Structural and functional insights into Ubl domain-mediated regulation of SARS-CoV-2 PLpro

Abstract

Background

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SARS-CoV-2 papain-like protease (PLpro) is essential for viral replication and immune evasion. It contains an N-terminal ubiquitin-like (Ubl) domain, whose involvement in enzymatic function remains poorly understood.

Results

In this study, we investigated the role of the Ubl domain in modulating the structural dynamics and catalytic efficiency of PLpro. Using molecular dynamics (MD) simulations, inhibitor binding assays, and steady-state kinetic analyses, we found that the Ubl domain stabilizes critical structural elements, notably the ridge helix in the thumb subdomain. Removal of the Ubl domain altered substrate processing, reducing catalytic efficiency of the enzyme. Interestingly, free ubiquitin enhanced enzymatic activity, likely via non-canonical binding sites distinct from the SUb1 and SUb2 sites.

Conclusion

These findings uncover a regulatory role for the Ubl domain in allosteric modulation of PLpro activity and reveal additional layers of enzymatic plasticity. Understanding these mechanisms could guide the design of future antiviral therapeutics targeting PLpro’s regulatory or allosteric sites.

Introduction

SARS-CoV-2, the causative agent of the COVID-19, relies on various structural, non-structural and accessory proteins for its replication and pathogenicity. Among the non-structural proteins, Nsp3 is the largest and the most complex protein, comprising multiple functional domains, including the papain-like protease (PLpro) domain that hydrolyzes peptide and isopeptide bonds of viral and cellular substrates. PLpro cleaves viral polyproteins to release Nsp1, Nsp2, and Nsp3 which is essential for viral replication [1, 2]. In addition, PLpro removes ubiquitin (Ub) and interferon-stimulated gene product 15 (ISG15) from the cellular protein substrates- a process known as deubiquitination (DUB) and deISGylation, respectively [2,3,4]. By acting as both a deubiquitinase and a deISGylase, PLpro enables SARS-CoV-2 to evade host immune responses. It impairs the interferon signalling pathway by removing Ub and ISG15 from key signalling proteins, thereby disrupting innate immunity [5,6,7]. Furthermore, PLpro-mediated deISGylation of the viral nucleocapsid protein enhances viral replication [8].

SARS-CoV-2 PLpro comprises an N-terminal ubiquitin-like (Ubl) domain (residues 1–60) followed by a catalytic core domain (residues 61–314). While the function of catalytic domain is well-characterized, the role of Ubl domain in catalysis remains largely unexplored. The Ubl domain shares ~ 17% sequence identity with the ubiquitin and structural studies, including cryo-EM analysis of full-length Nsp3 [9], show that the Ubl domain is spatially separated from other functional domains of Nsp3. Moreover, crystal structures of PLpro bound to ubiquitin (PDB ID: 7RBR, 5E6J and 7UV5) and ISG15 (PDB ID: 7RBS, 6XA9 and 6YVA) reveal no direct interactions between the Ubl domain and these substrates [4, 10,11,12]. Interestingly, certain PLpro inhibitors have been observed to bind at the interface between the Ubl and catalytic domains [13], raising questions about how ligand binding at distal sites can modulate enzymatic activity. A set of engineered ubiquitin variants (UbVs) were also shown to bind at a distal allosteric site on PLpro, potently inhibiting its enzymatic activity and reducing viral replication in cell culture [14]. Further, a single mutation near the Ubl domain (F69S) was shown to impair hydrolysis of K48-linked diUb-TAMRA and reduce cleavage efficiency of Ub‐TAMRA substrate [12] suggesting a possible regulatory role for the Ubl domain in catalysis. It was also shown recently that the catalytic cysteine (C111) of SARS-CoV-2 PLpro is essential for suppressing NF-κB signaling, while its Ubl domain is dispensable for interferon antagonism [5].

Previous studies on MERS-CoV PLpro suggest that its Ubl domain is dispensable for proteolytic, deubiquitinating or deISGylating functions [15, 16]. In contrast, deletion of Ubl domain in SARS-CoV PLpro leads to protein instability and a loss of enzymatic activity over time, indicating that this domain is critical for maintaining structural integrity in vitro [17]. The Ubl domain of SARS-CoV-2 PLpro shares 88% sequence identity and high structural similarity with SARS-CoV counterpart. In SARS-CoV-2, the significance of Ubl domain in proteolytic activity, substrate interaction, and inhibitor binding remains unclear.

Interestingly, parallels can be drawn from ubiquitin-specific proteases (USPs), a major subclass of deubiquitinases (DUBs), given their involvement in diverse processes such as ubiquitin homeostasis and protein regulation [18, 19]. Ubiquitin-like (Ubl) domains within USPs play pivotal roles in modulating their enzymatic activity. For example, the Ubl domain of USP14 promotes its association with the proteasome, thereby enhancing its catalytic performance [20, 21]. In contrast, USP4’s Ubl domain functions as an autoinhibitory element, mimicking ubiquitin to suppress activity until it is removed or masked [22]. USP7 contains five Ubl domains, with the last two enhancing catalysis allosterically by inducing favorable conformational changes [23]. These cases illustrate the diverse mechanisms through which Ubl domains regulate DUB activity ranging from inhibition to allosteric activation ensuring precise control over their cellular functions.

