First Archaeal PEPB-Serine Protease Inhibitor from Sulfolobus

First Archaeal PEPB-Serine Protease Inhibitor from Sulfolobus solfataricus with Noncanonical Amino Acid Sequence in the Reactive-Site Loop. Gianna Pal...
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First Archaeal PEPB-Serine Protease Inhibitor from Sulfolobus solfataricus with Noncanonical Amino Acid Sequence in the Reactive-Site Loop Gianna Palmieri,†,§ Giuliana Catara,†,§ Michele Saviano,*,‡ Emma Langella,‡ Marta Gogliettino,† and Mose` Rossi† Istituto di Biochimica delle Proteine, Via P. Castellino 111, 80131 Napoli, Italy, and Istituto di Biostrutture e Bioimmagini, Consiglio Nazionale delle Ricerche, Via Mezzocannone 16, 80134 Napoli, Italy Received August 1, 2008

The specific inhibition of serine proteinases, which are crucial switches in many important physiological processes, is of great value both for basic research and for therapeutic applications. In this study, we report the molecular cloning of the sso0767 gene from Sulfolobus solfataricus, and the functional characterization of its product, SsCEI, which represents the first archaeal phosphatidylethanolaminebinding protein (PEBP)-serine proteinase inhibitor, reported to date. SsCEI is a monomer protein with a molecular mass of 19.0 kDa and a pI of 6.7, which is able to inhibit the serine proteases R-chymotrypsin and elastase with Ki values of 0.08 and 0.1 µM, respectively. Moreover SsCEI is extremely resistant to both thermal inactivation and proteolytic attack suggesting compact folding of the protein. Within the I51 family, the archaeal inhibitor shows strong similarity to the human and murine members. The threedimensional model of SsCEI revealed a general β-fold and the presence of an anion-binding pocket, the hallmark of the PEBP family. Moreover SsCEI binds the cognate proteases according to a common, substrate-like standard mechanism. Point mutation experiments supported the prediction of the protease-binding site located on the surface at the C- terminal region of the protein. Interestingly, searches based on preidentified structural reactive loop motifs revealed the occurrence of a sequence (T123-N130) that is not represented in all serine-protease inhibitor families. This unique motif may provide new insights into both the inhibitor/protease binding mode and the specific biological functions of SsCEI within the PEBP family. Keywords: Serine protease inhibitor • PEBP • Sulfolobus solfataricus • protease inhibitor family • protease binding site • homology modeling

Introduction Organized into a number of distinct families, the protease inhibitors are ubiquitous proteins which have been identified in a wide variety of organisms. Considerable attention to date has been devoted to the eukaryotic serine protease inhibitors, which are assembled in gene families including from 10 up to 500 members.1 Comparative genome analyses have revealed that only three families of protease inhibitors are shared by prokaryotes and eukaryotes as well as the Archaea: these are the serpins (I4), chagasins (I42) and phosphatidylethanolaminebinding proteins (PEBP) (I51). The I51 family constitutes a distinct cluster characterized by the presence of a PEBP binding domain,2 and show no sequence similarity to other known proteinase inhibitors. The PEBP family contains over 500 members, only a small number of which have been individually characterized in terms of their specific biological function. They * To whom correspondence should be addressed. Michele Saviano, Phone: (39) 0812536648, Fax: (39) 0812534560, E-mail: [email protected] † Istituto di Biochimica delle Proteine. ‡ Istituto di Biostrutture e Bioimmagini. § These authors equally contributed to this paper. 10.1021/pr800587t CCC: $40.75

 2009 American Chemical Society

are multifunctional proteins, shown to be involved in signaling mechanisms during cell growth and differentiation via the modulation of catalytic activity of kinases or serine proteases, and by the recognition of lipids or nucleotides.3-7 Among the PEBP class, the TFS1 inhibitor from Saccharomyces cerevisiae8 and the mouse PEBP,2,9 the most studied members of the I51 family, are considered archetypal serine proteinase inhibitors. Recently, the human PEBP has been included as new member of this family as an inhibitor of proteasome chymotrypsin-like activity.10 The current study has been undertaken with the aim to isolate novel thermophilic serine-proteinase inhibitors from Archaea, in particular from the hyperthermophile Sulfolobus solfataricus, both for evolutionary studies and potential biotechnological applications. A computational genome analysis of S. solfataricus P2 revealed the occurrence of a unique and putative PEBP-protease inhibitor-encoding gene, sso0767. Through extensive S. solfataricus proteome investigation obtained via combined separation approaches,11 the Sso0767product was shown to be expressed in S. solfataricus cells in midexponential growth phase, representing one of the 1323 Journal of Proteome Research 2009, 8, 327–334 327 Published on Web 12/04/2008

