Foot-and-mouth disease virus leader proteinase: Structural insights into the mechanism of intermolecular cleavage

Abstract

Translation of foot-and-mouth disease virus RNA initiates at one of two start codons leading to the synthesis of two forms of leader proteinase L^pro (Lab^pro and Lb^pro). These forms free themselves from the viral polyprotein by intra- and intermolecular self-processing and subsequently cleave the cellular eukaryotic initiation factor (eIF) 4G. During infection, Lb^pro removes six residues from its own C-terminus, generating sLb^pro. We present the structure of sLb^pro bound to the inhibitor E64-R-P-NH₂, illustrating how sLb^pro can cleave between Lys/Gly and Gly/Arg pairs. In intermolecular cleavage on polyprotein substrates, Lb^pro was unaffected by P1 or P1′ substitutions and processed a substrate containing nine eIF4GI cleavage site residues whereas sLb^pro failed to cleave the eIF4GI containing substrate and cleaved appreciably more slowly on mutated substrates. Introduction of 70 eIF4GI residues bearing the Lb^pro binding site restored cleavage. These data imply that Lb^pro and sLb^pro may have different functions in infected cells.

Highlights

• •The leader proteinase (L^pro) of foot-and-mouth disease virus is a virulence factor.
• •L^pro can exist in three different forms in the infected cell.
• •Structural analysis reveals how L^pro can accept basic residues at P1 and P1′.
• •Isoform lacking six C-terminal residues is impaired in intermolecular cleavage.
• •Properties of the isoforms may modulate enzymatic activity during viral replication.

Introduction

Virally encoded proteinases play essential roles not only in the processing of the viral proteins but also in cleavage of host cell proteins in order to manipulate cellular processes to the advantage of the virus. One of the first such reactions to be documented was the modification of cellular translation factors during picornaviral replication leading to the shut-off of protein synthesis from capped cellular mRNA (Etchison et al., 1982, Leibowitz and Penman, 1971). This reaction was subsequently shown to be performed by the 2A proteinase (2A^pro) in enteroviruses (Kräusslich et al., 1987), a chymotrypsin-like cysteine proteinase (Petersen et al., 1999), whereas in aphthoviruses, the proteolysis is performed by the leader proteinase (L^pro, illustrated in Fig. 1) (Devaney et al., 1988), a papain-like cysteine proteinase (Guarné et al., 1998). The targets of both proteinases are the two homologues of the host protein eukaryotic initiation factor (eIF) 4G (Gingras et al., 1999). Cleavage of the eIF4G homologues prevents recruitment of capped mRNAs to the ribosome (Lamphear et al., 1995) whereas viral RNA can still be translated under these conditions as it initiates via an internal ribosome entry segment (IRES) (Martinez-Salas and Ryan, 2010). In addition, L^pro has been shown to be involved in impairing the host innate immune defence by influencing NF-κB activation and to have deubiquitinase activity (de Los Santos et al., 2007, de los Santos et al., 2009, Skern and Steinberger, 2014).

Fig. 1.

Schematic drawing of FMDV L^pro self-processing and eIF4G cleavage. (A) The FMDV RNA genome is shown as a black line, the single open reading frame as a box with the names of the mature proteins and the position of the IRES. L^pro, being expressed either as Lab^pro or Lb^pro, is indicated in red. (B) Synthesis of the polyprotein from the FMDV genome showing that Lb^pro can either be freed by an intramolecular or intermolecular reaction. sLb^pro (shown in orange) is generated by self-processing at the C-terminus of Lb^pro. (C) The effect of eIF4G cleavage by Lb^pro or sLb^pro. The cellular mRNA is shown as a black line with the cap structure as a filled circle. Lb^pro and sLb^pro are shown in red and orange, respectively. The 40S ribosomal subunit, the polyA-binding protein (PABP), eIF4G, eIF4E, eIF4A and eIF3 are shown in different shades of grey. Following cleavage of eIF4G by Lb^pro or sLb^pro, the capped mRNA is no longer connected to the 40S subunit and cannot be translated. In contrast, the viral RNA can bind to the C-terminal fragment of eIF4G and thus to the 40S subunit via eIF3.

Given these involvements in such different reactions as intramolecular and intermolecular self-processing, eIF4G cleavage and deubiquitination, it is not surprising that L^pro has unusual specificity determinants. These are well illustrated by the sequences of the three L^pro cleavage sites that have been determined directly by protein sequencing: KVQRKLK⁎GAGQSS for both intra- and intermolecular cleavage on the viral polyprotein between the C-terminus of L^pro and VP4 (Strebel and Beck, 1986), PSFANLG⁎RTTLST on eIF4GI (Kirchweger et al., 1994) and VPLLNVG⁎SRRSQP on eIF4GII (Gradi et al., 2004). Studies on L^pro intramolecular self-processing and cleavage of peptide substrates have revealed that L^pro can cleave before or after basic residues provided that the other amino acid before or after the scissile bond is glycine (Glaser et al., 2001, Nogueira Santos et al., 2012, Santos et al., 2009). However, a peptide that contained basic residues before and after the scissile bond was refractory to cleavage and was subsequently shown to be an inhibitor in the micromolar range (Santos et al., 2009). This information was then used to develop a nanomolar epoxide inhibitor based on E64, termed E64-R-P-NH₂ (Santos et al., 2009); the structure and inhibitor parameters are shown in Fig. 2, together with those of the other inhibitors used or referred to in this work. The slow formation of the tight enzyme–inhibitor complex indicates that inhibition follows slow-binding kinetics (Santos et al., 2009, Zhou et al., 1998).

