Tài liệu Báo cáo khoa học: C-Terminal extension of a plant cysteine protease modulates proteolytic activity through a partial inhibitory mechanism doc

C-Terminal extension of a plant cysteine protease modulates proteolytic activity through a partial inhibitory mechanism Sruti Dutta, Debi Choudhury, Jiban K Dattagupta and Sampa Biswas Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, Kolkata, India Keywords C-terminal extension; cysteine proteases; modulation of proteolytic activity; papain-like; thermostable Correspondence S Biswas, Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, ⁄ AF Bidhannagar, Kolkata 700 064, India Fax: +91 332 337 4637 Tel: +91 332 337 5345 E-mail: sampa.biswas@saha.ac.in (Received 14 March 2011, revised 16 May 2011, accepted 22 June 2011) doi:10.1111/j.1742-4658.2011.08221.x The amino acid sequence of ervatamin-C, a thermostable cysteine protease from a tropical plant, revealed an additional 24-amino-acid extension at its C-terminus (CT) The role of this extension peptide in zymogen activation, catalytic activity, folding and stability of the protease is reported For this study, we expressed two recombinant forms of the protease in Escherichia coli, one retaining the CT-extension and the other with it truncated The enzyme with the extension shows autocatalytic zymogen activation at a higher pH of 8.0, whereas deletion of the extension results in a more active form of the enzyme This CT-extension was not found to be cleaved during autocatalysis or by limited proteolysis by different external proteases Molecular modeling and simulation studies revealed that the CT-extension blocks some of the substrate-binding unprimed subsites including the specificity-determining subsite (S2) of the enzyme and thereby partially occludes accessibility of the substrates to the active site, which also corroborates the experimental observations The CT-extension in the model structure shows tight packing with the catalytic domain of the enzyme, mediated by strong hydrophobic and H-bond interactions, thus restricting accessibility of its cleavage sites to the protease itself or to the external proteases Kinetic stability analyses (T50 and t1 ⁄ 2) and refolding experiments show similar thermal stability and refolding efficiency for both forms These data suggest that the CT-extension has an inhibitory role in the proteolytic activity of ervatamin-C but does not have a major role either in stabilizing the enzyme or in its folding mechanism Structured digital abstract l ErvC cleaves ErvC by protease assay (View interaction) l trypsin cleaves ErvC by protease assay (View interaction) Introduction Papain-like cysteine proteases (EC 3.4.22) from plant sources are of industrial and biotechnological importance because these enzymes are better suited to various industrial processes [1] A cysteine protease is expressed as an inactive precursor in a pre-proenzyme form which contains a signal peptide (pre-), an inhibitory Abbreviations CT, C-terminal; E-64, 1-[L-N-(trans-epoxysuccinyl)leucyl]amino-4-guanidinobutane; Erv-C, ervatamin-C; pNA, p-nitroanilide; rmErv-C+CT, recombinant mature ervatamin-C with C-terminal extension; rmErv-CDCT, recombinant mature ervatamin-C without C-terminal extension; rproErv-C+CT, recombinant proervatamin-C with C-terminal extension; rproErv-CDCT, recombinant proervatamin-C without C-terminal extension 3012 FEBS Journal 278 (2011) 3012–3024 ª 2011 The Authors Journal compilation ª 2011 FEBS S Dutta et al pro-region and a mature catalytic domain [2–4] Following synthesis, the pre-peptide is removed during passage to the lumen of the endoplasmic reticulum [2] The inactive proenzyme subsequently undergoes proteolytic processing to produce an active mature enzyme by autocatalytic cleavage of the propeptide part at the N-terminus [2] It is known that the propeptide at the N-terminus of the protease acts as an intramolecular chaperone to mediate correct folding of the protease [2] The mature catalytic domain of the enzyme of this family has a molecular mass of  21–30 kDa and shares a common fold with papain, the archetype enzyme of the family These proteases are folded into two compact interacting domains of comparable size, delimiting a cleft which contains the active site residues cysteine and histidine, forming a zwitterionic catalytic dyad (Cys) His+) [5] Sometimes a larger precursor is also synthesized which contains a C-terminal (CT) extension ⁄ propeptide in addition to the abovementioned N-terminal propeptide flanking the mature protease domain [6,7] Unlike N-terminal propeptides, the role of CT-extensions (or propeptides) is not yet well established Sometimes an endoplasmic reticulum retention signal Lys-Asp-Glu-Leu (KDEL) is found in the CT-propeptide which regulates the delivery of protease precursor to other cellular compartments [8] Some other members of the papain family from plant sources also contain a larger CT-propeptide domain which shares a similarity with animal epithelin ⁄ granulin and the function of this domain is reported to be involved in leaf senescence [6] In addition, a CT-propeptide without any specific domain or motif is observed in some papain-like cysteine proteases from plants like Nicotiana tabacam (Q84YH7), Actinidia chinensis (P00785) and Vicia sativa (Q41696) In most of the cases, such a CT-extension contains the vacuolar sorting signal and is cleaved inter- or intramolecularly after sorting [9] No conserved sequence motif has been found in the vacuolar sorting signal at CT-propeptide, rather an amphipathic-like (hydrophobic and acidic) motif is generally observed [9,10] at the core of such peptides Other than plant systems, a CT-extension found in a lysosomal cysteine protease (Lpcys2) of Leishmania pifanoi, plays a role in the regulation of enzyme activity [11] CT-extension in mammalian and yeast bleomycin hydrolase [12,13] is a key factor which regulates their endo-peptidase or exo-aminopepetidase activity by blocking the unprimed subsites in the enzymes Ervatamin-C (Erv-C) is