In this study, we investigate the structural and functional significance of the Ubl domain in SARS-CoV-2 PLpro. By characterizing a truncated variant lacking the Ubl domain (PLpro TR), we examine its effects on enzymatic activity, stability, and substrate/inhibitor interactions. Our findings reveal that the Ubl domain plays a critical role in maintaining the catalytic efficiency and structural integrity of PLpro, providing new mechanistic insights into its regulatory function.

Materials and methods

Cloning, expression, and purification of PLpro WT and TR

The gene encoding wild-type (WT) SARS-CoV-2 PLpro was available in our laboratory [24]. Genes for truncated version of PLpro (TR) lacking the N-terminal 60 residues, human ISG15 and Ubiquitin (based on NCBI reference sequences NP_005092.1 & 1UBQ_A, respectively) were codon-optimized and synthesized for expression in Escherichia coli. All constructs were cloned into the pET28a vector (Novagen) using NdeI and BamHI restriction sites. Each included an N-terminal hexahistidine tag for affinity purification. A thrombin cleavage site was introduced downstream of the His-tag for PLpro and ISG15 constructs, while a TEV protease site was used for the Ubiquitin construct. Recombinant plasmids were transformed into E. coli BL21 (DE3) cells. Cultures were grown in Luria-Bertani (LB) medium at 37 °C until the optical density at 600 nm (OD₆₀₀) reached 0.8–1.0. Protein expression was induced by adding IPTG to a final concentration of 0.5 mM, followed by overnight incubation at 18 °C. Cells were harvested by centrifugation at 6000 Xg, resuspended in lysis buffer (50 mM Tris-HCl, pH 7.5; 300 mM NaCl; 10 mM imidazole; 10 mM β-mercaptoethanol; 1 mg/mL lysozyme), and lysed by sonication. The lysate was clarified by centrifugation at 10,000 Xg for 30 min at 4 °C. The resulting supernatant was incubated with Ni-NTA agarose resin (Roche) for 90 min at 4 °C with gentle rocking. The resin was washed twice with wash buffer (50 mM Tris-HCl, pH 7.5; 300 mM NaCl; 20 mM imidazole; 10 mM β-mercaptoethanol), and the bound protein was eluted using elution buffer (50 mM Tris-HCl, pH 7.5; 300 mM NaCl; 250 mM imidazole; 10 mM β-mercaptoethanol). Eluted proteins were dialyzed overnight at 4 °C against dialysis buffer (30 mM Tris-HCl, pH 7.5; 150 mM NaCl; 5 mM DTT).

Enzymatic assays

All enzymatic assays were performed in black, flat-bottom, 96-well, non-treated plates (Corning, USA) using a CLARIOstar Plus multi-mode microplate reader (BMG Labtech, Germany). Fluorescence measurements were recorded with an excitation wavelength of 355 nm and an emission wavelength of 430 nm. Reactions were carried out in assay buffer containing 50 mM MES, pH 6.5; 100 mM NaCl; 0.5 mM EDTA; 5 mM DTT and 0.1 mg/mL BSA [24, 25]. Three fluorogenic substrates were used to evaluate the catalytic activity of wild-type (WT) and truncated (TR) PLpro: Z-RLRGG-AMC (0–700 µM), Ub-AMC (0–2.33 µM) and ISG15-AMC (0-0.625 µM). All kinetic parameters were determined under identical assay conditions for each enzyme-substrate pair. Enzyme concentrations were optimized for each substrate: 50 nM for Z-RLRGG-AMC, 10 nM for Ub-AMC, and 5 nM for ISG15-AMC. For IC₅₀ determinations, assays were conducted with 50 nM enzyme and 40 µM Z-RLRGG-AMC, using GRL0617 and ISG15 at concentrations ranging from 0 to 100 µM, and Ubiquitin from 0 to 200 µM. To evaluate deubiquitination activity in the presence of free Ubiquitin, reactions included 0.25 µM Ub-AMC, 10 nM enzyme, and 0–20 µM Ubiquitin. For deISGylation assays, 0.15 µM ISG15-AMC and 5 nM enzyme were used, with Ubiquitin concentrations ranging from 0 to 20 µM. All IC₅₀ values were determined by non-linear regression analysis of initial velocity data using GraphPad Prism (www.graphpad.com). The K_m/V_max values were obtained from the slope of the Lineweaver–Burk plots (1/[V] vs. 1/[S]) via linear regression analysis and individual kinetic parameters (k_cat and K_m) were estimated using non-linear regression analysis using GraphPad Prism.