Fig. 2.

Chemical structures of inhibitors referred to in this work. The structures and kinetic parameters of the inhibitor E64-R-P-NH₂ (Nogueira Santos et al., 2012) crystallised with sLb^pro are shown together with those of the inhibitors NS-134 (Stern et al., 2004) and CA074 (Yamamoto et al., 1997) whose structures were determined in complex with cathepsin B. The correspondence of side-chains in the inhibitors to substrate side-chains is shown using the nomenclature of Schechter and Berger, (19670.

The structural basis for this unusual specificity has not been elucidated, as the present structures determined by X-ray crystallography and NMR (Cencic et al., 2007, Guarné et al., 1998, Guarné et al., 2000, Steinberger et al., 2013) only provide information on the S binding region but not on the S′ binding region of L^pro. The nomenclature for sites (S) on the enzyme binding to residues of substrate (P) is that of Schechter and Berger (1967); prime site residues are those C-terminal to the scissile bond. Indeed, information on the nature of the S′ region from related papain-like proteinases is also sparse (Turk et al., 2012), with structural information only being available for cathepsin B (Stern et al., 2004, Turk et al., 1995, Yamamoto et al., 1997) determined with inhibitors similar to E64-R-P-NH₂ (Fig. 2). However, cathepsin B is also unusual in being an exopeptidase, with an occluding loop that prevents access beyond the S2′ site, that is the site on the enzyme interacting with the P2′ residue of the substrate (Stern et al., 2004). Thus, any information on the S′ binding region of FMDV L^pro will shed light on the nature of this region in papain-like cysteine proteinases generally.

Understanding of the mechanism of L^pro is complicated by the presence of different forms of the protein in the infected cell (Sangar et al., 1987, Sangar et al., 1988). Two isoforms, Lab^pro and Lb^pro (Fig. 1), arise from the presence of two in-frame AUG codons for the initiation of protein synthesis on the viral RNA (Sangar et al., 1987). Consequently, the Lab^pro possesses an additional 28 amino acids at the N-terminus than Lb^pro. Cao et al. (1995) demonstrated in cell culture that Lb^pro was essential whereas Lab^pro was not; nevertheless, there may still be as yet unknown roles for Lab^pro during infection in the host organism. In addition, a shortened form of Lb^pro (sLb^pro) lacking 6 or 7 amino acids at the C-terminus has long been known (Sangar et al., 1988). The truncation arises through Lb^pro self-cleavage (Sangar et al., 1988) and can be observed in vitro when Lb^pro expressed in rabbit reticulocyte lysates (RRLs) is incubated for longer time periods (e.g. 1 h)(Mayer et al., 2008). A separate function for sLb^pro has not been identified; however, one report suggested that Lb^pro and sLb^pro may differ in their cleavage efficiencies in intermolecular cleavage of the polyprotein substrate (Cencic et al., 2007).

One clear difference between Lb^pro and sLb^pro is the ability of Lb^pro to form homodimers through interactions of the C-terminal extension (CTE) of one monomer and the substrate binding site of the neighbouring one and vice versa. sLb^pro cannot form homodimers in this way because it lacks the six most C-terminal residues. The Lb^pro homodimer has been observed by X-ray crystallography and NMR (Cencic et al., 2007, Guarné et al., 1998, Guarné et al., 2000, Steinberger et al., 2013) with the K_D being estimated from NMR analyses to be in the millimolar range (Cencic et al., 2007). Therefore, formation of the homodimer at concentrations of Lb^pro achieved when it is synthesised in the infected cell seems unlikely unless there is a high local concentration. In contrast, both Lb^pro and sLb^pro use an exosite featuring residues Tyr183 to Leu188 as well as Cys 133 to recognise binding sites located on the eIF4G homologues, located in both cases 20 to 30 amino acids from the cleavage site (Foeger et al., 2005). How this binding favours Lb^pro or sLb^pro cleavage of the eIF4G homologues is not known.

To investigate further the properties of sLb^pro, we set out to determine the structure of sLb^pro complexed with the inhibitor E64-R-P-NH₂ and to define differences in the cleavage of intermolecular polyprotein substrates by sLb^pro and Lb^pro.

Materials and methods

Materials

The bacterial expression plasmid pET-11d sLb^pro (FMDV residues 29–195) was created by site-directed PCR mutagenesis of pET-11d sLb^pro C51A, described earlier (Guarné et al., 1998, Kirchweger et al., 1994), to restore the catalytic cysteine.