research articles proteins identified from the soluble fraction, even though a functional role remains to be assigned. In this communication, we report on the recombinant expression and biochemical characterization of the sso0767 gene product, and on the discovery of the first archaeal protease inhibitor from S. solfataricus P2 belonging to the I51 family. Similar to its eukaryotic counterparts, the Sso0767 product was found to specifically inhibit R-chymotrypsin but not trypsin, a distinct feature of members of the I51 cluster. Moreover, owing to its ability to decrease elastase activity, it was named “Sulfolobus solfataricus-chymotrypsin-elastaseinhibitor” (SsCEI). Homology modeling of SsCEI and point mutation experiments demonstrated the functional importance of the predicted reactive-site loop with respect to the inhibition activity. Interestingly, the results of our studies lead to the discovery of a new motif for the protease binding site that cannot be classified among the canonical consensus sequences of the serine protease inhibitors.

Materials and Methods Gene Cloning. The gene sso0767 was obtained by PCR amplification of total genomic DNA from S. solfataricus strain P2 prepared according to Yoshida et al.,12 using the following oligonucleotides: P1 5′-CCATGGGCTTGAATAGTGAAAGTATATA3′ and P2 5′-GAGCTCTTTTCTCTTATATTTACCCATTACGAAT3′. The NcoI and XhoI restriction sites, underlined in the sequences, were introduced to allow ligation into the expression vector. The PCR amplification experiments were performed on thermal cycler PCR (Hybaid) in a 0.05 mL reaction mixture, containing 1× Finnzymes PCR buffer (Finnzymes), 0.2 mM of the deoxynucleoside triphosphates, 1 µM (each) primers, 2.5 U of Taq polymerase (Finnzymes) and 100 ng of genomic DNA template. The conditions used for the gene amplification were 95 °C for 5 min (one cycle), followed by 95 °C for 0.75 min, 60 °C for 1 min, 72 °C for 1 min (30 cycles), and final extension at 72 °C for 10 min. The PCR product was cloned by blunt ends into pMos vector (Promega) and the final construct verified by sequencing. All restriction enzymes were from New England Biolabs (NEB). The NcoI/XhoI 0.5 kb DNA fragment excised from the pMos vector was then subcloned into pET-28c expression vector (Novagen). Expression and Purification of the Recombinant Protein SsCEI. Escherichia coli cells strain BL21- CodonPlus RIL (Stratagene) harboring the pET28-0767 plasmid were grown at 37 °C in LB medium (50 mL) containing kanamycin (0.05 mg mL-1) until the optical density reached 0.6 OD600. Protein expression was induced by addition of 1 mM IPTG followed by further cultivation for 4 h. The harvested cells were suspended in 5 mL of 0.05 M sodium phosphate buffer, pH 8.0, containing 0.1 M phenylmethylsulfonyl fluoride (PMSF), and disrupted by French Press (American Instrument Company). After centrifugation, the supernatant fraction was recovered (5 mL, 9 mg of total proteins, 305 U of total inhibition activity) and the Sso0767-product identified by N-terminal sequencing. The sample was incubated at 70 °C for 30 min, and after centrifugation, the recombinant protein in the supernatant fraction (5 mL, 3.6 mg of total proteins, 478 U of total inhibition activity) was further purified by using the His-Select Spin Column (Sigma) following the manufacturer’s instructions. The sample eluted in 0.5 M imidazole (1.2 mL, 0.18 mg of total proteins, 1200 U of total inhibition activity) was then subjected to size exclusion chromatography on a Superdex G75 (1.6 × 328