Differential scanning fluorimetry (DSF)

DSF assays were conducted using an Analytik Jena qTOWER³G real-time PCR instrument. Each 25 µL reaction was prepared in a 96-well low-profile clear plate and contained 2.5–5 µM protein, 5X SYPRO Orange dye, and buffer composed of 30 mM Tris (pH 7.5), 150 mM NaCl, and 5 mM DTT. The concentration of GRL0617 prepared in the same buffer was varied from 0 to 100 µM for inhibition studies with PLpro WT and TR. Samples were incubated at 20 °C for 10 min before initiating the thermal scan. The temperature was increased from 20 °C to 80 °C at a rate of 5 °C per minute, with fluorescence measurements taken at 0.5 °C intervals. Background fluorescence was corrected by subtracting values obtained from control wells lacking protein.

Dynamic light scattering (DLS)

DLS measurements were performed using a Zetasizer Nano-ZS instrument (Malvern Panalytical Ltd., UK). Purified protein was prepared at a concentration of 2 mg/mL in buffer containing 30 mM Tris, pH 7.5; 150 mM NaCl and 5 mM DTT. Measurements were conducted in low-volume disposable sizing cuvettes (ZEN0112, Malvern). Samples were equilibrated at 10 °C for 30 s prior to analysis. Each measurement consisted of three replicates, with each replicate comprising 15 runs of 10 s. Light scattering was detected at a backscattering angle of 173°.

Crystal structure determination

Crystallization of wild-type (WT) PLpro was carried out using the hanging-drop vapor diffusion method. The protein was prepared in buffer containing 30 mM Tris, pH 7.5; 150 mM NaCl and 10 mM DTT, and concentrated to 8–10 mg/mL. Crystals were grown at 4 °C using a crystallization solution consisting of 0.1 M Tris (pH 8.0); 0.8 M NaH₂PO₄ and 1.2 M K₂HPO₄. X-ray diffraction data were collected at 100 K on Dectris Pilatus3 R 1 M detector mounted on mardtb platform using Excillum MetalJet X-ray source (Ga, Kα radiation, λ = 1.34 Å). Diffraction images were recorded in steps of 0.5° and exposure time of 30 s per frame. Data were indexed, integrated, scaled, and merged using the XDS software suite [26]. Initial phases were obtained by molecular replacement employing the SARS-CoV-2 PLpro structure (PDB ID: 6WRH) as the search model using PhaserMR [27] in the Phenix software suite [28]. The resulting solution was refined through a combination of rigid-body and restrained refinement using phenix.refine. Manual model building and adjustments were performed iteratively using COOT [29], followed by additional rounds of refinement with phenix.refine. During refinement, 5% of the reflections were set aside for cross-validation. A complete summary of the data processing and refinement statistics is provided in the Results section. The atomic coordinates and structure factors have been deposited in the Protein Data Bank (www.rcsb.org) under the PDB ID: 9VAO.

In-silico docking studies using ClusPro

To identify potential ubiquitin binding sites on wild-type papain-like protease (PLpro WT), protein–protein docking was performed using the ClusPro 2.0 web server (https://cluspro.bu.edu) [30, 31]. The prepared 3D structures of PLpro WT (as receptor) and ubiquitin (PDB ID: 3NS8, as ligand) were submitted for docking. The Balanced docking mode was selected to optimize the interplay of electrostatic, van der Waals, and desolvation energies, ensuring biologically relevant complex formation. Docked models were clustered based on root mean square deviation (RMSD) to identify dominant binding poses. The largest clusters were examined to determine the most likely ubiquitin binding sites on PLpro WT.

Molecular dynamics studies

Molecular dynamics (MD) simulations of wild-type PLpro (PLpro WT) and its truncated variant (TR) were performed using GROMACS version 2021.4 [32]. The simulation protocol included system preparation, energy minimization, equilibration, production MD, and post-simulation analysis. The starting structure for PLpro WT was derived from our experimentally determined model (PDB ID: 9VAO). The truncated variant was generated by removing the first 60 N-terminal residues from PLpro WT, and this modified structure served as input for the simulations. All non-standard residues including glycerol (GOL), phosphate ions (PO₄), zinc ions (Zn²⁺), and crystallographic waters, except the ones involved in mediating H-bonding interactions, were removed during system preparation. Topologies were generated using an all-atom OPLS (OPLS-AA/L) force field and water model. Each protein was placed centrally within a cubic simulation box, ensuring at least 1.0 nm distance from the box edges. The systems were solvated with water molecules, and counter-ions were added to neutralize the net charge on the protein by adding the correct number of negative ions. Energy minimization was performed using the steepest descent algorithm until the maximum force on any atom fell below 1000 kJ/mol/nm, yielding a relaxed starting structure. Equilibration was then carried out in two steps: an NVT ensemble to stabilize the temperature at 300 K, followed by an NPT ensemble to equilibrate the pressure at 1 bar. A 100 ns production MD run was subsequently performed under NPT conditions without position restraints. Post-simulation analyses included calculation of root-mean-square deviation (RMSD) for structural stability, root-mean-square fluctuation (RMSF) for residue flexibility, and hydrogen bond analysis, among other structural parameters. Visualization of results were done using Grace (https://plasma-gate.weizmann.ac.il/Grace/) and plotting of results were done using GraphPad Prism software (www.graphpad.com).

