Biochimica et Biophysica Acta 1844 (2014) 1749–1756

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta

j ourna l homepage: www.e lsev ie r .com/ locate /bbapap

Fungal β-trefoil trypsin inhibitors cnispin and cospin demonstrate theplasticity of the β-trefoil fold

Petra Avanzo Caglič a, Miha Renko b,c, Dušan Turk b,c, Janko Kos a,d, Jerica Sabotič a,⁎a Department of Biotechnology, Jožef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Sloveniab Department of Biochemistry, Molecular, and Structural Biology, Jožef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Sloveniac Centre of Excellence for Integrated Approaches in Chemistry and Biology of Proteins, Jamova 39, SI-1000 Ljubljana, Sloveniad Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, SI-1000 Ljubljana, Slovenia

Abbreviations: API-A, arrowhead protease inhibitosubtilisin inhibitor; Cnp, cnispin (Clitocybe nebularis serLentinus edodes serine protease inhibitor; PIC, cospin (Copinhibitor); PPI, papaya protease inhibitor; STI, soybeanbean α-chymotrypsin inhibitor⁎ Corresponding author. Tel.: +386 14773754; fax: +3

E-mail address: jerica.sabotic@ijs.si (J. Sabotič).

http://dx.doi.org/10.1016/j.bbapap.2014.07.0041570-9639/© 2014 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 8 April 2014Received in revised form 4 July 2014Accepted 8 July 2014Available online 14 July 2014

Keywords:TrypsinChymotrypsinBeta-trefoilProtease inhibitorMushroomSerine protease

The recently identified fungal protease inhibitors cnispin, from Clitocybe nebularis, and cospin, from Coprinopsiscinerea, are both β-trefoil proteins highly specific for trypsin. The reactive site residue of cospin, Arg27, is locatedon theβ2–β3 loop.We showhere, that the reactive site residue in cnispin is Lys127, located on theβ11–β12 loop.Cnispin is a substrate-like inhibitor and the β11–β12 loop is yet another β-trefoil fold loop recruited for serineprotease inhibition. By site-directed mutagenesis of the P1 residues in the β2–β3 and β11–β12 loops in cospinand cnispin, protease inhibitors with different specificities for trypsin and chymotrypsin inhibition have beenengineered. Double headed inhibitors of trypsin or trypsin and chymotrypsin were prepared by introducing asecond specific site residue into the β2–β3 loop in cnispin and into the β11–β12 loop in cospin. These resultsshow that β-trefoil protease inhibitors from mushrooms exhibit broad plasticity of loop utilization in proteaseinhibition.

r A; BASI, barley α-amylase/ine protease inhibitor); LeSPI,rinopsis cinerea serine proteasetrypsin inhibitor; WCI, winged

86 14773594.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Serine proteases of the chymotrypsin fold (family S1 in theMEROPSclassification) are the largest known family of proteases. Trypsin andchymotrypsin have been studied extensively, especially in view oftheir interaction with protein protease inhibitors [1–3]. Protein inhibi-tors that act according to the standard mechanism of inhibition arethe most studied group of protease inhibitors of S1 proteases. They be-long to many different structurally and evolutionarily unrelated fami-lies but all have an exposed protease-binding loop with acharacteristic canonical conformation. The loop conformationresults from an extensive system of disulfide bond(s), hydrogenbonds, and/or hydrophobic interactions that involve residues bothfrom the loop and the inhibitor scaffold. This loop binds into theactive site of the protease in a substrate-likemanner and the exposedP1–P1' peptide bond is hydrolyzed, but extremely slowly as aconsequence of the very high stability of the enzyme–inhibitorcomplex, and with very low dissociation rate constants. The P1residue is thus the primary determinant of specificity of the inhibitor

for a particular S1 protease. Arginine and lysine residues at P1 confertrypsin-like specificity and phenylalanine, tyrosine or leucineresidues confer chymotrypsin-like specificity [2–4]. Several inhibitorsbelonging to different families have been used to show that changingthe P1 residue from Leu to Arg changes the specificity of the inhibitorfrom chymotrypsin to trypsin [5,6]. Replacing the P1 residue by Leu,Met, Phe or Trp results in a potent chymotrypsin inhibitor showingweak trypsin inhibition [7–10].