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Palmieri et al. 88 cm) column eluted with 0.05 M Tris-HCl buffer, pH 8.0, containing 0.1 M NaCl. The recombinant protein (1 mL, 0.14 mg of total proteins, 1200 U of total inhibition activity) was desalted and then concentrated by ultrafiltration system for further characterization. Reverse Zimography. The detection of protease inhibition was achieved by reverse zymography SDS-PAGE analysis according to Le and Katunuma,13 with some modifications. After electrophoresis, the polyacrylamide gel was washed with 2.5% Triton X-100 for 1 h, rinsed with water and then equilibrated in Tris-HCl, 0.02 M CaCl2, pH 8.0. The gel was then soaked in equilibration buffer containing R-chymotrypsin (13 mg/mL) at 50 °C for 1 h. After removal of the enzymatic solution, the gel was incubated with fresh R-chymotrypsin (1 mg mL-1) dissolved in 0.05 M Tris-HCl, 0.02 M CaCl2, pH 8.0, at 37 °C overnight. After proteolysis, the gel was stained using Coomassie brilliant blue R-250. Chymotrypsin Inhibitory Activity of SsCEI. Three microliters of bovine TLCK-treated R-chymotrypsin (Sigma) (1 mg mL-1) and aliquots (0.08-2.5 µg) of SsCEI solution were preincubated at 50 °C for 30 min in Tris-HCl buffer, pH 8.0, containing 20 mM CaCl2 in 0.1 mL final volume. After the incubation, this sample was added to the reaction mixture containing 0.1 mM Suc-Ala-Ala-Pro-Phe-pN-anilide (Sigma), in Tris-HCl buffer, pH 8.0, in a final volume of 1 mL to assay the residual protease activity. Substrate hydrolysis was followed by absorbance increase at 410 nm (ε ) 8800 M-1 cm-1) against a blank (reaction mixture without the inhibitor). One protease inhibitory activity unity was defined as the amount of the inhibitor that produces a decrease of 0.01 OD/min of the protease activity under the described assay conditions. Serine Protease Inhibitory Activity of SsCEI. The enzymes and the related chromogenic substrates were all obtained from Sigma. Each reaction mixture, containing a 1:1 (pmol/pmol) molar ratio of SsCEI and the target protease, was preincubated at 50 °C for 30 min under the conditions described for chymotrypsin, in a final volume of 0.1 mL. Determination of Kinetic Properties. The kinetic parameters of SsCEI were determined by measuring the initial rate of the target protease activity. The inhibition constants Ki were determined by the Lineweaver-Burk equation. In these experiments, the residual enzymatic activity was measured after the protease (0.12 µM) and SsCEI (0.09 and 0.16 µM) were mixed and incubated at 50 °C as described above, and the samples were subsampled and assayed with increasing substrate concentrations (10-1000 µM). The values obtained were fitted with the Sigma plot software. Determination of Molecular Mass. The molecular mass of recombinant protease inhibitor was determined using a Superdex 75 PC 3.2/30 gel filtration column (Pharmacia) calibrated with LMW gel calibration kit Amersham. Mass Spectrometry Analysis. Purified SsCEI was loaded onto a C18 column by an HPLC system. The eluted protein was directly analyzed with a mass spectrometer QSTAR Elite (Applied Biosystems) equipped with an electrospray ionization source. Sequence Analysis. Automated N-terminal sequencing of the protein in solution was performed using a Perkin-Elmer Applied Biosystem 477A. The sequence database was searched using the BLAST-PSI program.14 Homology Modeling of the Serine Protease Inhibitor. An initial three-dimensional model of SsCEI was built using the SWISS-MODEL Protein Modeling Server. This model was

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completed using the “Homology” module of the INSIGHT/ DISCOVER package, running on a Silicon Graphics Octane2 workstation. Energy minimizations were carried out using the conjugate gradient algorithm to refine the model and to avoid high energy conformations of protein backbone and residue side chains. These procedures were stopped when the maximum derivative was e0.001 kcal/mol. All graphical analyses were also run on a SGI workstation, using the INSIGHT program. Generation of Point Mutations. Mutants of SsCEI were generated by Geneart (Regensburg, Germany). The synthetic gene mutants were assembled from synthetic oligonucleotides and/or PCR products and cloned into pGA4 (ampR). The final constructs were verified by sequencing. DNA fragment encoding mutations were cloned into the pET28c vector generating the plasmid pET28c_C259 mutant 1, mutant 2 and mutant 3 by NcoI and XhoI restriction sites.