Results

Purification, oligomeric state, stability, and aggregation behavior of PLpro TR

Recombinant SARS-CoV-2 PLpro TR (truncated form lacking the Ubl domain) was successfully expressed in E. coli BL21 (DE3) and purified to homogeneity, as confirmed by SDS-PAGE and size-exclusion chromatography (SEC) using Superdex 75 Increase column (Fig. 1A, B). From one liter of bacterial culture, approximately 2 mg of soluble protein was obtained. Initial purification of His-tagged PLpro TR resulted in noticeable protein aggregation. To mitigate this, the His-tag was removed via thrombin cleavage, and all subsequent experiments were conducted using tag-free protein.

SEC analysis showed that PLpro TR eluted primarily as a monomer, similar to the wild-type (WT) protein. PLpro TR displayed a slightly larger elution volume (12 mL) compared to the WT (11 mL), consistent with the smaller size of TR as compared to WT (Fig. 1B). A minor population of higher-order aggregates was also observed in the PLpro TR elution profile.

Thermal stability was evaluated using differential scanning fluorimetry (DSF). The melting temperature (T_m) of PLpro TR was 51.5 °C, closely matching that of the WT protein (52 °C) (Fig. 1C). This indicates that deletion of the Ubl domain does not significantly compromise the thermal stability of the protein. Dynamic light scattering (DLS) experiments confirmed that both PLpro WT and TR preparations were monodispersed with hydrodynamic diameters below 10 nm (Fig. 1D).

Fig. 1

Purification and characterization of SARS-CoV-2 PLpro TR. A 12% SDS-PAGE Lane 1: Eluted protein after Ni-NTA chromatography, Lane 2: Purified protein after His-tag removal, Lane 3: Prestained protein ladder (IRIS11; BR BIOCHEM). The gel was stained with coomassie blue. B Size-exclusion chromatography to determine the oligomeric state of PLpro WT (Black) and PLpro TR (Red). C DSF experiment showing raw thermal isotherm upon heating the PLpro WT (Black) and TR (Red) from 20–80 °C in presence of Sypro Orange dye. D DLS experiments showing particle size distribution at pH 7.5 after thrombin cleavage of PLpro WT (Black) and PLpro TR (Red)

Enzymatic activity of PLpro TR

To evaluate the functional role of the Ubl domain in PLpro enzymatic activity, we performed kinetic assays using three fluorogenic substrates representing its major enzymatic functions: a penta-peptide substrate (Z-RLRGG-AMC), a deISGylation substrate (ISG15-AMC), and a deubiquitination substrate (Ub-AMC). PLpro TR was capable of cleaving all three substrates, indicating that removal of the Ubl domain does not abolish enzymatic activity. However, catalytic efficiency was reduced compared to the WT enzyme (Fig. 2A–C, Supplementary Fig. 2). The kinetic parameters (k_cat, K_m and k_cat/K_m) for each substrate are summarized in Table 1. Although the 95% confidence intervals for k_cat and K_m values are relatively wide, the trend indicates that removal of the Ubl domain leads to a reduction in k_cat, while K_m remains largely unaffected. These results suggest that Ubl domain plays a modulatory role in optimizing PLpro enzymatic efficiency, with a more pronounced effect on peptide and ubiquitin substrates than on ISG15.

Table 1 Michaelis-Menten parameters of PLpro WT and TR for different substrates

Fig. 2

Lineweaver–Burk plots with k_cat/K_m values for enzymatic activity of PLpro TR and WT using A RLRGG-AMC, B Ub-AMC and C ISG15-AMC substrates

Crystal structure of PLpro WT and structural insights into the role of the Ubl domain

To investigate the structural impact of Ubl domain deletion, we attempted crystallization of both PLpro WT and the truncated variant (PLpro TR). While PLpro TR failed to yield diffraction-quality crystals, we successfully determined the crystal structure of PLpro WT at 1.82 Å resolution. Data processing and refinement statistics are summarized in Table 2.

Table 2 Data processing and refinement statistics for PLpro WT

The structure shows that the catalytic domain adopts a chair-like architecture that accommodates the Ubl domain. Specifically, the ridge helix (residues 61–68) serves as the backrest, and the base helix (residues 71–91) forms the seat of this structural platform (Fig. 3A). The Ubl domain rests atop this platform, with a buried surface area of approximately 708 Ų at the interface between the Ubl and catalytic domains. This interface is stabilized by a network of hydrogen bonds and van der Waals interactions (Fig. 3B, C). Residues of the Ubl domain engage in both direct and water-mediated interactions with residues in the ridge and base helices. These interactions are illustrated in Figs. 3D–F. Upon removal of the Ubl domain, as in PLpro TR, these stabilizing contacts are lost, potentially increasing the flexibility of the ridge and base helices and indirectly perturbing the catalytic domain.