One of the best studied trypsin inhibitors, theKunitz soybean trypsininhibitor (STI), is a β-trefoil protein and belongs to clan IC of theMEROPS classification of protein protease inhibitors. The β-trefoil foldis composed of 12 β-strands connected by 11 loops of different lengths,sequences and conformations. Trypsin and chymotrypsin inhibitorshaving the β-trefoil fold were first described only from plants (familyI3) and, in most members, the β4–β5 loop was identified as the trypsinor chymotrypsin inhibitory loop, depending on the P1 residue [3,11].A few plant β-trefoil trypsin/chymotrypsin inhibitors have beendescribed with double reactive sites. A second reactive site was initiallyobserved in winged bean α-chymotrypsin inhibitor (WCI) [12] andlater predicted to be in the β2–β3 loop [13] and confirmed by smallangle X-ray scattering experiment for papaya protease inhibitor (PPI)[14]. The protease inhibitor API-A from arrowhead has two distinctreactive sites that differ from those of all othermembers of the I3 familyand involve the β5–β6 and β9–β10 loops [15].

The versatility of β-trefoil fold loops involved in trypsin inhibitionwas broadened when representatives from fungi were identified and

1750 P. Avanzo Caglič et al. / Biochimica et Biophysica Acta 1844 (2014) 1749–1756

characterized (families I66 and I85), in which the β4–β5 loop was notinvolved in inhibition of S1 proteases. In macrocypin 4 (family I85), acysteine protease inhibitor from the parasol mushroom (Macrolepiotaprocera) [16], the β5–β6 loopwas shown to be involved inweak trypsininhibition [17]. Three trypsin-specific protease inhibitors from mush-rooms have been characterized and constitute family I66 in theMEROPS classification. They will be, analogously to mycocypins (fungalcysteine protease inhibitors, MEROPS families I48 and I85), collectivelyreferred to as mycospins (fungal serine protease inhibitors). The firstrepresentative of the I66 family, LeSPI, was isolated from fruiting bodiesof the shiitake mushroom (Lentinus edodes) and shown to be a stronginhibitor of trypsin and a weak one of chymotrypsin. An arginineresiduewas implicated in trypsin inhibition through chemicalmodifica-tion; however, its position in the primary structure was not determined[18]. Characterization of the trypsin-specific protease inhibitor cospin(PIC) from the inky capmushroom (Coprinopsis cinerea), which inhibitstrypsin with Ki in the picomolar range, revealed the β-trefoil fold ofmembers of the I66 family. The reactive site residue was determinedto be Arg27 in the β2–β3 loop [19]. Similar potent trypsin-specificinhibitors, the CnSPIs, were isolated from fruiting bodies of the cloudedagaric mushroom (Clitocybe nebularis) and a recombinant CnSPI versiontermed cnispin (Cnp) was characterized showing highly specific inhibi-tion of trypsin with Ki in the nanomolar range [20].

Here, we describe themechanism of inhibition of trypsin by cnispin,whichutilizes yet another loop of theβ-trefoil fold for trypsin inhibition.Additionally, by engineering inhibitors with different P1 residues,several cnispin and cospin variants exhibiting different inhibitorypatternswere produced and analyzed for their ability to inhibit differentproteases.

2. Materials and methods

2.1. Enzymes and substrates

Bovine trypsin (EC 3.4.21.4), chymotrypsin (EC 3.4.21.1), porcinekallikrein (EC 3.4.21.35), papain (EC 3.4.22.2) and porcine pepsin (EC3.4.32.1) were from Sigma, bovine thrombin (EC 3.4.21.5) fromCalbiochem, Bacillus subtilis subtilisin (EC 3.4.21.62) from BoehringerMannheim and porcine elastase (EC 3.4.21.36) from Serva. Legumain(EC 3.4.22.34) was isolated from pig kidney cortex [16]. SubstratesZ-Phe-Arg-MCA (7(4-methyl)-coumarylamide), Suc-Ala-Ala-Pro-Phe-MCA, Boc-Val-Pro-Arg-MCA, H-Pro-Phe-Arg-MCA, Suc-Ala-Ala-Ala-MCA, Z-Ala-Ala-Asn-MCA and N-benzoyl-DL-arginine-p-nitroanilide(BAPNA) were from Bachem, and MOCAc-Ala-Pro-Ala-Lys-Phe-Phe-Arg-Leu-Lys-(Dnp)-NH2 was from Peptide Institute Inc.

2.2. Expression and purification of cnispin and cospin mutants

Cnispin and its mutants (Cnp-R21A, Cnp-H27R, Cnp-K127N,Cnp-R21F, Cnp-H27F, Cnp-H27F/K127N, Cnp-H27R/K127N) and cospinand its mutants (PIC-R27N, PIC-R27F, PIC-P130R, PIC-R27N/P130R,

Fig. 1. SDS-PAGE analysis of cnispin, cospin and theirmutants. SDS-PAGE analysis under reducin

PIC-R27F/P130R) were expressed in Escherichia coli BL21(DE3) andpurified as described [19,20]. Inhibitory activities of samples againsttrypsin were determined against BAPNA substrate as described [20].