Results Isolation of the Gene sso0767 Encoding SsCEI. The S. solfataricus genome database was searched for the presence of genes homologous to serine protease inhibitors using the BLAST algorithm. A unique gene (ORF sso0767) encoding a hypothetical protein with strong similarity to the protease inhibitors belonging to the I51 family [27% Identity (I) to human PEBP1 NP_002558.1; 25% I to mouse PEBP NP_061346.2; 33% I to the yeast TFS1 NP_013279.1; 38% I to E. coli YBCL NP_415077.1; and 36% I to E. coli YBHB NP_415294.1 (Figure S1 in Supporting Information)], was identified. The open reading frame encoding SsCEI (NC 002754), was isolated by direct PCR amplification from total S. solfataricus genomic DNA by using the P1 primer, which included the putative start codon of sso0767, and the P2 primer lacking the stop codon at 3′ end of the coding sequence to obtain the recombinant protein Histagged at the carboxyl terminus. Expression of SsCEI in E. coli, Purification and Characterization of the Recombinant Protein. SsCEI was purified from the crude extracts of induced E. coli cells, by a three-step purification procedure as described in Materials and Methods following the protease inhibitory activity toward R-chymotrypsin. The homogeneity of the purified SsCEI was confirmed by SDS-PAGE, Edman degradation analysis and gel filtration chromatography. Through this procedure, about 265-fold purification was achieved, with a final yield of 95% and 3 mg L-1 production level of the recombinant protein. SsCEI is a monomer showing a pI value of 6.7 and a molecular mass of 19.0 and 16.0 kDa as revealed by SDS-PAGE analysis and gel filtration chromatography, respectively. A more accurate determination, obtained by electrospray mass spectrometry, yielded a molecular mass of 19 007 Da, exactly matching the theoretical mass value corresponding to the deduced amino acid sequence of the recombinant His-tagged protein. Substrate Specificity and Kinetics. To screen for the biological function of SsCEI, we tested the ability of this protein to inhibit a panel of serine proteinases (trypsin, R-chymotrypsin, elastase, carboxypeptidase Y, subtilisin, and thrombin). Because of the thermophilic nature of the SsCEI, its activity should be assayed in a temperature range of 70-80 °C. However, in these conditions, an increase of the autocatalytic activity and/or thermal inactivation of the target mesophilic proteases occurred; hence, the preincubation of SsCEI with each proteases was performed at 50 °C. Beside inhibition activity toward

Figure 1. (A) Binding of Sso0767 to R-chymotrypsin. The hyperbolic curve indicates the best fit for the percentage inhibition data obtained, and the IC50 value was calculated from the graph. (B) Inhibition kinetics analysis. R-Chymotrypsin (0.12 µM) was incubated, without (•) or with SsCEI at 0.09 µM (9) and 0.16 µM (2) concentrations and assayed at increasing substrate concentrations. The reciprocals of the rate of the substrate hydrolysis for each inhibitor concentration were plotted against the reciprocals of the substrate concentrations. Ki was determined from the formula as per the competitive type of inhibition.

R-chymotrypsin (inhibitor specific activity 8.5 × 103 U/mg), which is the best target protease among those tested, results showed a clear activity versus elastase (inhibitor specific activity 3.3 × 103 U/mg), while trypsin, carboxypeptidase Y, subtilisin or thrombin were not affected by SsCEI. The specific interaction between SsCEI and the target proteases was also examined by incubating SsCEI in a reaction mixture containing all the proteases investigated. In these conditions, the catalytic performance of the inhibitor was identical to that observed by assaying each protease alone. The inhibition of SsCEI versus R-chymotrypsin followed a hyperbolic pattern with increasing concentrations of the inhibitor (Figure 1A) and the IC50 value (50% inhibitory concentration) was 0.10 µM. Since the secondary plot (the slope of inhibition graph versus SsCEI concentration) was linear, it was suggested that the application of Michaelis-Menten inhibition kinetics was appropriate in this study. The inhibition constant Ki, determined by the double reciprocal plot was 0.08 ( 0.01 µM, revealing a higher affinity of SsCEI for R-chymotrypsin with respect to the cognate PEBPserine protease inhibitor from mouse.2 Moreover, the Lineweaver-Burk reciprocal plot (Figure 1B) showed that SsCEI was a competitive inhibitor for R-chymotrypsin. The SsCEI IC50 value versus elastase was 0.15 µM, while the inhibition constant Ki determined using the double reciprocal plot was 0.10 ( 0.01 Journal of Proteome Research • Vol. 8, No. 1, 2009 329