a papain-like cysteine protease (EC 3.4.22) with high stability purified from the latex of a tropical plant Ervatamia coronaria [14] The Role of C-terminal extension in a cysteine protease 3D structure of Erv-C reveals an extra disulfide bond, shorter loop regions and additional electrostatic interactions in the interdomain space, which are thought to be responsible for its high stability [15] Sequencing of the cDNA (from mRNA) of Erv-C from the leaf of the plant in our laboratory [16], and comparison of the cDNA-derived amino acid sequence with other members of the family reveal that Erv-C is synthesized as a precursor protein and in addition to the pre- (19 amino acids), pro- (114 amino acids) and mature (208 amino acids) parts, it contains an extension of 24 amino acids at the CT of the mature enzyme [16] (Fig 1A) This CT-extension was not observed, however, when the mature Erv-C was purified directly from the latex of the plant [15] In this article, we attempt to understand the role of this CT-extension in zymogen activation, enzyme activity, folding and stability in vitro at the molecular level from structural and functional points of view Results Cloning, expression, purification and refolding of rproErv-CDCT and rproErv-C+CT Both the proteins, recombinant proervatamin-C without the CT-extension (rproErv-CDCT) and recombinant proervatamin-C with the CT-extension (rproErvC+CT), were expressed in E coli as inclusion bodies with an apparent molecular mass of  41 and  43 kDa (Fig 1B), respectively, which is consistent with the estimated molecular masses of their deduced amino acid sequences Correct refolding was checked by gelatin gel assay The condition and efficiency of refolding were almost similar for both forms with > 90% recovery of the folded form from Ni-NTA purified protein for each (Table S1) Activation to mature protease The purified refolded rproErv-C+CT could be converted into its mature active form (rmErv-C+CT) by using cysteine (20 mM) as the activator in 50 mM Tris buffer, pH 8.00, at 60 °C for 25–30 min, whereas the purified refolded rproErv-CDCT could be converted into its mature form (rmErv-CDCT) by the same activator in 50 mM Na-acetate buffer, pH 4.5, at 60 °C for 45 (Fig 2A) Thus the zymogen activation process occurs at different pH and time of maturation for the two enzymes The molecular mass of the mature enzyme rmErv-C+CT is higher ( 27 kDa) than that of rmErv-CDCT ( 25 kDa) as observed in the SDS ⁄ PAGE analyses (Fig 2A) This difference in FEBS Journal 278 (2011) 3012–3024 ª 2011 The Authors Journal compilation ª 2011 FEBS 3013 Role of C-terminal extension in a cysteine protease 19 aa N-Pro 208 aa 34 aa CT - ex Stop 114 aa Xho1 365 aa 24 aa 208 aa N-Pro N Pro II 24 aa Protease domain BamH H1 Start I 114 aa Pre A S Dutta et al Protease domain CT - ex 34 aa III 208 aa N-Pro NP Xho1 114 aa Stop BamH1 Start 380 aa Protease domain 356 aa MSTLFIISILLFLASFSYAMDISTIEYKYDKSSAWRTDEEVKEIYELWLAKHDKVYSGLVEYEKRFEIFKDNLKFIDEH S S S S SS SG S NSENHTYKMGLTPYTDLTNEEFQAIYLGTRSDTIHRLKRTINISERYAYEAGDNLPEQIDWRKKGAVTPVKNQGKCG SCWAFSTVSTVESINQIRTGNLISLSEQQLVDCNKKNHGCKGGAFVYAYQYIIDNGGIDTEANYPYKAVQGPCRAAK KVVRIDGYKGVPHCNENALKKAVASQPSVVAIDASSKQFQHYKSGIFSGPCGTKLNHGVVIVGYWKDYWIVRNSW GRYWGEQGYIRMKRVGGCGLCGIARLPYYPTKAAGDENSKLETPELLQWSEEAFPLA IV B 66 kDa 45 kDa 36 kDa 29 kDa 24 kDa 20 kDa Fig (A) (I) Open reading frame of Erv-C precursor, pre-pro-ErvC (II) Recombinant ervatamin-C with C-terminal extension, rproErv-C+CT (III) rproErv-CDCT, recombinant ervatamin-C without C-terminal extension Red indicates vector portion and ‘aa’ stands for amino acids (IV) The amino acid sequence of the open reading frame The sequence of CT-extension is in red (B) Lanes and 2, Purified proteins rproErvC+CT and rproErv-CDCT, respectively; lane 3, Molecular mass markers molecular mass almost fits the theoretically calculated value for the 24-amino-acid CT-extension We expected, therefore, that the CT-extension continued to remain attached with the mature enzyme even after autocatalytic processing of rproErv-C+CT Gelatin gel 3014 assay (Fig 2B) and western blot analyses (Fig 2C) also confirmed the retention of the CT-extension in the mature rmErv-C+CT Because Erv-C isolated from the latex of the same plant does not show the CT-extension, the possibility FEBS Journal 278 (2011) 3012–3024 ª 2011 The Authors Journal compilation ª 2011 FEBS S Dutta et al Role of C-terminal extension in a cysteine protease A 97 kDa 66 kDa 66 kDa 43 kDa rproErv-C+CT 45 kDa 36 kDa 29 kDa rmErv-C+CT 20 kDa M 45 30 25 20 15 10 C rproErv-CΔCT 29 kDa 24 kDa rmErv-CΔCT 20 kDa M 45 30 25 20 15 10 C C B rproErv-C+CT rproErv-CΔCT rmErv-C+CT rmErv-CΔCT 2 Fig (A) Time course of activation to the mature form (I) rproErv-C+CT (II) rproErv-CDCT as discussed in Materials and methods Time intervals of 0–45 are indicated for the respective lanes Untreated proteins (control) are labelled as ‘C’, ‘M’ denotes the molecular mass marker (B) Gelatin gel assay of activated rproErv-CDCT (lane 1) and rproErv-C+CT (lane 2) (C) Western blot analysis Lane 1, Erv-C purified from the plant latex; lanes and 3, purified and refolded rproErv-C+CT and rproErv-CDCT [Correction added on 26 July 2011 after original online publication: in the figure, labelling for part C was changed from ‘rproErv-C+CT, rproErv-CDCT, rproErv-C+CT and rproErv-CDCT to rproErv-C+CT, rproErv-CDCT, rmErv-C+CT and rmErv-CDCT’] of removal of the CT-extension by other plant proteases in vivo could not be ruled out To explore this possibility, we performed a trans mode activation of rproErv-C+CT in vitro using seven different proteases (four cysteine proteases Erv-A, -B, -C and papain from the plant latex; two serine proteases trypsin and chymotrypsin from bovine pancreas; one aspartic protease pepsin from porcine stomach mucosa), each having sequences specific for their cleavage in the amino acid sequence of the CT-extension The activated mature protein thus generated in each case shows a band at the same position ( 27 kDa) like that in the autoactivated