The plasmids that were used as templates for in vitro transcription pCITE-1d Lb^pro (residues 29–201 of Lb^pro), pCITE-1d sLb^pro (residues 29–195 of Lb^pro) and pCITE-1d Lb^pro C51A VP4/VP2 (residues 29–201 of Lb^pro, all 85 residues of VP4 and 78 residues of VP2) have been described (Glaser et al., 2001). The constructs pCITE-1d Lb^pro C51A VP4/VP2 containing the mutations at position P1 and P1′ of the Lb^pro-VP4 cleavage site (VQRKLG⁎RAGQ, VQRKLK⁎RAGQ, VQRKLG⁎AAGQ) were created by site-directed PCR mutagenesis of pCITE-1d Lb^pro C51A VP4/VP2. The construct pCITE-1d Lb^pro C51A VP4/VP2 containing the eIF4GI sequence SFANLG⁎RTTL at the Lb^pro-VP4 cleavage site, termed pCITE-1d Lb^pro C51A VP4/VP2 SFANLG⁎RTTL (FMDV residues 29–195 of Lb^pro, residues 669–678 of eIF4GI, residue 5–85 of VP4 and 78 residues of VP2), has been described (Cencic et al., 2007). The construct pCITE-1d Lb^pro C51A VP4/VP2 containing residues 599–678 of eIF4GI, termed pCITE-1d Lb^pro C51A eIF4GI_599-668 VP4/VP2 SFANLG⁎RTTL, (FMDV residues 29–195 of Lb^pro, residues 599–678 of eIF4GI, residue 5–85 of VP4 and 78 residues of VP2) was created by PCR amplification of residues 599–678 of eIF4GI using the plasmid pKS eIF4GI 400–739 as template and cloning of this fragment into pCITE-1d Lb^pro C51A VP4/VP2 via the restriction sites Bpu10I and SacI.

The inhibitor E64-R-P-NH₂ was prepared as described (Nogueira Santos et al., 2012).

Protein expression and purification

Protein expression and purification were performed as described by Steinberger et al. (2013) with the following modifications. Proteins were expressed from the construct pET-11d sLb^pro transformed into BL21(DE3)LysE bacteria. To avoid degradation of the active protease, all purification steps were carried at a maximum of 10 °C.

Preparation of the sLb^pro-E64-R-P-NH₂ complex

Purified sLb^pro was incubated with a fivefold molar excess of E64-R-P-NH₂ over night at 4 °C to allow complex formation. Subsequently, the complex was dialysed against a buffer containing 50 mM NaCl, 10 mM Tris HCl pH 8, 1 mM TCEP, 5% glycerol to remove excess inhibitor. The concentration was adjusted to 18 mg/ml and centrifuged at 18,000g for 10 min at 4 °C to remove precipitated protein.

Crystallisation, data collection, structure determination and refinement

Crystals of the sLb^pro-E64-R-P-NH₂ complex were initially obtained in the Wizard I and II screen crystallisation screen (Emerald Bio), using the sitting-drop vapour diffusion technique and a nanodrop-dispensing robot (Phoenix RE; Rigaku Europe, Kent, United Kingdom), and optimised to 0.1 M sodium acetate pH 4.8, 0.9 M NaH₂PO₄ and 1.2 M K₂HPO₄ using the hanging drop vapour diffusion technique at 22 °C and seeding technique. The seed stock was produced by a “seed-bead” kit from Hampton Research (Luft and DeTitta, 1999). The crystals were flash-frozen in liquid nitrogen in a reservoir solution supplemented with 25% glycerol prior to data collection.

Diffraction data sets were collected at the ESRF Synchrotron (Grenoble) at beamline ID14-1 at 100 K using a wavelength of 0.93 Å to 1.6 Å resolution, processed using the XDS package (Kabsch, 2010), converted to mtz format using POINTLESS and scaled with SCALA (Winn et al., 2011).

The crystal structure was solved by difference Fourier techniques using the protein atomic coordinates of the inactive mutant of sLb^pro from the Protein Data Bank (accession code 1QMY). Model building and refinement steps were performed with REFMAC and COOT. The structure was refined using the programs REFMAC (Murshudov et al., 1997) and Phenix Refine (Adams et al., 2010) and model building was done with the program Coot (Emsley and Cowtan, 2004). Data collection and refinement statistics are shown in Table 1. Stereo-chemistry and structure quality were checked using the MolProbity web server (Davis et al., 2007).

Table 1.

X-ray parameters and refinement statistics.

Data collection
Source	ID14-1, ESRF
Wavelength (Å)	0.93
Resolution (Å)	45.35–1.6 (1.69–1.6)a
Space group	P21
Unit cell (Å, °)	a=45.81 b=110.68, c=56.77
	α=γ=90, β=98.12
Molecules / a.u.	3
Unique reflections	72906 (10232)
Completeness (%)	99.0 (95.0)
Rmergeb	0.037 (0.174)
Rmeasc	0.041 (0.213)
Multiplicity	4.9 (2.9)
I/sig(I)	29.9 (5.6)
BWilson (Å2)	22.5
Refinement
Rcrystd/ Rfreee	16.9/20.1
R.m.s.d. bonds (Å)	0.011
R.m.s.d. angles (°)	1.4
Ramachandran plot (%)
favored/allowed/outliers	96.9/3.1/0

^aValues in parentheses are for the highest resolution shell.

^b$R_{merge}={\displaystyle \frac{{\sum }_{hkl}{\sum }_{i=1}^N|I_{i(hkl)}-{\overline{I}}_{(hkl)}|}{{\sum }_{hkl}{\sum }_{i=1}^N{I}_{i(hkl)}}}$

^c$R_{meas}={\displaystyle \frac{{\sum }_{hkl}\sqrt{N/(N-1)}{\sum }_{i=1}^N|I_{i(hkl)}-{\overline{I}}_{(hkl)}|}{{\sum }_{hkl}{\sum }_{i=1}^N{I}_{i(hkl)}}}$ where ${\overline{I}}_{(hkl)}$ is the mean intensity of multiple $I_{i(hkl)}$ observations of the symmetry-related reflections, N is the redundancy

^d$R_{cryst}={\displaystyle \frac{\sum \left|\left|F_{obs}\right|-\left|F_{calc}\right|\right|}{\sum \left|F_{obs}\right|}}$

^eR_free is the cross-validation R_factor computed for the test set of reflections (5%) which are omitted in the refinement process.