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Figure 2. (A) Reverse zymography pattern of the inhibitor analyzed by the 15% SDS-PAGE. Lane 1, molecular weight marker; lane 2, the reverse zymography of SsCEI inhibitor; lane 3, purified inhibitor. (B) Resistance of SsCEI toward proteolytic attack. The purified inhibitor was incubated with different proteases at 37 °C for 30 min in 50 mM Tris-HCl buffer pH 8.0. After chilling on ice, each reaction mixture, containing inhibitor/protease with a 1:1 molar ratio, or protease without inhibitor, was subjected to SDS-PAGE analysis. For each experiment, the solution containing only the enzyme not incubated at temperature was run as control. Gel on the left, lanes 1-7 refer to reaction mixtures containing: 1, R-chymotrypsin; 2, R-chymotrypsin/inhibitor; 3 trypsin; 4, trypsin/inhibitor; 5, elastase; 6, elastase/ inhibitor; 7 inhibitor; 8, R-chymotrypsin control; 9, trypsin control; 10, elastase control. Gel on the right, lanes 1-5 refer to reaction mixtures containing: 1, carboxypeptidase Y; 2, carboxypeptidase Y/inhibitor; 3, thrombin; 4, thrombin/inhibitor; 5, inhibitor; 6, thrombin control; 7, elastase control.

µM. On the basis of these results, it can be claimed that SsCEI substrate specificity reflects that of the I51 family typically inhibiting R-chymotrypsin, but not trypsin. SsCEI-Protease Interaction. To assess the complex formation between SsCEI and the target proteases, a reverse zimography assay was performed. As shown in Figure 2A, following incubation at 37 °C in R-chymotrypsin solution, a unique band was visualized on the gel migrating at the molecular mass corresponding to the inhibitor analyzed under denaturing conditions. Experiments aimed to evaluate the susceptibility of SsCEI to proteolysis clearly indicated that the purified inhibitor was not hydrolyzed by the serine proteases tested (Figure 2B), but it plays a key role in preventing its target proteases from autocatalysis, thus, suggesting formation of a complex between SsCEI and R-chymotrypsin or elastase (Figure 2B, gel on the left side). Moreover, SsCEI was found to be extremely resistant to thermal inactivation (100% of residual inhibitory activity after 3 days of incubation at 90 °C). Three-Dimensional Model of SsCEI. The SsCEI preliminary model obtained from the SWISS-MODEL Protein Modeling Server Sequence was built using the X-ray structure of E. coli YBCL as a template, a bacterial homologue of the Raf Kinase Inhibitor Protein (PDB Code: 1FUX)15 This model was subsequently refined for alignment and completed for the N-terminal residues and for two loops with the “Homology” module of the INSIGHT/DISCOVER program suite, using the secondary structure prediction tools of ExPASy server (www.expasy.ch/ tools/). Preliminary energy minimization was then carried out keeping all backbone atoms fixed to refine the spatial position 330

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of side-chains, followed by a full minimization to obtain the final model. The alignment data between SsCEI and YBCL (Figure 3) illustrate that the two structures, on an alignment length of 167 residues, show 30% identity. A ribbon representation of SsCEI is reported in Figure 4. The general β-fold of the SsCEI is shared with all the PEPB members.15,16 As seen for YBCL, SsCEI possesses a single disulphide bridge linking C29 and C114. The comparative analysis of the SsCEI model with the YBCL structure15 reveals the presence of typical conserved regions among the PEBP-like sequences. In particular: (a) the GXG loop (residues 24-27) located in proximity to the ligand binding pocket, which may affect the substrate accessibility; (b) the CR1 region (residues 54-64), which contains the residues including the signature sequence DPDxP, the semiconserved F62 and the conserved H64, which face the inside of the binding pocket, but lacks any cis peptide bond, the key characteristic of the eukaryotic PEBPs structures; (c) the CR2 region (residues 100-114), which shows the typical eukaryotic consensus sequence of the G residue replaced by a proline (PxHRx); (d) the salt bridge between the residues D56 and R113 linking the regions CR1 and CR2, a conserved characteristic of the PEBP family; (e) the presence of the unique R-helix H1 which is conserved among all the PEBP structures. Further inspection reveals that the residues W62, R96 and S137, involved in the dimer interface stabilization in YBCL are replaced with the residues I63, V92 and A133, respectively, in