enzyme, as observed in SDS ⁄ PAGE analyses (Fig S1), indicating that these external enzymes can not cleave the CT-extension Even with a prolonged digestion time (24 h), the same result was obtained for all proteases except trypsin (Fig S1) Trypsin digestion for 24 h resulted in a truncated protein at  26 kDa, slightly above the activated mature rmErv-CDCT ( 25 kDa) This result probably indicates that trypsin has some accessibility to its sites of specificity (Lys, which is in position of the CT-extension) (Fig S2) and only after a prolonged incubation time can it result in a band at a slightly lower molecular mass position, as observed in the SDS ⁄ PAGE analysis (Fig S1) Specific activity and optimum temperature of activity The optimum temperature of activity, Topt (Fig 3), for both forms is 65 °C Interestingly, it was observed that rproErv-C+CT shows no activity below 45 °C and then activity rises sharply from 60 °C onwards, reaching a maximum at 65 °C In the case of rproErv-CDCT, a gradual increase in activity with temperature was observed until it reached its maximum at 65 °C At Topt (65 °C), the specific activity of rmErv-CDCT was found to be almost double that of rmErv-C+CT (Table 1) At 37 °C, however, rproErv-CDCT shows measurable proteolytic activity, whereas no activity was seen for rmErv-C+CT Kinetic measurements of the recombinant proteins Kinetic constants of rproErv-C+CT and rproErv-CDCT were measured at room temperature against N-benzoyl-Phe-Val-Arg-p-nitroanilide (pNA), a tripeptide substrate with a valine at the P2 position which is known to act as a substrate for Erv-C [17] The kinetic constants of the two recombinant enzymes (Table 1) clearly show that rproErv-CDCT has almost 10 times FEBS Journal 278 (2011) 3012–3024 ª 2011 The Authors Journal compilation ª 2011 FEBS 3015 Role of C-terminal extension in a cysteine protease Table Kinetic stabilities ND, not determined 110 rproErv-C+CT 100 Tmax (°C) rproErv-CΔCT 90 Residual activity (%) S Dutta et al 80 rproErv-C+CT (activity at 65 °C) rproErv-CDC (activity at 65 °C) Native mature Erv-C (latex) [14,32] 70 60 50 40 T50 (°C) t1 ⁄ at 65 °C (min) Topt (°C) 50 76  400 65 45 72  400 65–70 70 76 ND 50 30 20 10 100 Residual activity (%) 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Temperature (°C) Fig Determination of optimum temperature of activity (Topt) of rproErv-C+CT and rproErv-CDCT Purified proenzymes (10–20 lg) were converted to their respective mature forms and the percentage residual enzyme activities were determined with respect to the maximum activity using an azocasein assay at different temperatures, as described in Materials and methods Each data point is an average of three independent experiments having similar values (Table S3) higher activity than rproErv-C+CT One can probably conclude that the CT-extension has some inhibitory effect on the activity of the enzyme against a small peptide Thermal stability Temperatures of maximum proteolytic activity (Tmax) for rproErv-C+CT and rproErv-CDCT are 50 and 45 °C (Table and Fig 4) and they retain > 90% activity up to 65 and 60 °C, respectively These data indicate a good thermotolerance for these enzymes The pattern of retention and fall in activity beyond Tmax was more or less the same for both enzymes The T50 values for rproErv-C+CT and rproErv-CDCT were 76 and 72 °C 80 60 40 20 rproErv-C+CT rproErv-CΔCT 40 45 50 55 60 65 70 75 80 85 90 Temperature (°C) Fig Effect of temperature on activity of rproErv-C+CT and rproErv-CDCT Each purified proenzyme (10–20 lg) was treated for 10 at different temperatures followed by activation of the proproteins to their respective mature forms The percentage residual enzyme activities (at each temperature) were determined with respect to the maximum activity using an azocasein assay at 65 °C as described in Materials and methods Each data point is an average of three independent experiments having similar values (Table S3) (Fig 4), respectively The half lives (t1 ⁄ 2) at 65 °C were  400 (Fig 5) for both enzymes Molecular modeling studies To gain insight into the stability and dynamic properties of the structure, solvent MD simulation was Table Kinetic constants using the substrate N-benzoyl-Phe-Val-Arg-pNA Specific activity using azocaesin and IC50 value for the inhibitor E-64 ND, not determined kcat (s)1)a rproErv-C+CT rproErv-CDCT Latex Erv-C [17] Km (lM)a kcat ⁄ Km (s)1ỈmM)1) Specific activity at 37 °Cb (mg)1) Specific activity at 65 °Cb (mg)1) IC50 against E-64 (nM)c 0.0170 ± 0.004 0.2295 ± 0.057 9.312 88.33 ± 56.77 127.3 ± 48.46 1063 0.193 1.803 8.76 No activity 13.21 ± 2.32 75.0 15.87 ± 1.36 35.27 ± 1.78 ND 482.5 ± 108.0 349.1 ± 62.0 225.0 a Given standard errors were calculated based on nonlinear fitting of the Michaelis–Menten saturation curve using the software Graphpad (http://www.graphpad.com/prism) b Each value of specific activity of rproErv-C+CT and rproErv-CDCT is a mean of three independent experiments ± SD c Given standard deviations were calculated from linear regression plot of residual activity and inhibitor concentration PRISM 3016 FEBS Journal 278 (2011) 3012–3024 ª 2011 The Authors Journal compilation ª 2011 FEBS S Dutta et al Role of C-terminal extension in a cysteine protease 100 Residual activity (%) 90 rproErv-C+CT 80 rproErv-CΔCT 70 60 50 40 30 20 420 360 300 240 180 0 10 20 30 40 50 60 120 10 Time in Fig Time course of thermal inactivation of rproErv-C+CT and rproErv-CDCT at 65 °C An aliquot of the purified proenzymes (10–20 lg) was treated at 65 °C for to h followed by activation of the pro-proteins to their respective mature forms At each indicated time, the residual enzyme activity was determined using an azocasein assay at 65 °C, as described in Materials and methods, and values are expressed as percentage of the initial activity of the respective enzymes The experiment was carried out in duplicate for each data point (Table S3) performed The total energy of the whole system and root mean square deviation (RMSD) from the starting structure are