In vitro transcription and translation

In vitro transcription reactions were performed as described (Neubauer et al., 2013) with the following modifications. The plasmids were cleaved with BamHI for the expression of active proteinases (pCITE-1d Lb^pro and pCITE-1d sLb^pro) and SalI for the substrates (pCITE-1d Lb^pro C51A VP4/VP2 and derivatives thereof).

In vitro translation reactions were performed as described (Cencic et al., 2007) with the following modification. To translate substrate proteins and proteinases, RNA was added to the reaction at concentrations of 14 ng/µl.

Electrophoresis and immunoblotting

Electrophoresis and immunoblotting for protein analysis were performed as described (Neubauer et al., 2013), except for the separation of translation products when SDS-PAGE gels containing 17.5% acrylamide were used (Dasso and Jackson, 1989).

Structural comparisons

Structural alignments and superimpositions were done using Coot (Emsley and Cowtan, 2004, Krissinel and Henrick, 2004). All drawings were created using PyMOL (DeLano, 2002). The electrostatic potential of sLb^pro was calculated using the Adaptive Poisson-Boltzmann Solver package (Baker et al., 2001) within PyMOL.

Accession numbers

Coordinates for the structure determined here have been deposited in the protein data bank (pdb accession code 4QBB). The PDB identifiers of the structures used for comparisons were 1QOL for Lb^pro, 1GEC for glycyl endopeptidase-complex with benzyloxycarbonyl-leucine-valine-glycine-methylene, 3CH3 for SERA5 from plasmodium falciparum, 1SP4 for bovine cathepsin B-complex with NS-134, 1QDQ for bovine cathepsin B-complex with CA074.

Results and discussion

The crystal structure of the inhibitor E64-R-P-NH₂ bound to sLb^pro has been determined. The correspondence of the side-chains in the inhibitor to substrate side-chains is illustrated in Fig. 2; a portion of the electron density in the final model of the inhibitor bound to the active site is shown in Fig. 3. Three chains, termed A, B and C were found in the asymmetric unit of the crystal lattice. Electron density was visible for sLb^pro residues 29–187 of chain A, 29 to 184 of chain B and 29–185 of chain C. For the inhibitor, electron density was visible for all atoms except for those of the P3 amino-alkyl guanidinium group (referred to as Arg-m in the text and figures) and P1′ Arg. For P3 Arg-m, density was visible up to the C_β atom for chain A, for all atoms of chain B (due to favourable interactions with an Asp residue from a symmetry related molecule) and to atom N_ε for chain C. For the P1′ arginine residues, density up to the C_β atom for chain A was visible whereas for chains B and C density was observed to the C_γ atom. The remaining atoms of these side-chains including the guanidinium group were modelled in Fig. 4, Fig. 5, Fig. 6, Fig. 7 after the side-chain trace of C_α to C_γ in the most likely conformation. Density for the covalent bond between the active site cysteine and the inhibitor (atom C1) was very clear in all three chains. Superimposition of the structure of sLb^pro bound to E64-R-P-NH₂ with the unbound Lb^pro structure of sLb^pro C51A C133S (PDB ID 1QMY, chainB) (Guarné et al., 2000) gave an r.m.s.d. of 0.35 Å over 156C_α atoms superimposed. Given that the best resolution of the inhibitor was found in chain B, all structural analysis is based on this chain.

Fig. 3.

Stereo view of the arrangement of the inhibitor E64-R-P-NH₂ and the substrate binding site of sLb^pro. 2F₀–F_c maps contoured at 1 σ are shown as grey mesh for the inhibitor and the sLb^pro residues Asp49, Cys51, Glu96 and Glu147. The inhibitor is shown as green sticks. Residues of sLb^pro interfacing with the inhibitor are shown as grey sticks. Oxygen, nitrogen and sulphur atoms are coloured red, blue and yellow, respectively. Due to the lack of electron density, no structure is shown for the P1‘ Arg residue of E64-R-P-NH₂ from the Cδ atom onwards.

Fig. 4.

Comparison of the binding of E64-R-P-NH₂ and P1-P3 of the CTE. (A) The inhibitor (green sticks) is shown in the substrate binding site of sLb^pro. Side-chains of the inhibitor are labelled. In Fig. 4, Fig. 5, Fig. 6, Fig. 7, the atoms of the P1′ Arg residue from C_δ onwards are modelled based on the most favourable conformation. Residues of the active site (Cys51, His148, Asp163) as well as the three acidic residues discussed in the text are shown as sticks. (B) As in A, with the P1-P3 residues (in yellow and labelled) of the CTE superimposed for comparison.

Fig. 5.