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Figure 3. Comparison of the amino acid sequence of Sso0767 with that of YBCL from E. coli (1FUX), a member of “I51” family. Regions highlighted in black are the conserved regions among SsCEI and the analyzed protein, while those in gray correspond to segments with accepted amino acid substitution. Gaps are denoted by dashes.

Figure 4. Stereo drawing of the ribbon representation of SsCEI as obtained after homology modeling The CR1, CR2 loops and the reactive site loop are reported.

SsCEI. The occurrence of these mutations could be responsible for the monomer nature of SsCEI, since the residues W62, R96 and S137 in the YBCL structure are involved in the formation of three hydrogen-bonds cross-linking the two polypeptide chains of the dimer. However, crystallographic structures of eukaryotic PEBPs show a predominantly homodimer form for these proteins, whereas the structures of calf brain PEBP, mouse PEBP-2 and yeast TFS1 alone have been solved in monomer form. Nevertheless, the E. coli YBHB and YBCL are the only proteins able to associate into homodimers in solution. Hence, the oligomer aggregation of PEBPs observed in the 3-D structure may not be integral to biological activity. Anion Binding Site of SsCEI. In SsCEI, one small pocket is defined by residues located on the surrounding loops GXG, CR1, CR2 and the R-helix (Figure 5). Several conserved residues are found15 by the sequence alignment and the structure comparison of SsCEI and YBCL. In particular, the conserved residues, D54/52, P55/53, A57/55, P58/56, H64/63, F97/99, G102/106, P105/109, P106/110, H112/116, Y114/118, and L136/ 140 are present in the binding sites of both proteins, though a few differences are related to the nonconserved residues: P23/ F20, K25/G22, T27/G24, L139/Y143, and E141/N146. Nevertheless, the SsCEI and YBCL binding pockets show high similarity, indicating that even the SsCEI binding site can accommodate various small anionic groups but not molecules such as Hepes, probably due to steric hindrance. Protease Binding Site of SsCEI. Though a number of ExPASy similarity search tools were used, pattern and profile searches

were not able to identify the amino acid sequence responsible for the SsCEI’s inhibitor specificity. Nevertheless, careful inspection of the SsCEI model and of the structural requirements occurring in chymotrypsin/elastase specific inhibitors16-21 allowed us to propose that the reactive site loop encloses the sequence T123-N130, in which the scissile peptide bond is L126 (P1)-E127 (P′1) (Figure 6; Table 1). This sequence is located on an external, protruding loop located far from the anion binding site and possessing an extended conformation similar to other reactive sites in serine protease inhibitors. The presence of residues I128 at the P2′ site and K129 at the P3′ site is in agreement with the occurrence of a noncharged and a positively charged amino acid required for strong binding to R-chymotrypsin (Table 1).20-23 On the other hand, as previously reported,24 four clear conserved patterns within P3-P3′ loop can be distinguished by analyzing several sequences of serine protease inhibitors belonging to 10 different families. Notably, 91% of all serine protease inhibitor sequences available include one of the four canonical motifs.24 On the basis of this structural investigation, it is clear that the proposed reactive site loop in SsCEI consists of a noncanonical amino acid sequence which cannot be classified within any of the consensus patterns of serine protease inhibitors (Table 2). As previously reported, the exposed binding loop is always stabilized by additional interactions between residues flanking the reactive site and the inhibitor core by main-chain protein interactions.18 Interestingly, in the SsCEI model, the side chain amide group of N69 forms two hydrogen bonds with the Journal of Proteome Research • Vol. 8, No. 1, 2009 331

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Figure 5. Stereo drawing detail of SsCEI reporting the conserved regions among the PEBP-like sequences.