2.3. The methylation of lysine for cnispin active site determination

Chemical modification of lysine residues in cnispin, macrocypin 4and cospin (each at 0.6 mg/mL) was performed by overnightmethylation in 50 mM HEPES, 250 mM NaCl buffer, pH 7.5 at 4 °Cusing dimethylamine-borane complex (Fluka) in the presence offormaldehyde (Fluka). Proteins were dialyzed against 50 mM Tris–HCl, 200 mM NaCl, pH 7.5, and inhibition of trypsin was assayedusing Z-Phe-Arg-MCA as a substrate.

2.4. Site-directed mutagenesis

Sequences of cnp cDNA (GenBank: GQ141891) and pic1 cDNA(GenBank: GQ903329)were used to design themutagenic oligonucleo-tides (Supplementary Table S1) used in PCR site-directed mutagenesisusing KODHot Start DNAPolymerase (Novagen) as described for cospinmutants [19].

2.5. Determination of inhibition constants

Inhibition kinetics of trypsin were determined under pseudo-firstorder conditions in continuous assays and data were analyzed bynonlinear regression analysis according to Morrison [21] as described[20]. Inhibition kinetics for the inhibition of chymotrypsin, subtilisin,kallikrein, elastase, thrombin and legumainwere determined accordingto Henderson [22], as described [16,19]. Inhibitory activity againstcysteine protease papain was assayed as described [19] and inhibitoryactivity against aspartic protease pepsin was assayed using substrateMOCAc-Ala-Pro-Ala-Lys-Phe-Phe-Arg-Leu-Lys-(Dnp)-NH2 at 5 μMfinal concentration in citric acid phosphate buffer, pH 4.5.

2.6. Analysis of inhibitor–enzyme complexes

To characterize trypsin–cnispin complex formation, trypsin andcnispin were mixed in molar ratio 1:2 (final concentrations 0.02 mMand 0.04 mM) and incubated at 37 °C. Samples were taken at timesranging from 5 min to 7 days and analyzed by Blue Native PAGE usingthe Novex NativePAGE™ Bis–Tris Gel System (Life Technologies) andmass spectrometry. Prior to analysis on a MALDI-TOF massspectrometer (Bruker Daltonics) the samples were desalted usingC18 resin embedded in ZipTips (Millipore) following themanufacturer's recommendations. For analysis of complex forma-tion, cnispin or cospin mutants Cnp-H27F or PIC-R27F were mixedwith chymotrypsin in 2:1 molar ratio (final concentrations:0.02 mM and 0.01 mM, respectively) and analyzed by isoelectric fo-cusing using the Novex pH 3–10 IEF Protein Gels (Life Technologies).

g conditions of cnispin, cospin and theirmutants. LaneM, proteinmolecularmassmarkers.

Fig. 2. Alignment of fungal β-trefoil trypsin inhibitors belonging to MEROPS family I66. Amino acid sequences of cnispin (CNP), cospin (PIC) and LeSPI were aligned with BLOSUM62ma-trix. Cnp shares 30% and 31% of sequence identity with PIC and LeSPI, respectively, and PIC shares 37% sequence identity with LeSPI. Identical amino acid residues are shaded dark gray andsimilar ones in light gray. The β-strands of PIC secondary structure are indicated by lines above the sequences. The arrows indicate the trypsin reactive P1 residue in cospin (white) andcnispin (black).

1751P. Avanzo Caglič et al. / Biochimica et Biophysica Acta 1844 (2014) 1749–1756

For triple complex formation between trypsin, Cnp-H27F andchymotrypsin, Blue Native PAGE analysis was used.

2.7. Cospin–trypsin complex crystallization and modeling

Cospin–trypsin complex was purified by gel filtration on SephacrylS100 equilibrated in 20 mM Tris–HCl, 300 mM NaCl, pH 7.5, dialyzedin 10 mM Tris–HCl and concentrated to approximately 25 mg/mL.After initial crystallization, attempts using commercial screens and,after numerous optimizations, crystals with high anisotropy andmosaicity were produced. The collected data were not suitable for thecrystal structure determination.