essential to determine the sustainability and convergence of MD simulation Figure 6A shows the RMSD of backbone atoms of the CT-extension in association with a mature domain as well as in an isolated form The graph shows that the RMSD reached ˚ below  0.4 A when the extension is attached to the ˚ mature domain and is > A when the extension is on its own The fluctuation in the radius of gyration was also analyzed (Fig 6B) as a measurement of the overall stability of the CT-extension for both forms These analyses show that the extension achieves a relatively more stable conformation when it is attached to the mature domain The modeled structure of the CT-extension with the mature catalytic domain (rmErv-C+CT) shows that the extension peptide blocks some of the unprimed subsites of the enzyme (Fig 7) The interface area of the CT-extension and the mature catalytic domain is ˚ 1037 A2, which is  56% of the total surface area of the CT-extension The Leu side chain at the position 23 of the CT-extension occupies specificity pocket S2 of the catalytic domain and is stabilized mainly by hydrophobic interactions with S2 subsite residues A67, F68, A131, L155 and L201 (Fig 8) [17] Residue Phe21 of the CT-extension is buried inside a hydrophobic pocket of mature domain formed by V69, L201 Fig ns molecular dynamics trajectory of CT-extension part in association with the mature Erv-C domain (red) and in an isolated form (black) (A) Backbone RMSD and (B) radius of gyration A B Fig Surface presentation of mature Erv-C (A) Mature Erv-C without the CT-extension (Protein Data Bank ID 2PNS), the catalytic cleft is marked in red (B) Mature Erv-C with modeled CT-extension, the CT-extension is displayed in magenta and Y203 (Fig 8B) Other residues of the CT-extension are stabilized by electrostatic and hydrophobic interactions with the mature catalytic domain of the enzyme Superposition of the crystal structure of the complex of mature Erv-C (without CT-extension) with the inhibitor 1-[L-N-(trans-epoxysuccinyl)leucyl]amino4-guanidinobutane (E-64; Protein Data Bank ID 2PRE) and the modeled structure of Erv-C with CT-extension (rmErv-C+CT) reveals that the Leu of FEBS Journal 278 (2011) 3012–3024 ª 2011 The Authors Journal compilation ª 2011 FEBS 3017 Role of C-terminal extension in a cysteine protease S Dutta et al E-64 at P2 position of the inhibitor lies close to Leu23 of the CT-extension of the rmErv-C+CT molecule with ˚ a minimum distance of 1.9 A between two atoms of the two leucines (Fig 8A) Because the Leu23 residue of the CT-extension of the rmErv-C+CT molecule is buried inside the S2 pocket, it restricts access of Leu at the P2 position of E-64 and results in a higher IC50 value (482.5 nM) of E-64 inhibition compared with rmErv-CDCT (349.1 nM) (Table 1) in which the extension is truncated Modeling studies also reveal that although this CT-extension blocks some of the unprimed subsites beyond S2, access to the catalytic centre (Cys-His dyad) is not totally blocked A Discussion B Fig (A) The structure of the catalytic cleft and unprimed subsites region of mature Erv-C with modeled CT-extension The mature catalytic domain is represented as an electrostatic potential surface and the CT-extension as a stick model with C atoms in green The inhibitor E-64 (taken from the structure of Erv-C and E-64 complex; Protein Data Bank ID 2PRE) is also docked for comparison and is represented as a stick model with C atoms in brown The catalytic cysteine residue (C25) of Erv-C is represented as a ball and stick model (light blue) and the S2 subsite residues are represented as a stick model (light pink) The minimum distance ˚ (1.9 A) between P2 (Leu23) of the CT-extension and P2(Leu) of E-64 are indicated by the dotted line (B) The last four residues, F21P22L23A24, of the CT-extension domain (C atoms are shown in magenta) and the neighboring residues of Erv-C mature domain ˚ within 4.2 A (C atoms are shown in deep bottle green) Interdomain ˚ distances within 4.2 A are shown in green 3018 It is known that the autocatalytic processing of papain-like cysteine proteases from pro- to mature form generally occurs at acidic pH [18] The 3D structures of papain-like cysteine proteases in the pro-form [19–23] reveal that N-terminal propeptide part adopts a specific globular structure which is conserved among the family despite a relatively low homology in their amino acid sequences Previous reports suggest that an acidic pH induces a conformational change in the N-terminal propeptide domain, resulting in a molten globule state and thereby the activation process is triggered [24] The molten globule state of the N-terminal propeptide domain results in a reduction in the association affinity of the propeptide towards the protease domain and cleavage of the propeptide occurs leading to a mature active enzyme To date, there has been practically no detailed report available in the literature on the role of the CT-extension (or CT-propeptide) in the maturation process for proteases in this family There is one study on kiwifruit cysteine protease, actinidin, which shows that its C-terminal propeptide is required for correct processing [7] In our studies, we have observed that for the precursor rproErvC+CT, in vitro autoactivation essentially removes the N-terminal propeptide part leading to a mature active protease (rmErv-C+CT) with a molecular mass of  27 kDa (Fig 2) with the CT-extension remaining attached Moreover, this autocatalytic processing does not occur at acidic pH, instead autoactivation is found to occur at a basic pH of 8.0 By contrast, in rproErvCDCT, in vitro activation to mature enzyme (rmErvCDCT) occurs at an acidic pH of 4.5, like in other members of the family [17] The 24-amino-acid CT-extension contains six negatively charged residues (one aspartate and five glutamates) (Fig 1A) and our previous molecular modeling studies [16] indicated that this negatively charged region of the C-terminus could FEBS Journal 278 (2011) 3012–3024 ª 2011 The Authors Journal compilation ª 2011 