Figure 6. Stereo drawing detail of the proposed reactive site loop for SsCEI. Active site residues are labeled. The hydrogen bonds formed by N69 are represented as dashed lines. Table 1. Comparison of Residues in SsCEI from P4-P4′ of the Reactive-Site Loop with Equivalent Residues in a Selection of Protein Inhibitors of Chymotrypsin/Elastase Proteinases residue position inhibitor

P4

P3

P2

P1

P1′

P2′

P3′

P4′

SsCEI C/E-1a OMTKY3b PCI-1c EG-Cd

T123 P28 A15 A89 S42

I124 C29 C16 C90 V43

L125 P30 T17 P91 T44

L126 L31 L18 L92 L45

E127 M32 E19 N93 D46

I128 C33 Y20 C94 L47

K129 R34 R21 D95 R48

N130 R35 P22 P96 Y49

Table 2. The Four Conserved Motifs Found within the Reactive Site Loops (P3-P3′) of Several Serine Protease Inhibitors Are Compared with That of SsCEIa motif

P3

P2

P1

P′1

P′2

P′3

A: Cys-Pro B: Thr C: Cys D: Pro SsCEI

Cys Aaa Aaa Pro Ile

Pro Thr Cys Aaa Leu

Aaa Aaa Aaa Aaa Leu

Aaa Aaa Aaa Aaa Glu

Aaa Aaa Aaa Aaa Ile

Aaa Aaa Aaa Aaa Lys

a Chymotrypsin/elastase inhibitor from Ascaris.20 b Ovomucoid inhibitor third domain from turkey 1.21 c Chymotrypsin inhibitor-1 from potato.22 d Leech inhibitor eglin-c.23

a The four conserved motifs (A-D) are indicated with the canonical residue/s found in the reactive site loop as previously reported.24 Each of the conserved sequence motif is representative of hundreds of serine protease inhibitors belonging to different families.24 Aaa denotes any amino acid.

backbone of reactive site loop residues (Figure 6). This feature has been widely described in the Kunitz family of serine protease inhibitors,25-28 in which the interaction of N residue with the trypsin/chymotrypsin inhibitory loop plays a key role in the stability and in correct orientation of the reactive site loop for proper inhibitory activity. A structural comparison between the SsCEI model and the crystal structure of mouse PEBP-29 allowed the identification

of a putative reactive site loop for mouse PEBP-2 enclosing the sequence 128DKPLRCDE135. It is noteworthy that the side chain amide group of N91 forms two hydrogen bonds with the backbone of reactive site loop residues, in a similar fashion described previously for SsCEI. Characterization of the Effect of the P1 Position on the Interaction with Proteases by Mutant Analysis. The specificity of protein inhibitors of serine proteinases, which obey the

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Figure 7. Inhibition activity of the wild-type (WT) SsCEI (white bars), and the mutants M1 (gray bars) and M2 (black bars) versus R-chymotrypsin. Three micrograms of the protease was incubated with different inhibitor concentrations and the residual protease activity was measured.

“standard mechanism,” is significantly, but by no means exclusively, determined by the nature of the residue occupying the P1 position of the reactive site.29,30 Specifically in inhibitorchymotrypsin complexes, the interactions of the P1 side chain of the inhibitor with the S1 subsite of the protease are considered the primary determinants31 of specificity of the inhibitory activity. To verify the functional importance of the predicted SsCEI reactive-site loop, the biochemical properties of the mutants M1 (L125A/L126S), M2 (L126S) and M3 (N69S/ L126S) were analyzed. Amino acid sequencing, mass spectrometry and CD spectroscopic analyses revealed that purified M1 and M2 had the primary structure shown in Figure S2 in Supporting Information and that the overall conformation was similar to that of the wild-type (data not shown). However, the inhibition of R-chymotrypsin by M1 and M2 mutants was abolished (Figure 7), thus, confirming that the sequence T123-N130 is located in the protease-binding site and that the Leu126 is the P1 residue responsible for the specific inhibition activity toward R-chymotrypsin, similarly to that observed for R-chymotrypsin and elastase inhibitors (Table 1). With regards to the M3 mutant, the protein was expressed in E. coli but lost through the purification procedure, possibly due to aggregation/precipitation and/or proteolysis. This behavior could be explained by uncorrected folding of M3, in which the additional mutation of N69 residue in S69 respect to the M2 could contribute to protein destabilization.