A model of cospin mutant P130R was created on the basis of nativecospin structure (PDB ID: 3N0K) [19] by replacing proline residuewith arginine in software MAIN [23]. A model of the ternary complexof one cospin molecule with two trypsin molecules was created inmultiple stages. Firstly, a crystal structure of soybean trypsin inhibitor(STI) in complex with trypsin [24] was reduced to trypsin molecule incomplex with P3–P3′ residues of the inhibitory loop of STI. Secondly,P3–P3′ residues of theβ2–β3 inhibitory loop in cospinwere structurallyaligned with P3–P3′ residues of the inhibitory loop of STI in complexwith trypsin to create 1:1 model of trypsin, interacting with theβ2–β3 inhibitory loop. In the next step, P3–P3′ residues of theβ11–β12 inhibitory loop were aligned with P3–P3′ residues of theinhibitory loop in the second STI/trypsin reduced complex to obtainthe ternary complex. The ternary complex was subjected to real-spaceminimization in MAIN [23] in order to remove sterical clashes betweenthe side chains of cospin and trypsin molecules.

Table 1Kinetic constants for the interaction of cnispin, cospin and their mutants with various proteaseorder conditions in a continuous kinetic assay according to Morrison [21]. Equilibrium constapapain and pepsin were determined according to Henderson [22]. Experiments were performdetermined.

Trypsin Chymotrypsin Subtilisin Kal

Cnpa 3.10 ± 0.66 120 ± 20 N1000 N10Cnp R21A 15.2 ± 2.1 NI N1000 NICnp R21F 25.0 ± 2.3 NI N1000 NICnp K127N NI N1000 NI NICnp H27F 16.2 ± 1.2 2.4 ± 0.1 N1000 N10Cnp H27R/K127N 1.12 ± 0.19 N1000 NI NICnp H27F/K127N N1000 0.17 ± 0.01 NI NIPICb 0.022 ± 0.002 116 ± 8 N1000 N10PIC R27Nb 480 ± 30 77 ± 12 NI NIPIC R27F 120 ± 30 0.52 ± 0.06 N1000 N10PIC R27N/P130R 4.72 ± 0.96 68 ± 15 N1000 N10PIC R27F/P130R 4.03 ± 0.75 0.15 ± 0.07 545 ± 52 N10

a Reported previously [20].b Reported previously [19].

3. Results

3.1. Expression and purification of cnispin and cospin mutants

Cnispin mutants (R21A, R21F, H27R, H27F, K127N, H27F/K127N,H27R/K127N), like the wild type recombinant cnispin [20], wereexpressed in E. coli as insoluble inclusion bodies. Cospin mutants R27F,R27N, P130R, R27N/P130R and R27F/P130R were expressed as solubleproteins [19]. All purified proteins exhibited a single band onSDS-PAGE under reducing conditions (Fig. 1).

3.2. Determination of the cnispin inhibitory binding site and mechanism oftrypsin inhibition

The initialmutantswere selected on the basis of sequence alignmentbetween cospin and cnispin (Fig. 2). The closest arginine or lysineresidue in the vicinity of the Arg27 in cospin is Arg21. It was thereforeselected as a potential P1 residue for mutagenesis of cnispin to abolishtrypsin inhibition (mutant Cnp-R21A) and convert it to a chymotrypsininhibitor (mutant Cnp-R21F). These mutants inhibited trypsin to asimilar extent as did the wild type cnispin, with Ki in the nanomolarrange. Chymotrypsin was not inhibited, indicating that the position ofthe inhibitory reactive loops in cospin and cnispin differs. Furthermore,similar inhibitory patterns of cnispin and both mutants were observedfor other proteases analyzed (Table 1). Thus, cnispin and cospin donot utilize the same inhibitory reactive loop and a lysine residue wasnext considered as the P1 reside for trypsin inhibition by cnispin.

Methylation of Lys residues abolished the inhibitory activity ofcnispin, indicating that, in contrast to cospin and LeSPI, a lysine rather

s. Equilibrium constants for the inhibition of trypsin were determined under pseudo-firstnts for the inhibition of chymotrypsin, subtilisin, kallikrein, elastase, thrombin, legumain,ed at 25 °C. Standard deviation is given where appropriate; NI, no inhibition; n.d., not

likrein Elastase Thrombin Legumain Papain Pepsin

Ki (nM)

00 NI NI NI NI NINI NI n.d. NI NINI NI n.d. NI NINI NI NI NI NI

00 NI NI n.d. NI NINI NI n.d. NI NINI NI n.d. NI NI

00 N1000 NI NI NI NINI NI NI NI NI

00 N1000 NI n.d. NI NI00 NI NI n.d. NI NI00 N1000 NI n.d. NI NI

Fig. 3. Structural superimposition of the crystal structure of cospin (cyan) and model ofcnispin (green). P1 residues are shown as sticks. Model of cnispin was created withModeller [30] on the basis of the crystal structure of cospin (PDB ID: 3N0K). The modelshows that cnispin is structurally similar to cospin (RMSD for 148 CA atoms of 0.48 Å).