Electrostatic interactions involved in sLb^pro interaction with E64-R-P-NH₂ and the P1-P3 residues of the CTE. The electrostatic potential of sLb^pro was calculated using the Adaptive Poisson–Boltzmann Solver package (Baker et al., 2001) within PyMOL (DeLano, 2002). The surface is coloured according to the electrostatic potential ranging from −5 (red) to +5 (blue) kT/e. (A) The inhibitor E64-R-P-NH₂ is shown as green sticks, (B) residues P1-P3 of the CTE as yellow sticks. The representations on the right are rotated 90° on the x-axis relative to those on the left.

Fig. 6.

Comparison of arrangement of negatively charged residues in the substrate binding sites of sLb^pro, glycyl endopeptidase and SERA5. (A) sLb^pro bound to E64-R-P-NH₂ (green sticks). (B) Substrate binding site of SERA5. (C) Glycyl endopeptidase bound to the inhibitor benzyloxycarbonyl-leucine-valine-glycine-methylene (yellow sticks). (D) Stereo view of the superimposition of the three structures in A–C. Residues referred to in the text are shown as sticks.

Fig. 7.

Comparison of the S1′ and S2′ binding sites of FMDV sLb^pro and cathepsin B. (A and B) E64-R-P-NH₂ (green sticks) bound in the substrate binding site of Lb^pro with the inhibitors NS-134 (A, blue sticks) and CA074 (B, magenta sticks) superimposed. (C and D) NS-134 (C) and CA074 (D) bound to the substrate binding site of cathepsin B with E64-R-P-NH₂ superimposed. The colour coding is as in A and B. The structure of Trp221 for which there is no equivalent in Lb^pro is shown as sticks as are residues referred to in the text.

To determine the binding of E64-R-P-NH₂ to sLb^pro, we first compared its arrangement in the substrate binding site of sLb^pro to that of the last three residues of the CTE observed in the crystal structure of Lb^pro (Guarné et al., 1998). Fig. 4 shows that the positions of the P3 Arg-m side-chain of the inhibitor and the P3 Lys side-chain of the CTE occupy similar positions in the two structures. The C_γ atom of the P1′ Arg residue of the inhibitor lies between the side-chains of Asp49 (distance from C_γ to carboxy group of Asp49 is 4.2 Å) and Glu147 (distance from C_γ to C_β of Glu147 is 4.5 Å). Given the uncertainty in the position of the guanidinium group (as mentioned earlier, the remaining atoms were modelled as no density was observed), a closer localisation is not possible. Nevertheless, the superimposition in Fig. 4B shows that the P1 Lys of the CTE lies almost equidistant between Asp49, Glu96 and Glu147. The disorder of the P1′ Arg in the structure of the inhibitor presented here indicates that the side-chain is flexible; in contrast, in the previously published structure of Lb^pro C51A, good density was observed to the P1 Lys residue in the substrate binding site of Lb^pro (Guarné et al., 1998). Given that the polypeptide chain is fully extended in both the CTE and E64-R-P-NH₂ bound structures, this explains how a peptide containing Lys and Arg at P1 and P1′ can be refractory to cleavage (Nogueira Santos et al., 2012). If the Lys at P1 points away from the globular domain, an Arg side-chain at P1′ would have to point towards it. Thus, on oligopeptide substrates at least, the enzyme can only accommodate a basic residue at one of the positions, presumably because it requires a glycine with its greater freedom of rotation at the other. However, the data do not answer the question why a peptide containing Lys and Arg at P1 and P1′ can inhibit Lb^pro (Nogueira Santos et al., 2012). This implies that the inhibitor may bind in a mode that has not yet been observed that moves the scissile bond out of the active site. However, additional structural information will be required to elucidate the nature of the binding of this peptide.

Overall, comparison of the binding of the E64-R-P-NH₂ and the CTE residues (Fig. 5) show that the P1/P1′ binding area is a deep cleft surrounded by the acidic residues Asp49, Glu96 and Glu147. We set out to determine whether other papain-like cysteine proteinases have been identified that have a similar arrangement of three acidic residues in the vicinity of the S1/S1′ binding sites. Berti and Storer (Berti and Storer, 1995) compared the sequences of 48 representative papain-like cysteine proteinases. Only one, SERA5 (Serine repeat antigen 5, termed PfalI in (Berti and Storer, 1995)) from P. falciparum showed acidic amino acids at the equivalent positions to those in sLb^pro; these are Asp594, Glu638 and Asp761 which are equivalent to Asp49, Glu96 and Glu147 of sLb^pro ((Hodder et al., 2009); Fig. 6A and B). However, little is known about the biochemistry of this protein; indeed, proteolytic activity has not been shown. Furthermore, the putative active site residue is serine, not cysteine. In addition, the authors suggested that Asp594 (equivalent to Asp49) of SERA5 is too near to the substrate binding site to allow substrate to bind.

A second enzyme, glycyl endopeptidase (ppiv in Berti and Storer, (1995)), also possesses two acidic residues, Glu23 and Asp158, equivalent to Asp49 and Glu147. The third residue (Asn64, equivalent to Glu96 in sLb^pro) is however not acidic and is followed by Arg65. As can be seen in Fig. 6C, the presence of Glu23 and Arg65 preclude the entry of any substrates with amino acids larger than glycine at P1, thus conferring the specificity referred to in the name glycyl endopeptidase.