Discussion and Conclusion Among the classes of protease inhibitors characterized to date, the PEBP family represents a novel and highly conserved cluster of proteins,2 which possesses homologues in a wide variety of organisms. As the number of genomes being sequenced increases and further studies on these proteins are undertaken, it has become apparent that many organisms contain several forms of PEBP, thus, implicating the involvement of such proteins in fundamental cellular processes.7 The PEBPs represent a multifunctional protein family, although the three-dimensional structures show an extensive fold conservation.16 In this study, we identified a protease inhibitor, SsCEI, which represents the first archaeal PEBP-serine proteinase inhibitor from S. solfataricus reported to date. The inhibitor was found to be specific for the serine proteases R-chymotrypsin and elastase, and did not show any activity against serine proteases belonging to other families. Furthermore, the inhibition of R-chymotrypsin followed a hyperbolic pattern, and since SsCEI

research articles interacts with the protease in a 1:1 molar ratio, it can be classified under the “tight binding inhibitor” group. The inhibition specificity of SsCEI looks different with respect to the two eukaryotic counterparts of the I51 family, the mouse PEBP2 and the yeast TFS1.8 In fact, the inhibition data clearly indicate that SsCEI exclusively affects serine proteases (Rchymotrypsin/elastase) that hydrolyze hydrophobic amino acids at the P1 position, while the mouse PEBP and the yeast TFS1 interact with proteases (R-chymotrypsin/thrombin or R-chymotrypsin/carboxypeptidase, respectively) that cleave after hydrophobic or basic amino acids. However, the high level of sequence identity between SsCEI and the cognate PEBP inhibitor members indicated that they share some common structural and possibly functional properties, but that their functional specificity may ultimately be determined by regions of amino acid divergence. The general fold of SsCEI is a β-fold consisting of two antiparallel β-sheets and long strand connecting loops similar to the human and bovine PEBPs. The major differences are located within the loops and at the N/C-terminal ends. The peptide containing the first 20 amino acid residues of the mammalian PEBPs, neurotransmitter peptide, is absent in SsCEI and YBCL. In addition, none of these proteins exhibit a C-terminal helix that in the mammalian PEBPs partially covers the ligand binding site and is presumed to regulate its accessibility.16 The SsCEI model also gives some important insights into the active site loop of the inhibitor, located on the protein surface at the C-terminal region, that possesses a noncanonical amino acid sequence, including the scissile peptide bond L126 (P1)-E127 (P′1) as confirmed by point mutation experiments. Many inhibitors bind with their cognate enzyme(s) according to a common, substrate-like standard mechanism.18 They are all relatively small (from 29 to 190 amino acids) and share an exposed, rigid binding loop with a characteristic ‘canonical’ conformation, which inserts into the active site cleft of the enzyme in a substrate-like manner. At least 18 different families of canonical inhibitors showing this standard mechanism have been defined based on structural criteria.22 While they all share the combining loop conformation, each has its own global three-dimensional structure. Although the mode of action of SsCEI appears to be in a substrate-like manner, the identification of a noncanonical amino acid sequence in the reactive site loop of SsCEI, not classifiable in any of the characterized consensus motifs, may indicate that the SsCEI has developed a different protease recognition geometry which could extend the knowledge of the protease/inhibitor complexes. These findings are clearly in conflict with those observed for the yeast PEBP TFS1, which interacts with its target in a nonsubstratelike manner by two protease binding sites located at the N-terminal region of the protein.32 Also of note, SsCEI’s high resistance to both extreme temperatures and to hydrolysis by mesophilic proteases appears to be unique and very advantageous for new biotechnological therapeutic approach.33 Moreover, preliminary results showed that SsCEI was able to inhibit an endogenous serine protease (data not shown), and further studies are in progress to better elucidate the structural and biological implications of the S. solfataricus protease-SsCEI interaction. In conclusion, our work underlines that SsCEI is the first archaeal PEBP-serine protease inhibitor, in which the coexistence of the distinctive structural motifs both from Journal of Proteome Research • Vol. 8, No. 1, 2009 333

research articles bacterial and eukaryotic organisms reflects the early evolutionary linkage of the archaeal protein.

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Acknowledgment. This work was supported by ASI project MoMa No, 014/06/0 (M.R.). We thank Mr. Luca De Luca and Mrs. Immacolata Fiume for technical assistance.

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