1752 P. Avanzo Caglič et al. / Biochimica et Biophysica Acta 1844 (2014) 1749–1756

than an arginine is involved in inhibiting trypsin by cnispin. Twoβ-trefoil inhibitors were used as controls in the Lys methylationanalysis. Macrocypin 4, which utilizes Lys74 for trypsin inhibition [17],lost inhibitory activity by Lys methylation while cospin, in whichArg27 is the key residue for trypsin inhibition [19], retained itsinhibitory activity.

Lys127, that is unique to cnispin (Fig. 2) and, according to the cnispinmodel (Fig. 3), is present in the β11–β12 loop, was selected formutagenesis as a candidate P1 site residue for trypsin inhibition.Cnp-K127N did not inhibit trypsin and, together with its very weakinhibition of chymotrypsin, proved Lys127 to be the reactive site residuefor binding and inhibition of trypsin. Other proteaseswere not inhibited(Table 1).

Additional confirmation that Lys127 is indeed the P1 residue wasprovided by mass spectrometry analysis of cnispin preincubated withtrypsin at a 2:1 ratio. While cnispin incubated at 37 °C underwent nodegradation, similar incubation in the presence of the trypsineventually resulted in the proteolytic removal of approximately2 kDa, corresponding to the C-terminal part of the protein (Ser128–Asp146), confirming cleavage after Lys127 (Fig. 4A). Absence ofadditional non-specific cleavages of cnispin by trypsin and offormation of a cnispin–trypsin complex (Fig. 4B), as shown bynative-PAGE analysis, demonstrates that cnispin is a substrate-likeinhibitor of trypsin-like serine proteases, with which it forms tight-binding complexes and undergoes slow proteolytic cleavage(Fig. 4C). However, cnispin is a weaker inhibitor of trypsin thancospin. While cospin shows inhibition in the picomolar range andits complex with trypsin is stable for more than two weeks at 37 °C[19], cnispin shows inhibition in the nanomolar range and iscompletely degraded by trypsin after 24 hour incubation at 37 °C(Fig. 4B, C).

3.3. Engineering β-trefoil loops in cnispin and cospin for trypsin inhibition

Since the P1 residue for trypsin inhibition is located in different loopsin cnispin and cospin and since sequence alignment suggests that theirsecondary structure and loop length, orientation and arrangement aremost probably similar (Fig. 3), we sought to determine whether theβ2–β3 loop in cnispin and the β11–β12 loop in cospin, neither ofwhich possesses inhibitory properties in the native protein, can betransformed to inhibitory loops by simple replacement of the P1residues.

A cnispin mutant with potential dual trypsin inhibitory sites wasprepared by introduction of an additional P1 residue in the β2–β3loop (Cnp-H27R). Titration of trypsin with Cnp-H27R showed that

complete inhibition is achieved at 2:1 molar stoichiometry (Fig. 5),confirming the presence of two inhibitory binding sites for trypsin, onthe β2–β3 and β11–β12 loops. Similarly, a double-headed cospinmutant (PIC-P130R) was produced by introducing an arginine residueto the β11–β12 loop. The titration experiments were inconclusive forthis mutant, possibly because of the large difference in strengths oftrypsin inhibition by the two loops. Due to the absence of bindingcooperation information, the kinetic constants were not determinedfor these double-headed trypsin inhibitors. The inhibitory profileswere similar to those for the wild type cnispin and cospin (Table 2),with strong inhibition of trypsin and weak inhibition of chymotrypsin,and even weaker inhibition of subtilisin and kallikrein.

In addition, a cnispin double mutant (Cnp-H27R/K127N) with theβ2–β3 loop trypsin inhibitory reactive site only was produced and,analogously to cospin, showed an even more pronounced trypsin-specific inhibitory profile than wild type cnispin (Tables 1, 3). Further,a cospin double mutant (PIC-R27N/P130R), with the reactive site inthe β2–β3 loop inactivated and the P1 residue introduced to theβ11–β12 loop, showed an inhibitory pattern similar to that of thewild type cnispin, inhibiting trypsin in the nanomolar range andchymotrypsin in the micromolar range (Table 1). Kinetics of trypsininhibition by cnispin and cospin double mutants are presented inTable 3.