It should be noted that only these three papain-like enzymes have an amino acid other than glycine at the position equivalent to Gly23 in papain (equivalent to Asp 49 in sLb^pro). Superimposition of the three structures (Fig. 6D) shows that Asp49 in sLb^pro is further away from the substrate binding site than Glu23 or Asp594 in glycyl endopeptidase (O’Hara et al., 1995) and SERA5 (Hodder et al., 2009). This is due to the presence of only four residues in sLb^pro lying between the oxyanion hole defining residue (Asn46) and the active site Cys51. In all other papain-like cysteine proteinases, five residues are present between the oxyanion-hole residue Gln19 and the active site nucleophile Cys25. Interestingly, Glu23 of glycyl endopeptidase is closer to the substrate binding site than Asp594 in SERA5, suggesting that the substrate binding site of SERA5 may be more open than previously thought. In contrast, Glu96 does not superimpose well with Glu638, with the C_α lying 3.7 Å apart (Fig. 6D). Finally, as an important control for the accuracy of the structural superimposition, we note that the C_α of the catalytic histidines (H148, H762 and H159) superimpose well (Fig. 6D).

Comparisons with inhibitor complexes from cathepsin B

Information on the structural details of the S1′ and S2′ binding sites in papain-like cysteine proteinases is limited, especially for the S2′ site (Turk et al., 1998, Turk et al., 2012). Indeed, structures of compounds with residues bound in the S2′ position are only available for cathepsin B complexed with the inhibitors CA030, CA074 and NS-134 (Fig. 2; (Stern et al., 2004; Turk et al., 1995; Yamamoto et al., 1997)). To compare the binding of the inhibitors CA074 and NS-134 to cathepsin B (CA030 differs only in the length and chemical bond of the N-terminal aliphatic moiety (Turk et al., 1995)) and that of E64-R-P-NH₂ to sLb^pro, the structures of cathepsin B complexes with CA074 and NS-134 were superimposed on that of sLb^pro using the SSM tool of Coot (Krissinel and Henrick, 2004). The r.m.s.d. values were 2.44 Å for sLb^pro superimposed on cathepsin B complexed to CA074 (1QDQ) and 2.31 Å for sLb^pro superimposed on cathepsin B complexed to NS-134 (1SP4). Fig. 7A and B show the positions of the inhibitors NS-134 and CA-074 relative to E64-R-P-NH₂ on sLb^pro; Fig. 7C and D show the relationships on the structure of cathepsin B.

The P1′ residues of CA074 and NS-134 are Ile and Leu, respectively, in contrast to the Arg found in E64-R-P-NH₂. Despite the difference in chemical composition, however, the side-chains of these residues superimpose well and occupy the same relative space. The specificity of cathepsin B for these large hydrophobic residues is derived from the presence of a hydrophobic pocket made up of residues Phe174, Val176, Phe180, Leu181, M196 and Trp221 (Figs. 7C and D). In contrast, in sLb^pro, there is no equivalent loop to those in cathepsin B bearing residues Phe174 to Leu181 and Met196 or Trp221. This region is thus open and, as mentioned before, the presence of Glu147 and Asp49 enable sLb^pro to accept well the arginine residue.

At the P2′ position, all three inhibitors have a proline residue. It is clear that the positions of the proline residues from CA074 and NS-134 on the one hand and sLb^pro on the other are different. In sLb^pro, the proline P2′ residue of E64-R-P-NH₂ lies closer to Asp163, the third member of the catalytic triad. Two factors appear to be responsible for this. The first is the absence of a residue equivalent to Trp221 in cathepsin B (Trp177 in papain) that pushes the proline residue away from Asn219 (equivalent to Asp163 in sLb^pro). Second, albeit only in chain A, the sLb^pro residue Asp164 forms a hydrogen bond (2.7 Å) to the terminal nitrogen on the proline residue whereas in cathepsin B, the terminal carboxyl group of the proline is co-ordinated by His110 and His111 in the occluding loop, a structure unique to cathepsin B that is responsible for its exopeptidase activity. It is clear from the structure that there is no binding pocket for the P2′ residue in sLb^pro.

Lb^pro and sLb^pro differ in cleavage efficiencies on intramolecular substrates

The structural analysis illustrates how sLb^pro can bind to an inhibitor bearing Leu, Gly and Arg at the P2, P1 and P1′ sites, respectively, the very residues found at the eIF4GII cleavage site. Nevertheless, a peptide corresponding to the eIF4GI peptide (SFANLG⁎RTTL) was a poor substrate for both Lb^pro and sLb^pro ((Santos et al., 2009); unpublished data). However, the eIF4GI cleavage site when introduced into the background of the polyprotein substrate was efficiently cleaved by Lb^pro but was still refractory to cleavage by sLb^pro (Cencic et al., 2007). Examination of the state of the endogenous eIF4GI in RRLs used for the experiments showed that it was cleaved by both Lb^pro and sLb^pro (Cencic et al., 2007).

To understand these observations and illuminate differences in cleavage efficiencies between Lb^pro and sLb^pro, we decided to investigate further the cleavage of intermolecular polyprotein substrates using the system described by Cencic et al. (2007). Here, a fragment of the FMDV polyprotein encoding an inactive form of Lb^pro, VP4 and part of VP2 (termed Lb^pro C51A VP4/VP2) is labelled with ³⁵S methionine by translation in RRLs (Cencic et al., 2007). Subsequently, cold methionine is added and an mRNA encoding an active, mature Lb^pro or sLb^pro is added. The enzyme was synthesised from an RNA molecule rather than adding purified recombinant proteinase for two reasons. First, preparations of purified active Lb^pro always contain some sLb^pro that arises from self-processing, even when all purification steps are done at 4 °C (Kirchweger et al., 1994). Second, the translation of the RNA followed by Lb^pro cleavage of the eIF4G isoforms in the RRLs resembles more closely the in vivo situation during an FMDV infection. The labelled substrate and products are separated by SDS-PAGE and detected by fluorography. Although it is very difficult to vary either the enzyme or substrate concentrations in this assay, it still provides qualitative information on differences in the rates of reaction between different forms of L^pro.