FEBS S Dutta et al be positioned structurally in such a way that it could interact with a positively charged zone of the N-terminal propeptide in the zymogen Therefore, one may postulate that at acidic pH, this electrostatic interaction between the N- and C-terminal propeptides ⁄ extensions may be strong enough to restrict the conformational change in the N-terminal propeptide, required for the activation process But at basic pH, this interaction may become weak, leading to a free and flexible C-terminus, and the presence of the six acidic residues in this region may locally mimic an acidic environment to trigger the activation process This may be the explanation for autocatalytic activation at a higher pH for rproErv-C+CT which is not observed when the protein is expressed without the extension part in rproErv-CDCT Because the C-terminal extension of 24 residues remains intact in the mature form of the rmErv-C+CT molecule (Fig 2) after in vitro processing, autocatalytic trimming does not occur in this case It should be mentioned, however, that in some vacuolar papain-like cysteine proteases the CT-extension (propeptide), carrying a vacuolar sorting signal, is cleaved by intermolecular proteolysis by a different vacuolar protease in vivo [9,10] So, in this study, we performed an in vitro transactivation experiment using three latex enzymes Erv-A, -B and -C from the same plant, and papain from papaya latex and two other serine (trypsin and chymotrypsin) and one aspartic (pepsin) proteases The results showed that this extension is also not cleaved by any of these proteases in their optimum condition of activity However, because no experiment has been carried out in vivo, the possibility of intermolecular cleavage of the CT-extension by a different vacuolar protease under specific in vivo conditions can not be ruled out The results of enzyme kinetic studies show that the proteolytic activities of the two recombinant forms (rproErv-C+CT and rproErv-CDCT) are not of the same order (Table 1) In optimum temperature determination using azocasein as a substrate, rmErv-C+CT does not show any proteolytic activity when activity is assayed below 45 °C, but activity increases suddenly beyond 60 °C and sufficient activity is retained in the temperature range 65–75 °C (Fig 3) with the highest being at 65 °C This activity profile is different for rmErv-CDCT where activity increases systematically with temperature, although the highest activity in this case is also at 65 °C (Fig 3) However, the specific activity of rmErv-CDCT is almost twice as high as that of rmErv-C+CT at their optimum temperature of activity (65 °C) (Table 1) When the activity is measured with a small peptide like N-benzoyl-Phe-Val-Arg-pNA, Role of C-terminal extension in a cysteine protease both forms, rmErv-C+CT and rmErv-CDCT, show activity at room temperature although the former has a 10 times lower Kcat ⁄ Km value (Table 1) Thus, for a small peptide, enzymatic activity is observed at room temperature for rmErv-C+CT (around 25 °C), which is totally absent when this form is assayed with azocaesin (Table 1) These data suggest that the CT-extension interferes with a protein substrate at a lower temperature and the enzyme active site is not accessible to the protein substrate But at a higher temperature (beyond 60 °C), this interference is partly removed and the enzyme can show proteolytic activity to the protein substrate This behavior of the enzyme differs for a smaller peptide substrate, where the enzyme can work on this peptide even at lower temperatures, although to a lesser extent Molecular modeling studies show that the C-terminal tail blocks the unprimed subsites beyond S2 of the enzyme thus inhibiting the endopeptidase activity at temperatures below 55 °C for azocaesin Perhaps, at a higher temperature, a reduction of the association affinity for this tail towards the unprimed subsites occurs leaving the subsites partially free for substrate binding This is in conformity with a report [25] that the CT-extension sometimes has an inhibitory property towards some enzymes Blocking of unprimed subsites is also found in other papain-like proteases by different mechanisms either by a minichain like cathepsin-H [26] or an exclusion domain in cathepsin-C [27] or by C-terminal extension in bleomycin hydrolase [12,13] In all these cases, each protease loses its endo-peptidase activity and functions as an aminopeptidase because their unprimed subsites are blocked as found here A superposition of the structures of cathepsin-H and bleomycin hydrolase on the modeled structure of rmErv-C+CT revealed that although the blocking strategy of these peptides is similar, in the first two proteases the blocking peptides extend closer to the active site than in rmErv-C+CT (Fig 9) We also note from this study that the catalytic activity and maturation kinetics (both pH of activation and time of activation) of the two forms vary (Fig 2A) Alternative splicing in plant systems has been reported [28] to affect the stability and translatability at the RNA level and produce truncated or extended proteins with altered (increased, decreased or loss of) activity, cellular localization, regulation and ⁄ or stability Here, we used the clone of Erv-C which was constructed from the cDNA (mRNA) of the leaf of the plant [16] and the deletion mutant of that has been generated by eliminating the last 24 amino acids at the C-terminus The mature Erv-C isolated from the latex of the plant also does not have the CT-extension [15] Because both FEBS Journal 278 (2011) 3012–3024 ª 2011 The Authors Journal compilation ª 2011 FEBS 3019 Role of C-terminal extension in a cysteine protease S Dutta et al Fig Superposition of the catalytic cleft and the unprimed subsite region of cysteine proteases blocked by different peptides like the mini-chain in cathepsin H (Protein Data Bank ID 8PCH; sky blue), CT-extension in bleomycin hydrolase (Protein Data Bank ID 1CB5; green) and the modeled CT-extension in Erv-C (magenta) The E-64 molecule (brown) in complex with Erv-C (Protein Data Bank ID 2PRE) is also used in the superposition for comparison The respective catalytic domains of the proteases are shown in Ca presentation in corresponding colors The Ca atoms of the catalytic dyad residues Cys and His are shown in yellow and blue, respectively enzymes, latex Erv-C and recombinant mature rmErvCDCT without a CT-extension, appear to be the same, having a similar molecular mass (Fig 2C) and similar enzymatic properties [29], we cannot ignore the possibility that Erv-C may exist in two isoforms (splice variants) in different plant tissues, and that the activation requirements of the two isoforms are different depending on the presence or absence of a 24-amino-acid negatively charged CT-extension We have noted that both recombinant protease forms can fold efficiently in vitro So the CT-extension does not appear to have any noticeable effect on proper folding of the protein in vitro However, our observations also indicate that this extension has some effects in the maturation process and activity of the protease It suggests two possibilities for the presence of the CTextension in the enzyme: either it carries the signal for vacuolar sorting and after that is cleaved by another enzyme in vivo, or an mRNA (cDNA) exists in the leaf of the plant that essentially encodes an isoform of ErvC which is present in the latex of the plant Materials and methods vector [16] A fragment of the original cDNA encoding the prodomain, the mature domain and CT-extension of Erv-C was PCR-amplified from this clone using primers (Forward: 5¢-CCCGGATCCATGGACATATCTACC-3¢ and Reverse: 5¢-GGTCTCGAGTTAAGCAAGTGGAAAAGCT-3¢) designed to delete the pre-peptide (the signal peptide) and to include the restriction sites for BamHI and Xho1 (underlined) to facilitate cloning into pET-28a(+) expression vector (Novagen, Madison, WI, USA) The amplified product was then subcloned into respective restriction sites of pET28a(+) expression vector The resultant plasmid was transformed in E coli strain DH10B Insertion of the correct gene ⁄ transcript was confirmed by DNA sequencing, restriction digestion and colony PCR with gene ⁄ vector-specific primers This subcloned transcript was named rproErvC+CT and it was expressed using E coli strain BL21(DE3) Hexa-His-tagged recombinant protein expression was carried out as described earlier [29] except that the cells were grown for h after induction with 0.5 mM isopropyl b-Dthiogalactoside instead of overnight The overexpressed recombinant rproErv-C+CT was purified by Ni-NTA affinity chromatography (Qiagen, Hilden, Germany) under a denaturing condition and refolding of the eluted purified protein was done as for rproErv-CDCT by dialysis method [29] The refolded protein was concentrated by Amicon Ultra-4 (10 kDa cut-off) for further studies The generation of rproErv-CDC clone and its expression and purification was done as described previously [29] Conversion of rproErv-C+CT to its mature form Autocatalytic processing In vitro conversion of the purified and refolded rproErvC+CT into its mature and active form was performed by optimizing several parameters like proper activator (reducing agent), concentration of the activator, pH, temperature and time At a designated time interval, aliquots of the sample were collected from the reaction mixture and mixed with an equal volume of · SDS ⁄ PAGE gel loading sample buffer containing 2–3 mM irreversible inhibitor E-64 and analyzed by SDS ⁄ PAGE gel to optimize the time required for complete maturation Proenzyme processing of rproErv-C+CT was compared with rproErv-CDCT by SDS ⁄ PAGE analysis Purity of the mature forms of rproErv-C+CT and rproErv-CDCT was established by western blot analysis using rabbit antiserum raised against pure and mature Erv-C from the plant latex as primary antibody (Bangalore Genei, Bangalore, India) using the protocol described earlier [29] Wild-type Erv-C from plant latex was also used for comparison Cloning, expression, purification and refolding of rproErv-C+CT Effect of external proteases on C-terminal processing The entire open reading frame of the Erv-C precursor had been cloned previously in our laboratory in pTZ57R ⁄ T To determine whether the CT-extension of rproErv-C+CT can be cleaved by external proteases in trans mode, four 3020 FEBS Journal 278 (2011) 3012–3024 ª 2011 The Authors Journal compilation ª 2011 FEBS S Dutta et al cysteine proteases [Erv-A, -B and –C (isolated from the latex of Ervatamia coronaria in our laboratory) and papain from Carica papaya (Merck, Kenilworth, NJ, USA)], two serine proteases [trypsin and chymotrypsin from bovine pancreas (Sigma-Aldrich, St Louis, MO, USA)], one aspartic protease [pepsin from porcine stomach mucosa (SRL, Mumbai, India)] were used Purified rproErv-C+CT (3 mg) was digested using the abovementioned proteases (90 lg) at the optimum pH and temperature of activity of each protease In brief, digestion by Erv-A, Erv-B, Erv-C were carried out at pH 8.0 and 37 °C for h and blocked by E-64; digestion by papain was carried out at pH 6.5 and 60 °C for 0.5 h with 20 mM cysteine, mM EDTA and blocked by E-64; trypsinization was carried out at pH 8.0 and 37 °C for h and blocked by complete mini EDTA free cocktail inhibitor (Roche, Mannheim, Germany); digestion by chymotrypsin was carried out at pH 8.0 at 50 °C for h with mM CaCl2 and blocked by complete mini EDTA free cocktail inhibitor; pepsinization was carried out at pH 4.0 and 37 °C for h and blocked by E-64 and pepstatin Purified rproErv-C+CT was also digested using the abovementioned enzymes separately at 20 °C for 24 h under the same conditions The proteolytic digestion in each experiment was checked by SDS ⁄ PAGE analysis Measurement of proteolytic activity Substrate gel zymography using 0.1% gelatin as a substrate was used to demonstrate the protease activity of recombinant refolded proteases (rproErv-CDCT and rproErv-C+CT) using a protocol described previously [29] The specific activity of the recombinant proteases