3.4. Engineering β-trefoil loops in cnispin and cospin for chymotrypsininhibition

Replacing the P1 Arg in the β2–β3 loop of cospin by Phe (mutantPIC-R27F) replaced its trypsin inhibitory activity by inhibition ofchymotrypsin with a Ki value in the low nanomolar range. Its complexwith chymotrypsin was confirmed by isoelectric focusing (Fig. 6A).The Ki value for the inhibition of trypsin was in the micromolar range,and those for subtilisin, kallikrein and elastase significantly higher still(Table 1). An additional P130R mutation resulted in a double-headedtrypsin/chymotrypsin inhibitor (PIC-R27F/P130R), with trypsin bindingto the P1 residue in the β11–β12 loop and chymotrypsin to the β2–β3loop. Both chymotrypsin and trypsin were inhibited in the nanomolarrange (Table 1). Kinetics of trypsin inhibition by this mutant arepresented in Table 3.

A similar approach to introducing chymotrypsin inhibitory activitywas attempted for cnispin. Inhibition of both trypsin and chymotrypsin,utilizing separate binding sites, was achieved by introducing achymotrypsin targeted P1 residue into the β2–β3 loop (Cnp-H27F)while retaining the trypsin inhibitory P1 residue in the β11–β12 loop.Ki values for inhibition of trypsin and chymotrypsin were in thenanomolar range and for subtilisin and kallikrein in the micromolarrange (Table 1). Complex formation of the Cnp-H27F mutant withchymotrypsin alone (Fig. 6B) and a complex with both chymotrypsinand trypsin (Fig. 6C) were both demonstrated. Cnispin was convertedto a chymotrypsin inhibitor by replacing the trypsin targeted P1 residuein the β11–β12 loop (Cnp H27F/K127N), while retaining thechymotrypsin targeted P1 residue Phe27. The resulting mutantCnpH27F/K127N is a very strong inhibitor of chymotrypsin with Ki inthe low nanomolar range. Inhibition of trypsin was very weak and noinhibition of subtilisin or kallikrein was detected (Table 1).

3.5. Cospin–trypsin complex model

Well diffracting crystals of cospin or of cnispin in complex withtrypsin could not be obtained, despite extensive optimization of theconditions for crystallization. A ternary model of the complex betweencospin and trypsin was therefore created using the crystal structuresof cospin [19] and trypsin [24]. The two inhibitory loops in cospin arepositioned on opposite sides of the molecules, both extending awayfrom the protein surface (Fig. 7A). The P1 Arg27 residue in the β2–β3loop and the P1 Arg130 residue in the β11–β12 loop are both located

Fig. 4. Cnispin–trypsin complex formation andmechanismof inhibition. (A)MALDI-TOF analysis of cnispin and the cnispin–trypsin complex after 1 h incubation at 37 °C. BlueNative PAGEanalysis (B) and SDS-PAGE analysis (C) of cnispin–trypsin complex formation incubated at 37 °C for the indicated periods of time. M, molecular mass marker. In Native PAGE cnispinappears as two bands and a smear. In addition to the major cnispin band, one band is its dimer and the other is probably the result of rate-limited self-association [31].

1753P. Avanzo Caglič et al. / Biochimica et Biophysica Acta 1844 (2014) 1749–1756

on the exterior ends of the loops, and thus available for binding to the S1pocket of trypsin. While the main chain and side chains in the β2–β3loop around the S1 residue Arg27 fitted well to the active site cleft ofthe trypsin molecule (Fig. 7B), indicating the necessity for only smallconformation changes needed for binding to trypsin [19], the positionof the β11-β12 inhibitory loop and more importantly, the position ofthe side chains, as they were observed in the crystal structure of freecospin, must undergo more substantial changes. In addition to aP130R mutation in the β11–β12 loop of cospin that is required to gaintrypsin inhibitory activity, the positions of the side chains of Tyr132and Arg135 also have to adapt to trypsin (Fig. 7C). Although, in our

model, no extensive clashes are present between other side chains inthe β11–β12 loop of cospin and trypsin, it is clear from the significantlystronger inhibition of trypsin bywild type cospin than by the PIC R27N/P130R mutant that the sequence in the vicinity of P1 residues is betteroptimized for trypsin binding in the β2–β3 loop. In addition to theprimary sequence, scaffolding residues might play a key role in definingthe strength of complex formation and longevity of the inhibitor.The β2–β3 loop is stabilized by a hydrogen-bond network, involvingresidues Lys32, which forms a hydrogen bond with carbonyl oxygenatom of Leu30 and two hydrogen bonds with Ser28, and Glu25 formstwo hydrogen bonds with Arg21 (Fig. 7D). On the other hand,

Fig. 5. Titration of trypsin with Cnp and Cnp H27R.