Closer analysis of the PLpro WT structure identified three distinct hydrogen-bonding networks (“trails”) that connect the Ubl domain to the catalytic site. Trail 1 originates from Asp37 in the Ubl domain and extends through a series of hydrogen bonds to Trp106 near the active site (Fig. 4A). Trail 2 begins with Val11 and Tyr56 of the Ubl domain and also terminates at Trp106 (Fig. 4B). Trail 3 starts from His17 and Asp12 and leads directly to the catalytic cysteine residue, Cys111 (Fig. 4C).

Fig. 3

A Domain architecture of PLpro highlighting the Ubl domain (blue), Thumb (red), Palm (green) and Fingers (orange). The thumb sub-domain features a chair-like structure that cradles the Ubl domain, with the ridge helix (residues 61–68) acting as the backrest and the base helix (residues 71–91) forming the seat. B Residues showing van der Waals interactions at the interface of Ubl domain and ridge helix. C Residues showing van der Waals interactions at the interface of Ubl domain and base helix. D–F H-bonding interactions between the Ubl domain and the catalytic domain are illustrated. The Ubl domain is depicted in blue cartoon representation, while the catalytic domain is shown in orange. Interacting water molecules are shown as red spheres

Fig. 4

Trail of interactions from Ubl domain to the active site. Ubl domain residues (blue sticks) and catalytic domain residues (orange sticks). Active site triad residues are shown as pink sticks. Water molecules are depicted as red spheres. The (2 F₀–F_c) electron density map is contoured at the 1.0σ level

These trails suggest that the Ubl domain may influence the catalytic machinery through long-range interactions. Interestingly, mutational data reported by Ferreira et al. [33] show the effect of mutating some of these trail residues. Mutation of some of the trail 2 (Y72T and Y83A) and trail 3 (E67A) residues led to significant loss in PLpro activity, while the mutation D37A (trail 1) has limited effect - likely because its interaction is mediated via the backbone carbonyl oxygen rather than a side chain-specific contact (Fig. 4A).

Further, five apo structures of SARS-CoV-2 PLpro WT have been deposited to the PDB (PDB IDs: 6WZU, 6W9C, 7NFV, 9BF8, and 8FWO) till date, in addition to our structure (9VAO) [13, 34, 35]. Among these, 6W9C has a relatively lower resolution (2.7 Å) limiting visualization of water molecules. In the remaining structures, the hydrogen bonding trails are largely conserved, except in 6WZU where two conserved water molecules (corresponding to HOH669 and 754 in our structure) are absent despite clear electron density for at least one of them. However, comparative structural analysis with the MERS-CoV PLpro (PDB ID: 4RNA), which lacks dependence on the Ubl domain for activity, revealed significant differences [36]. The Ubl-mediated trails observed in SARS-CoV-2 PLpro are largely disrupted in MERS PLpro due to multiple residue substitutions. In Trail 1, the Asp37-Lys91 interaction is lost in MERS due to the substitution of Lys91 with His, while the Thr90-Tyr95 hydrogen bond is abolished due to Val and Met substitutions. In Trail 2, interactions such as Leu56-Tyr72, Leu56-Thr146, and Phe83-Thr146 are disrupted by substitutions at the corresponding positions. In Trail 3, extensive substitutions (His17→Thr, Glu67→Ala, Asp12→Gly, Tyr71→Leu, Asp134→Gln, Tyr136→Phe) eliminate the hydrogen-bonding network between the Ubl and catalytic domains. These structural differences underscore why the Ubl domain is dispensable for MERS-CoV PLpro activity, in contrast to SARS-CoV-2 PLpro, where it regulates catalytic efficiency, potentially via an extended hydrogen-bonding network.

Taken together, our structural analysis indicates that the Ubl domain contributes to PLpro function by stabilizing the ridge and base helices, which in turn may regulate the positioning and activity of catalytic residues through extended hydrogen-bonding networks. These findings provide a mechanistic rationale for the reduced enzymatic activity observed in the Ubl-deleted PLpro TR.

Molecular dynamics simulations

To understand the effect of Ubl domain deletion on PLpro conformational dynamics, we performed 100 nanosecond molecular dynamics (MD) simulations for both PLpro WT and the truncated variant (PLpro TR) and compared the results of three independent runs. The root mean square deviation (RMSD) trajectories of both proteins plateaued around 2 Å (Fig. 5A, Supplementary Fig. 3), indicating that both systems maintained overall structural stability throughout the simulation. Minor deviations observed during the early phases of the trajectory suggest subtle conformational differences between the WT and TR structures at the onset of simulation.

