, T. & Bricogne, G. (2011). Acta Cryst. D67, 293–302. Web of Science CrossRef CAS IUCr Journals Google Scholar
von Soosten, L. C., Edich, M., Nolte, K., Kaub, J., Santoni, G. & Thorn, A. (2022). Crystallogr. Rev. 28, 39–61. CrossRef CAS Google Scholar
Wolff, G., Limpens, R. W. A. L., Zevenhoven-Dobbe, J. C., Laugks, U., Zheng, S., de Jong, A. W. M., Koning, R. I., Agard, D. A., Grünewald, K., Koster, A. J., Snijder, E. J. & Bárcena, M. (2020). Science, 369, 1395–1398. Web of Science CrossRef CAS PubMed Google Scholar
Wolff, G., Melia, C. E., Snijder, E. J. & Bárcena, M. (2020). Trends Microbiol. 28, 1022–1033. CrossRef CAS PubMed Google Scholar
World Health Organization (2026). COVID-19 Deaths|WHO COVID-19 Dashboard. Geneva: World Health Organization. https://data.who.int/dashboards/covid19/cases. Google Scholar
Zhao, Y., Du, X., Duan, Y., Pan, X., Sun, Y., You, T., Han, L., Jin, Z., Shang, W., Yu, J., Guo, H., Liu, Q., Wu, Y., Peng, C., Wang, J., Zhu, C., Yang, X., Yang, K., Lei, Y., Guddat, L. W., Xu, W., Xiao, G., Sun, L., Zhang, L., Rao, Z. & Yang, H. (2021). Protein Cell, 12, 877–888. CrossRef CAS PubMed Google Scholar
This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.
| STRUCTURAL BIOLOGY COMMUNICATIONS |