Table 3Kinetic constants for the interaction of cnispin, cospin and their mutants with trypsin.Equilibrium constants for the inhibition of trypsin were determined under pseudo-firstorder conditions in a continuous kinetic assay according to Morrison [21]. Standarddeviation is given. Experiments were performed at 25 °C.

Ki

(nM)10−6 × ka(M−1 s−1)

104 × kd(s−1)

Cnpa 3.10 ± 0.66 0.310 ± 0.055 9.50 ± 0.47Cnp H27R/K127N 1.12 ± 0.19 1.35 ± 0.13 14.9 ± 1.6PICb 0.022 ± 0.002 5.28 ± 0.50 1.26 ± 0.07PIC R27N/P130R 4.72 ± 0.96 0.97 ± 0.12 46 ± 4PIC R27F/P130R 4.03 ± 0.75 1.37 ± 0.30 54 ± 9

a Reported previously [20].b Reported previously [19].

1754 P. Avanzo Caglič et al. / Biochimica et Biophysica Acta 1844 (2014) 1749–1756

stabilization of the longer β11–β12 loop involves three hydrogen bonds(Fig. 7E). However, new hydrogen bonds between scaffolding residueseither direct or water-mediated might be formed upon binding totrypsin as the loops most likely undergo rearrangements.

4. Discussion

The number of loops in the β-trefoil fold known to be involved ininhibiting S1 proteases has been increased by the demonstration thatcnispin inhibits trypsin through its β11–β12 loop, with Lys127 as theP1 residue. Previously known loops are β4–β5, β2–β3, β5–β6 andβ9–β10 in inhibitors from plants [13–15,24] and β5–β6 and β2–β3 ininhibitors of fungal origin [17,19] (Fig. 8). Sequence alignment (Fig. 2)suggests that the β2–β3 loop in another member of the I66 family,LeSPI, is responsible for trypsin inhibition, with Arg24 the most likelyP1 residue,making cnispin the unique knownmember of the I66 family.

The successful introduction of trypsin inhibition into originallyinactive loops confirms that the initial hypothesis of similar looplengths, orientation and arrangement in cospin and cnispin was correct.The β2–β3 and β11–β12 trypsin inhibitory loops in cospin and cnispincan be easily exchanged by the mutagenesis of P1 residues Arg27 andLys127. We have introduced second trypsin binding sites in the β2–β3loop in cnispin and in the β11–β12 loop in cospin, and engineered apotent double-headed trypsin inhibitor (Cnp-H27R and PIC-P130R)with a trypsin inhibitory site in each of the β2–β3 and β11–β12 loops.Furthermore, the cnispin mutant CnpH27R/K127N, which lost thewild type cnispin P1 residue Lys127 in the β11–β12 loop, still retainedtrypsin inhibition with nanomolar Ki through the introduced Arg27 inthe β2–β3 loop (Table 2). Modeling of cospin in complex with the twotrypsin molecules confirmed that inhibitory loops are located on

Table 2Trypsin and chymotrypsin inhibitory profile of cnispin, cospin and their mutants. Very stronginhibition by−.

β2–β3 loopP1 residue

β11–β12 loopP1 residue

Cnp His LysCnp K127N His AsnCnp H27R/K127N Arg AsnCnp H27F Phe LysCnp H27F/K127N Phe AsnCnp H27R Arg LysPIC Arg ProPIC R27N Asn ProPIC R27N/P130R Asn ArgPIC R27F Phe ProPICR27F/P130R Phe ArgPIC P130R Arg Arg

opposite sides of the inhibitor molecule (Figs. 3, 7), indicating that adouble-headed β-trefoil trypsin inhibitor can be engineered by simpleaddition of an appropriate P1 residue. The strength of inhibition,however, is influenced by the residues in the loop surrounding the P1residue. It appears that in cospin, the β2–β3 loop is better optimizedfor trypsin inhibition, which could, with the exception of cnispin, betrue also for other members of the I66 family, as it comprises moreevolutionarily conserved residues than the β11–β12 loop (Fig. 2). Ithas been shown for canonical serine protease inhibitors belonging todifferent families that strong inhibition requires amino acids aroundthe reactive site having appropriate electrostatic characteristics [25].Residues in the P4–P3′ segment of the loop contribute to the proteaseinhibitor–protease interaction [25,26] and certain other residues tothe structural rigidity of the loop [11,27]. Furthermore, residues fromother loops could take part in stabilization of the complex. For example,residues from the N-terminus and the β7–β8 loop contribute to thecomplex stabilization in the tamarind Kunitz inhibitor [28].