Analysis of the root mean square fluctuation (RMSF) profiles of Cα-atoms revealed two regions with pronounced differences in flexibility between the WT and TR proteins (Fig. 5B). The first region corresponds to the ridge helix (residues 61–68), which exhibited increased flexibility in PLpro TR. This observation is consistent with our structural findings, suggesting that the Ubl domain contributes to the stabilization of this helix. Enhanced mobility in the ridge helix of PLpro TR may perturb the hydrogen bonding interaction networks (trails 1–3) that connect the Ubl domain to the catalytic core, potentially impacting catalytic efficiency.

The second region showing notable difference in the RMSF values is the BL2 loop (residues 266–271), which displayed reduced flexibility in PLpro WT relative to TR. This region is important for substrate access and binding. Ferreira et al. also noted that dynamic motion of the Ubl domain correlates with opening and closing of the BL2 loop [37]. Although the BL2 loop appears more flexible in the truncated protein, further biochemical characterization (described in the following section) suggests that this increased flexibility has only a limited impact on catalysis.

Closer inspection of RMSF values for the trail residues revealed consistently higher fluctuations in Tyr71 and Met84 in PLpro TR compared to WT (Fig. 5E–F, Supplementary Fig. 4), suggesting that Ubl deletion enhances the dynamic behavior of these connecting elements. Tyr71 forms a bridging interaction via water molecule HOH522, linking the main-chain carbonyl of Asp12 and the hydroxyl oxygen of Tyr71 (trail 3). Increased fluctuations were observed in both the main-chain nitrogen and aromatic ring carbons of Tyr71, which engage in van der Waals interactions with Asp12. Similarly, Met84, a key residue in trail 2, stacks with Tyr56 and connects the Ubl and catalytic domains. In contrast, no significant differences were observed in the trail 1 residues, consistent with Ferreira et al., who found that K91A and D37A mutations in trail 1 residues had minimal impact on PLpro activity, whereas mutations in trail 2 and 3 residues (e.g., Y56, D12, Y71) significantly affected catalytic efficiency [33]. This suggests that trails 2 and 3 are more critical for maintaining catalytic communication between the Ubl domain and the active site.

Finally, we examined hydrogen bonding distances involving trail residues and coordinated water molecules (Supplementary Fig. 5). While most hydrogen bonds and water-mediated interactions are conserved between PLpro WT and TR, highlighting their essential role in catalysis, a key exception was observed: the hydrogen bond between Glu67 and water molecule HOH551 is absent in the TR variant. In the WT, HOH551 mediates a stabilizing H-bond network involving Asn115, Thr10, Val11, and Glu67 (Fig. 3F). Deletion of the Ubl domain in TR disrupts this network, rendering HOH551 more mobile and weakening its stabilizing interactions. This increased water mobility likely contributes to altered local flexibility, impairing the structural communication between the Ubl interface and the catalytic core. Such dynamic changes may underlie the reduced enzymatic efficiency observed in the TR variant.

Fig. 5

A RMSD of backbone and B RMSF of Cα atoms of PLproWT (Black) and PLproTR (Red) over 100 ns trajectory. C and D PLpro WT and TR coloured based on RMSF of Cα atoms, highlighting the regions of increased flexibility. E and F RMSFs of Tyr71 and Met84 which show large differences between PLpro WT (Black) and TR (Red)

Inhibition studies with GRL0617

Since the S3–S4 pocket of PLpro is partially formed by the BL2 loop and our MD simulations revealed increased flexibility of this loop in the TR variant, we investigated whether removal of the Ubl domain impacts inhibitor binding at this site. GRL0617, a well-characterized non-covalent inhibitor that targets the S3-S4 pocket, was used to compare binding interactions between PLpro WT and TR.

Differential Scanning Fluorimetry (DSF) revealed a concentration-dependent increase in the melting temperature (T_m) for both PLpro WT and TR upon GRL0617 binding (Fig. 6A, B; Table 3). The T_m shift was comparable between the two proteins, ranging from ~ 52 to 60 °C across increasing GRL0617 concentrations. This indicates that GRL0617 binds both variants with similar stability and specificity.

Table 3 Melting temperatures (T_m) of PLpro WT and TR upon GRL0617 binding

Fig. 6

Effect of GRL0617 binding at S3-S4 site in PLpro WT and TR. Change in T_m upon A GRL0617 binding to PLpro WT; B GRL0617 binding to PLpro TR; Thermodynamic and kinetic parameters of GRL0617 binding to PLpro. Top panel: Raw data of calorimetric titration showing exothermic heat changes with successive injections; bottom panel: Integrated binding isotherm plotted against molar ratio (GRL0617: PLpro); ITC experiment showing heat changes upon C GRL0617 binding to PLpro WT and D GRL0617 binding to PLpro TR. E Concentration-dependent inhibition of PLpro WT and TR enzymatic activity by GRL0617 for IC₅₀ determination; [PLpro] = 50 nM, [Z-RLRGG-AMC substrate] = 40 µM

To further evaluate binding affinity of GRL0617, Isothermal Titration calorimetry (ITC) was performed, and data were analyzed using one-site binding model. The calculated binding affinities (K_D) were 1.9 µM for PLpro WT and 1.6 µM for PLpro TR (Fig. 6C, D, Table 4), suggesting that removal of the Ubl domain does not significantly impact the thermodynamics of GRL0617 binding.