A typical experiment is shown in Fig. 8A, with Lb^pro cleaving the wild-type sequence between 15 and 30 min. The Lb^pro moiety has four methionine residues compared to only two in the VP4/VP2 part, providing a partial explanation for the lower intensity of the latter band (Glaser et al., 2001). In addition, we have evidence that the VP4/VP2 part is degraded in the RRLs (data not shown), with degradation being enhanced when a residue other than the wild-type glycine is present at the N-terminus of VP4 (see Fig. 8B–E). We first investigated the effect of substituting residues at the P1 and P1′ positions with residues (underlined in Fig. 8B–D), several of which had been shown to be detrimental to peptide cleavage (Guarné et al., 2000, Nogueira Santos et al., 2012, Santos et al., 2009). However, none of the modifications affected the cleavage efficiency of Lb^pro (Table 2). These results show that there are clear differences between the activity of Lb^pro on peptides and polyprotein substrates, indicating that the conformation of the substrate may be different in the background of the polyprotein. In addition, as previously observed, replacement of the P5-P4′ residues of the polyprotein cleavage sequence with those of the eIF4GI site (Fig. 8E) also did not affect the efficiency of Lb^pro cleavage.

Fig. 8.

Effects of P1 or P1′ site mutations on the intermolecular cleavage efficiency of Lb^pro. Intermolecular processing of the precursor Lb^pro C51A VP4/VP2 (A) and variants thereof (B–E) by Lb^pro. The cleavage sequence present in the background of the polyprotein is shown in grey boxes. Differences from the wild-type sequence of the precursor are underlined. RRLs were programmed with RNA (14 ng/µl) coding for the polyprotein Lb^pro C51A VP4/VP2. Translation was performed for 20 min at 30 °C in the presence of [³⁵S]-Met and terminated by the addition of unlabelled Met for 10 min. RNA (14 ng/µl) coding for Lb^pro was added and translation was continued at 30 °C. The reaction was terminated by placing the samples on ice and the addition of Laemmli sample buffer containing excess unlabelled Met and Cys. 10 µl aliquots were analysed by 17.5% SDS-PAGE gels, followed by fluorography. Uncleaved precursor Lb^pro C51A VP4/VP2 and cleavage products Lb^pro C51A and VP4/VP2 are indicated. The asterisk (*) indicates an aberrant cleavage product. Negative controls devoid of any RNA (-sub, -prot) or comprising only RNA encoding the precursor (+sub, -prot) are shown on the right of each gel. Protein standards are shown on the left. Each cleavage reaction was performed twice; a representative autoradiogram for each is shown.

Table 2.

Summary of the mutational analysis of the intermolecular cleavage efficiency of Lb^pro and sLb^pro. Data are taken from Fig. 8, Fig. 9 and Glaser et al., 2001 for cleavage of eIF4GI by Lb^pro. The experiments were performed twice.

	50% cleavage (min)
	pCITE LbproC51A VP4/VP2					endogenous eIF4GI
	VQRKLK⁎GAGQ	VQRKLG⁎RAGQ	VQRKLK⁎RAGQ	VQRKLG⁎AAGQ	SFANLG⁎RTTL (eIF4GI)	SFANLG⁎RTTL
Lbpro	15–30	0–15	15–30	0–15	15–30	0–15
sLbpro	30	30	45–90	90	No cleavage	0–15

We next investigated the ability of sLb^pro to cleave the same five substrates. In all cases, sLb^pro cleavage was delayed compared to the Lb^pro cleavage. 50% cleavage of the wild-type substrate and a substrate bearing P1 Gly and P1′ Arg occurred at 30 min (Fig. 9A and B) compared to 15–30 min and 0–15 min, respectively, with Lb^pro (Table 1). 50% cleavage of the substrate bearing two basic amino acids at the scissile bond was observed between 45–90 min whereas the substrate lacking basic amino acids at the cleavage site only showed 50% cleavage after 90 min. Finally, as was shown previously by Cencic et al. (2007), the substrate with the eIF4GI cleavage site was not cleaved at all, even after 120 min of incubation. Thus, the cleavage efficiencies of sLb^pro on modified polyprotein substrates are similar to those on analogously modified oligopeptides, in contrast to those of Lb^pro. As a control, we examined the state of the endogenous eIF4GI present in RRLs by performing a Western blot of the cleavage reaction using an anti-eIF4GI antiserum (Fig. 9F). Endogenous eIF4GI was cleaved within 15 min after the start of incubation, an efficiency comparable to that previously observed with Lb^pro and sLb^pro (Glaser et al., 2001). This cleavage efficiency on endogenous eIF4GI was also observed in all other reactions in Fig. 9 (data not shown).

Fig. 9.