rproErv-CDCT and rproErv-C+CT was determined using substrate azocasein The specific activity of the native mature Erv-C isolated from the plant latex was also determined using the same protocol for comparison (Table 1) For this assay, a reaction mixture containing 0.5 mL of 0.2% azocasein in Tris ⁄ HCl buffer (pH 8.0), 0.5 mL of recombinant proenzyme (10–20 lg) activated in Tris ⁄ HCl buffer (pH 8.0) and incubated for 30 The reaction was then terminated with 5% trichloroacetic acid The mixture was centrifuged at 9300 g for to remove precipitate and the absorbance of the supernatant was measured at 366 nm to determine the amount of released azopeptides using the specific absorption coefficient (A1%366 = 40) for azocasein solution [30] One enzyme unit was defined as the amount of soluble protease required to release lg of soluble azopeptidesỈmin)1 The specific activity was the number of units of activity per milligram of protein For specific activity measurements the concentration of pro-protease has been used in the calculations because once the proteases are autocatalytically activated as described above, it is difficult to isolate the active mature enzyme from degraded propeptide parts Role of C-terminal extension in a cysteine protease Determination of optimum temperature for enzymatic activity of rproErv-CDCT and rproErv-C+CT Topt or ‘temperature optima’ is the temperature at which the enzyme shows maximum activity Proteolytic activity of both the recombinant proteins (rproErv-CDCT and rproErvC+CT) was measured in the range 30–90 at °C intervals to determine the optimum temperature of activity (Topt) for both recombinant proteins The pro-proteases were first converted to mature enzymes and the proteolytic activity was then measured using azocasein as a substrate as described above at specific temperatures Kinetic measurements using a chromogenic peptide The mature form of rproErv-C+CT, activated from lM proenzyme rproErv-C+CT, was used for this assay An Erv-C-specific chromogenic substrate N-benzoyl-Phe-ValArg-pNA (Sigma-Aldrich) [17] was used in this study for kinetic measurements of the recombinant proteins Liberated pNA was monitored for 15 at 410 nm on a UV ⁄ Vis spectrophotometer (Nicolet Evolution 100; Thermo Electron Corporation, Rockville, MD, USA) Conditions for the measurement of the kinetic parameters of rproErv-C+CT are as described earlier [29] Km and Vmax values of rproErv-C+CT were calculated by nonlinear fitting of the Michaelis–Menten saturation curve using the software Graphpad PRISM (http://www.graphpad.com/ prism) The kcat value was calculated by using the equation kcat =Vmax ⁄ [E]T where [E]T is the total concentration of the active enzyme, the values of which were measured by active-site titration with the irreversible inhibitor, E-64 using the above mentioned pNA containing substrate, as described below An extinction coefficient of 8800 M)1Ỉcm)1 at 410 nm for pNA was used for the calculations Measurement of IC50 value of E-64 Aliquots of lM proenzyme (rproErv-C+CT and rproErvCDCT) were converted into their respective mature forms The irreversible inhibitor, E-64 was added in increasing concentrations to the aliquots until the residual activity reached The residual activity (DA410 nmỈmin)1) was determined with respect to the activity of the enzyme (carried out without any inhibitor) as described in the previous section, against the peptide substrate (N-benzoyl-Phe-Val-ArgpNA) for both the proteases This residual activity of the enzyme was plotted against the inhibitor concentration The inhibitor concentration required for half-maximal inhibition (IC50) of E-64 for these enzymes were determined from these plots FEBS Journal 278 (2011) 3012–3024 ª 2011 The Authors Journal compilation ª 2011 FEBS 3021 Role of C-terminal extension in a cysteine protease S Dutta et al Thermal stability of rproErv-CDCT and rproErv-C+CT The effect of temperature on the kinetic stability of the purified enzymes was investigated by incubating the proteins at different temperatures Purified proenzyme was incubated at different temperatures in the range of 40–90 °C at °C intervals for 10 each and then immediately chilled on ice The activity of the proteases was measured at 65 °C (optimum temperature of activity for the proteases) against azocasein as substrate, as described previously Tmax was expressed as the temperature of incubation at which the enzyme showed maximum activity and T50 was expressed as the temperature of incubation at which the enzyme showed 50% of maximum activity The half-life (t1 ⁄ 2) of an enzyme is the time it takes for the residual activity to reduce to half of the original activity after the enzyme is incubated at a particular temperature, generally at Tmax The t1 ⁄ values of two enzymes were estimated by incubating the proteins at 65 °C (Tmax for both the enzymes) for periods between and h and then the residual enzymatic activity was determined by azocasein assay at 65 °C Molecular modeling of the mature Erv-C with CT-extension (rmErv-C+CT) The X-ray structure of mature domain of Erv-C, without the CT-extension, was solved previously in our laboratory (Protein Data Bank ID 2PNS) We have now performed a BLASTP (http://blast.ncbi.nlm.nih.gov/) protein sequence search of the amino acid sequence of CT-extension part using RCSB Protein Data Bank (http://www.rcsb.org/pdb) and found no significant sequence homology with any structure in the database Therefore, homology modeling to generate the structure of the CT-extension domain did not seem to be possible However, we did find sequence homology for the three stretches: the first residues, residues 11– 19, and the last four residues (21–24) of the CT-extension, separately with some corresponding stretches of three different proteins having Protein Data Bank IDs 2X7X, 1PV8 and 3OHM respectively (Table S2) We therefore decided to build the structures of the three stretches of the CTextension from the corresponding parts of the abovementioned Protein Data Bank structures after