Table 4 Thermodynamic parameters of GRL0617 binding to PLpro TR and WT

Additionally, enzymatic inhibition assays were conducted using the fluorogenic substrate Z-RLRGG-AMC to assess the functional effect of GRL0617. The IC₅₀ values were 1.3 µM for PLpro WT and 1.5 µM for PLpro TR (Fig. 6E), further confirming that inhibitor binding and inhibition potency remain largely unaltered in the absence of the Ubl domain.

These results demonstrate that although the BL2 loop becomes more flexible upon Ubl domain removal, as suggested by MD simulations, this increased flexibility does not adversely affect GRL0617 binding. The similarity in T_m shifts, K_D, and IC₅₀ values between WT and TR forms indicates that the structural changes in the TR variant have minimal impact on ligand recognition and inhibition at the S3–S4 pocket.

Steady state kinetic Inhibition assays

To assess whether removal of the Ubl domain influences the binding of natural substrates ISG15 and ubiquitin, we conducted steady-state kinetic assays using competitive inhibition. ISG15 and ubiquitin proteins were used as competitors in cleavage reactions of three fluorogenic substrates: Z-RLRGG-AMC (penta-peptide-AMC), Ub-AMC, and ISG15-AMC, using both PLpro WT and TR enzymes.

Inhibition by ISG15

In the first set of experiments, increasing concentrations of ISG15 were used to inhibit cleavage of each fluorogenic substrate. As expected, ISG15 inhibited the cleavage of all three substrates in a concentration-dependent manner (Fig. 7A–C). The IC₅₀ values for inhibition of penta-peptide-AMC cleavage were nearly identical for WT (4.12 µM) and TR (4.05 µM). Similarly, for Ub-AMC, the IC₅₀ values were 1.59 µM (WT) and 1.06 µM (TR), and for ISG15-AMC, ~ 2.05 µM for both variants. These comparable IC₅₀ values indicate that the binding affinity of ISG15 is largely unaffected by the deletion of the Ubl domain. Despite increased flexibility of the BL2 loop observed in TR via MD simulations, ISG15 binding appears unimpaired. Thus, the reduced catalytic efficiency of the TR protein is more likely attributable to increased flexibility of the ridge helix rather than altered substrate binding.

Interestingly, although IC₅₀ values were similar, the inhibition curves for penta-peptide-AMC and Ub-AMC (Fig. 7A and B) initiated at higher initial velocities for the WT enzyme compared to TR, reflecting the inherently higher catalytic efficiency of the WT protein. However, for ISG15-AMC, both WT and TR showed comparable initial velocities (Fig. 7C). This suggests that ISG15-AMC binding may stabilize the ridge helix in TR via interactions between its distal domain of ISG15 (Fig. 8), thereby restoring catalytic efficiency. In contrast, penta-peptide-AMC and Ub-AMC do not contact the ridge helix, leaving it flexible in TR and thus reducing activity. These findings support a model in which ridge helix stabilization either by the Ubl domain in WT or by the distal domain of ISG15-AMC in TR enhances catalytic function.

Inhibition by ubiquitin

In a second set of experiments, increasing concentrations of ubiquitin were tested for their ability to inhibit cleavage of the three fluorogenic substrates (Fig. 7D–F). Contrary to expectations, ubiquitin had no significant effect on cleavage of the penta-peptide-AMC substrate by either WT or TR (Fig. 7D). More surprisingly, ubiquitin enhanced the cleavage of both Ub-AMC and ISG15-AMC substrates in a dose-dependent manner for both protein variants, WT and TR (Fig. 7E, F). This enhancement suggests that ubiquitin may be interacting with PLpro at sites other than the canonical SUb1 site, which is known to mediate competitive inhibition.

Fig. 7

Effect of increasing concentrations of ISG15 and Ubiquitin on penta-peptide cleavage (A and D), deubiquitination (B and E) and deISGylation (C and F) activities of PLpro WT (Black) and TR (Red)

Fig. 8

Interaction of distal domain of hISG15 (cyan) with ridge helix of thumb sub-domain of PLpro (orange)

To identify the potential binding sites of ubiquitin which may result in activation of PLpro activity, ubiquitin was docked with PLpro WT using ClusPro [30, 31]. Since, there is no prior knowledge of the dominant forces in the protein–protein interaction, we used the Balanced mode to calculate the weighted scores. We used a cut off of atleast 25 members in the cluster group to evaluate the poses. The results suggested that ubiquitin can bind PLpro at four different sites other than the SUb1 and SUb2 sites with varying affinities (Fig. 9). The probable binding sites and docking parameters are listed in Table 5. Crystal structure of SARS-CoV-2 PLpro in complex with a ubiquitin variant (PDB ID: 8CX9) showed that dimeric ubiquitin variant can bind to PLpro on the surface, away from the catalytic site [14]. Interestingly, one of the binding sites overlaps with our predicted site (Site 4) by ClusPro.