Effects of P1 or P1′ site mutations on the intermolecular cleavage efficiency of sLb^pro. The intermolecular processing of the precursor Lb^pro C51A VP4/VP2 (A) and variants thereof (B–E) by sLb^pro. The cleavage sequence present in the background of the polyprotein is shown in grey boxes. Differences from the wild-type sequence of the precursor are underlined. The translation and analysis was done as shown in Fig. 8. (F) The intermolecular cleavage of endogenous eIF4GI present in the RRLs from panel E. 10 µl aliquots were analysed on a 6% Dasso & Jackson SDS-PAGE (Dasso and Jackson, 1989), followed by immunoblotting with an antiserum detecting the N-terminal part of eIF4GI. Uncleaved eIF4GI and cleavage products (cp_N) are indicated. Negative controls devoid of any RNA (−sub, −prot) or comprising only RNA encoding the precursor (+sub, −prot) are shown. Protein standards are shown on the left. Each cleavage reaction was performed twice; a representative autoradiogram for each is shown.

How can we explain the differences observed above between Lb^pro and sLb^pro in their behaviour towards oligopeptide and polyprotein substrates (Table 2)? Why do the introduced mutations only affect sLb^pro cleavage and why is sLb^pro not capable of recognising the eIF4GI cleavage site in the background of the polyprotein? The difference in the cleavage efficiencies between Lb^pro and sLb^pro on polyprotein protein substrates can be explained by the ability of Lb^pro to bind to the cleavage site on the substrate with its canonical substrate binding site and through its own CTE to the “substrate binding site” of the substrate as shown in Fig. 10A and B. In contrast, sLb^pro can only make one of these interactions, as it lacks an intact CTE (Fig. 10C and D).

Fig. 10.

Model of the intermolecular cleavage of polyprotein substrates by Lb^pro and sLb^pro. (A and C) Cleavage of wild-type Lb^pro C51A VP4/VP2 by Lb^pro and sLb^pro, respectively. (B and D) Cleavage of Lb^pro C51A VP4/VP2 containing the eIF4GI cleavage sequence by Lb^pro and sLb^pro, respectively. E. Cleavage of Lb^pro C51A eIF4GI_599-668 VP4/VP2 SFANLG*RTTL by sLb^pro. Lb^pro is in light blue, sLb^pro in dark blue, VP4 in light green, VP2 in dark green and the eIF4GI_599-668 fragment in red.

sLb^pro can efficiently cleave the eIF4GI site on the native protein present in the RRL because this involves residues Cys133 and Asp184-Leu188 of the CTE but not the last six residues of the CTE (Foeger et al., 2005). Accordingly, we introduced 80 amino acids from eIF4GI containing the L^pro binding and cleavage sites into the Lb^pro C51A VP4/VP2 substrate (Fig. 10E). This modified substrate (termed Lb^pro C51A eIF4GI_599-668 VP4/VP2 SFANLG⁎RTTL) was cleaved by sLb^pro between 30 and 90 min, indicating that the availability of the two binding sites had allowed cleavage to take place (Fig. 11A).

Fig. 11.

eIF4GI_599-668 is essential for the cleavage of the sub-optimal eIF4GI cleavage site SFANLG*RTTL by sLb^pro. A-C, left panels. Intermolecular processing of Lb^pro C51A eIF4GI_599-668 VP4/VP2 by sLb^pro and sLb^pro C133S Q185R E186K and Lb^pro C51A VP4/VP2 by sLb^pro C133S Q185R E186K. The cleavage sequence present in the background of the polyprotein is shown in grey boxes. Translation reactions and analyses of products were as in Fig. 8. Uncleaved precursors Lb^pro C51A eIF4GI_599-668 VP4/VP2 and Lb^pro C51A VP4/VP2 as well as cleavage products Lb^pro C51A eIF4GI_599-668, Lb^pro C51A and VP4/VP2 are indicated. A–C, right panels. The intermolecular cleavage of endogenous eIF4GI present in the RRL. Analysis of the state of eIF4GI was as shown in Fig. 9. Uncleaved eIF4GI and cleavage products (cp_N) are indicated. Negative controls lacking added RNA (−sub, −prot) or comprising only of RNA encoding the precursor (+sub, −prot) are shown. Protein standards are shown on the left.

As a control, we examined the ability of the variant sLb^pro C133S Q185R E186K that had previously been shown to be unable to cleave endogenous eIF4GI because the variant cannot recognise its binding site on this factor (Foeger et al., 2005). This variant could also not cleave the Lb^pro C51A eIF4GI_599-668 VP4/VP2, but maintains the ability to cleave the wild-type substrate Lb^pro C51A VP4/VP2 (Figs. 11B and C, left panels). As previously reported, sLb^pro C133S Q185R E186K was however not able to cleave endogenous eIF4GI, even after 120 min of incubation (Figs. 11B and C, right panels).

Concluding remarks

The structural data presented here reveal that sLb^pro uses three acidic residues to bind to basic residues at the P1 or P1′ positions of a substrate and that this represents a unique arrangement that is not found in cellular papain-like proteinases. Differences in the cleavage efficiency of Lb^pro and sLb^pro were observed on modified polyprotein substrates. The presence of sLb^pro in infected cells (Sangar et al., 1988) suggests that differences in the properties of Lb^pro and sLb^pro will be relevant to the success of viral replication. Hence, the removal of six C-terminal residues 40 Å from the active site may represent a unique mechanism to modify the properties of a proteolytic enzyme during viral replication.