replacing mismatched residues appropriately As a next step, we fitted the three structural stretches individually at the mature domain of Erv-C (Protein Data Bank ID 2PNS), as described below, and finally the stretches were threaded by the connecting amino acid residues (8–10 and 20) to generate the entire structural model of the CT-extension in association with mature Erv-C The last four residues (24–21) generated from the template (Protein Data Bank ID 3OHM, 444–447) were initially docked manually into the unprimed subsites of Erv-C with Leu23 in the S2 pocket, 3022 considering the specificity of Erv-C towards a Leu residue [17] The mode of interactions of mini-chain of human lysosomal cysteine protease cathepsin H (Protein Data Bank ID 8PCH) and CT-extensions of human and yeast cysteine proteases bleomycin hydrolases (Protein Data Bank IDs 2CB5 and 1GCB) with the unprimed subsites of their cognate enzymes were also considered during this manual docking The docked tetrapeptide fragment of the CTextension was used as an anchor position and the next stretch of 19–11 residues, which showed a helical nature from both the template conformation (Protein Data Bank ID 1PV8, residues 320–329 of chain B) and the secondary structure prediction using the program DSC (Discrimination of Protein Secondary Structure Class) [31] implemented in DISCOVERY STUDIO 2.5 (Accelrys Inc., San Diego, CA, USA), were positioned and fitted, taking into account charge compatibility and hydrophobic interactions with respect to the structure of mature Erv-C The template (Protein Data Bank ID 2X7X, residues 44–50 of chain A) of the first seven residues of the CT-extension is a connecting polypeptide between a b sheet and an a helix In the structure of the mature domain of Erv-C (Protein Data Bank ID 2PNS) the C-terminus ends with a b sheet and therefore we connected this seven-residue fragment of the CT-extension to this b sheet of Erv-C here also, and oriented it towards the previously fitted helical stretch (19–11) Finally, these three fragments were joined by the connecting sequences (residues 8–10 and 20) and the rotamer conformation of the side chain of each residue was optimized The resulting complex of Erv-C together with the CT-extension was then globally optimized using ‘Smart Minimizer’ protocol in DISCOVERY STUDIO 2.5 (Accelrys Inc.) keeping the Ca position of the mature domain of Erv-C fixed The ˚ model was then solvated with a 30 A water sphere A few cycles of minimization were performed without any restriction to optimize the model and the position of the water molecules In the last step, the entire assembly was simulated at 300 K for 2.6 ns using ‘Standard Dynamic Cascade’ in DISCOVERY STUDIO (40 ps heating from 50 to 300 K, 20 ps equilibration at 300 K and ns production run at 300 K), keeping all Ca atoms of the mature part of the enzyme fixed Simulations were carried out at constant volume and temperature (NVT ensemble) through the Leapfrog Verlet integrator A time-step of fs for integration was used and a bond constraint was applied for all covalent bonds with H atoms through the SHAKE algorithm implemented in the program Coordinates were saved at 10-ps intervals for a 2-ns product run for analysis All simulations were carried out with CHARMm force field implemented in DISCOVERY STUDIO 2.5 with a non-bonded ˚ cut-off of 14 A The last 100-ps average structure from the simulation was further optimized for 100 steps Steepest Descent without any restraint and used as the model for structural interpretations in this study In the model thus generated, the length of the helix of CT-extension was FEBS Journal 278 (2011) 3012–3024 ª 2011 The Authors Journal compilation ª 2011 FEBS S Dutta et al reduced (residues 12–16) from the starting structure (residues 11–19) At the end of the simulation, the CT-extension was isolated by deleting the mature domain and the extension was allowed to relax in a same simulation protocol as described above for comparison The simulation trajectory was analyzed by ‘Analysis’ protocol of DISCOVERY STUDIO 2.5 The geometrical and structural consistencies of the model structure of Erv-C together with CT-extension were evaluated by different approaches The stereochemical quality of the model was evaluated using PROCHECK [32] and VERIFY3D [33] This structure was further validated using web-based software QMEAN [34], PROQ [35] and SOLX [36] All these validations (Table S2) signify that the model is of good quality and is reliable Acknowledgement The work was partially supported by 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Protein Sci 12, 1073–1086 Holm L & Sander C (1992) Evaluation of protein models by atomic solvation preference J Mol Biol 225, 93–105 Supporting information The following supplementary material is available: Fig S1 SDS ⁄ PAGE showing C-terminal processing of rproErv-C+CT by external proteases Fig S2 The mature Erv-C with CT-extension Table S1 Yield of rproErv-C+CT and rproErv-CDCT from L culture at different purification steps from inclusion bodies Table S2 CLUSTALW alignment and structural validations of the model structure of CT-extension Table S3 Raw data sets related to thermal stability experiments This supplementary material can be found in the online version of this article Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 278 (2011) 3012–3024 ª 2011 The Authors Journal compilation ª 2011 FEBS ... extension in a cysteine protease 19 aa N-Pro 208 aa 34 aa CT - ex Stop 114 aa Xho1 365 aa 24 aa 208 aa N-Pro N Pro II 24 aa Protease domain BamH H1 Start I 114 aa Pre A S Dutta et al Protease domain CT... protease acts as an intramolecular chaperone to mediate correct folding of the protease [2] The mature catalytic domain of the enzyme of this family has a molecular mass of  21–30 kDa and shares... characterization of a highly stable cysteine protease from the latex of Ervatamia coronaria Biosci Biotechnol Biochem 62, 1947–1955 15 GuhaThakurta P, Biswas S, Chakrabarti C, Sundd M, Jagannadham MV & Dattagupta