Table 5 ClusPro parameters for Ubiquitin binding to PLproWT

Fig. 9

Probable binding sites of Ub on to PLpro as determined using ClusPro

These findings align with a recent report that ubiquitin can bind to deubiquitinases at multiple allosteric sites, where it may promote catalysis through cooperative interactions [38]. Thus, the observed enhancement in deISGylation and deubiquitination activities in the presence of ubiquitin suggests that PLpro may possess multiple functional ubiquitin-binding sites beyond SUb1 and SUb2. The formation or utilization of these additional sites appears to be largely independent of the Ubl domain.

Discussion

SARS-CoV-2 PLpro is a multifunctional protease with critical roles in viral replication and immune evasion, owing to its ability to cleave both viral polyproteins and host ubiquitin and ISG15 conjugates. Structurally, PLpro comprises an N-terminal ubiquitin-like (Ubl) domain followed by a catalytic core subdivided into thumb, finger, and palm subdomains. The active site, located between the thumb and palm is partially covered by a flexible loop, called the BL2 loop (residues 266–271). Within the active site, a conserved catalytic triad composed of Cys111, His272, and Asp286 orchestrates peptide bond cleavage. The enzymatic mechanism involves the formation of a tetrahedral intermediate, stabilized by an oxyanion hole, with Trp106 playing a key role in its dynamic stabilization [39] .

In this study, we investigated the functional and structural contributions of the N-terminal Ubl domain to PLpro’s catalytic activity. Our molecular dynamics (MD) simulations revealed that removal of the Ubl domain increases the flexibility of the ridge helix within the thumb subdomain and alters the dynamics of residues involved in the structural communication between the Ubl interface and the catalytic core. Our biochemical data further support this regulatory role of the Ubl domain. While binding of competitive inhibitor GRL0617 remained largely unaffected by Ubl domain removal, substrate processing was significantly altered. The catalytic efficiency (k_cat/K_m) of PLpro TR was lower than that of WT, mainly due to reduced k_cat values without major changes in K_m, consistent with an allosteric role of the Ubl domain that primarily influences catalytic turnover rather than substrate binding. The reduction in catalytic efficiency of PLpro TR was more pronounced for substrates such as penta-peptide-AMC and Ub-AMC, which lack extended interactions with the ridge helix. In these cases, the absence of Ubl-mediated stabilization reduced catalytic turnover. By contrast, the effect was less pronounced with ISG15-AMC, as the distal domain of ISG15 engages the ridge helix at the SUb2 site, compensating for Ubl loss. This highlights a substrate-specific requirement for ridge helix stabilization, which can be provided either by intrinsic structural elements like the Ubl domain or via substrate-mediated interactions. Interestingly, mutations of the residues in the interaction trail between Ubl domain and catalytic domain affect the catalytic efficiency of the enzyme to varying degrees [33] .

The unexpected activation of PLpro activity in the presence of excess free ubiquitin was another notable finding. While ubiquitin was expected to act as a competitive inhibitor, it instead enhanced cleavage of Ub-AMC and ISG15-AMC substrates. Our docking analysis revealed potential alternative ubiquitin-binding sites besides the canonical SUb1 and SUb2 regions. This suggests a model wherein ubiquitin may allosterically enhance PLpro activity by engaging additional regulatory surfaces, consistent with cooperative mechanisms observed in other deubiquitinases [38]. These allosteric sites appear to function independently of the Ubl domain, as both WT and TR variants showed similar activation.

Conclusion

Our study highlights the multifaceted regulatory role of the N-terminal Ubl domain of SARS-CoV-2 PLpro, which, through allosteric communication pathways, fine-tunes the enzyme’s catalytic efficiency. While removal of the Ubl domain reduces activity for penta-peptide-AMC and Ub-AMC substrates by destabilizing the ridge helix, this effect is mitigated for ISG15-AMC due to compensatory interactions of ISG15 distal domain with the ridge helix. Though our MD simulations suggest that some interactions in trails 2 and 3 are affected on Ubl removal, the precise mechanism underlying the catalytic differences between PLpro WT and the TR variant remains unclear. Additionally, the unexpected activation of catalysis by free ubiquitin suggests auxiliary allosteric sites for ubiquitin beyond the canonical binding pockets, SUb1 and SUb2. Moreover, although our molecular docking study suggests potential allosteric Ub-binding sites, it remains to be determined whether these sites or the canonical pocket possess higher affinity and primarily mediate the observed catalytic enhancement by free Ub. These findings offer new insights into PLpro regulation by the Ubl domain and open new avenues for antiviral targeting beyond the active site.