We demonstrated that both loops (β2–β3 and β11–β12) can befunctional as trypsin or chymotrypsin inhibitory loops in both inhibi-tors, depending only on the nature of the P1 residue. Furthermore,formation of complexes with one and with both proteases, stressingthe independence of the two sites, has been separately confirmed.Introduction of Phe27 converted both cospin and the inactive cnispinmutant CnpK127N to strong chymotrypsin inhibitors, both formingstrong inhibitor–chymotrypsin complexes. In addition, a singlemutation (CnpH27F) converted cnispin from a specific trypsin inhibitorto a strong inhibitor of both trypsin and chymotrypsin forming a ternarycomplex with two reactive sites, namely Phe27 in the β2–β3 loop forchymotrypsin binding and Lys127 in the β11–β12 loop for trypsinbinding. Similarly, the addition of the P130R mutation to the PIC-R27Fmutant converted a chymotrypsin inhibitor to a strong inhibitor ofboth trypsin and chymotrypsin (Table 2).

inhibition is indicated by +++, strong inhibition by ++, weak inhibition by + and no

Trypsin inhibition Chymotrypsin inhibition

++ +− −++ −++ ++− ++++++ ++++ ++ +++ ++ +++++ ++++++ +

Fig. 6. Analyses of the complex formation between chymotrypsin and cospin (A) and between chymotrypsin and cnispin (B) and of the triple complex formation between chymotrypsin,cnispin and trypsin (C). (A) Isoelectric focusing analysis of PIC-R27F with chymotrypsin at 2:1 molar ratio. M, standard protein IEF markers. (B) Isoelectric focusing analysis of Cnp-H27Fwith chymotrypsin at 2:1molar ratio. (C) BlueNative PAGE analysis of complex formation between trypsin andCnp-H27F and between chymotrypsin and Cnp-H27F at 2:1molar ratio andof triple complex between trypsin, Cnp-H27F and chymotrypsin at 1:2:1 molar ratio.

1755P. Avanzo Caglič et al. / Biochimica et Biophysica Acta 1844 (2014) 1749–1756

Simple engineering of protease inhibitor specificity by one aminoacid replacement was successful for trypsin/chymotrypsin inhibitionin mycospins and for trypsin/legumain inhibition in mycocypins [17],indicating a similar inhibitory mechanism to S1 proteases also for C13proteases [11]. In legumain (asparaginyl endopeptidase) Asn is the P1residue that confers substrate-like binding inhibition [29]. Althoughreplacement of the P1 residue in the reactive site for legumain inhibitionin the clitocypin's β5–β6 loop, Asn70Lys, did not lead to gain of trypsininhibitory activity [17], exchanging the β5–β6 loop betweenmacrocypins 1 and 4 leads to exchanged legumain/trypsin inhibition[17]. Therefore, potential introduction of a legumain inhibitory reactivesite was investigated in cnispin and cospin mutants, where the P1residue was changed to Asn. With both mutants (CnpK127N and PICR27N) no gain of legumain inhibition was detected (Table 1). Inaddition to the pertinent P1 residue, the conformation of the loopcarrying it appears to be very important for strong inhibition of trypsinand legumain. While replacement of the P1 residue N70K in the β5–β6

Fig. 7. Cospin–trypsin complex model. (A) A model of ternary complex between cospin (greencospin, binding to S1 pocket of trypsin. Trypsin molecule is shown as gray surface with the(D) and β11–β12 (E) loops by hydrogen bonds (red dashed lines). P1 residues and residues, in

loop of clitocypin did not produce a trypsin inhibitor, replacing theβ5–β6 loop in macrocypin 1 (FRIDNSI) with that in macrocypin 4(FRADKSI) did. Furthermore, in the cnispin β11–β12 loop theLys127Asn mutation annulled trypsin inhibition. On the other hand,the β2–β3 loop in cospin still retained weak trypsin inhibitory activityafter the Arg27Asn mutation, albeit the strength of inhibition wasdiminished 2 × 104 fold, indicating that in addition of favorable P1residue also other residues in the β2–β3 loop of cospin play animportant role in effective trypsin inhibition. This is also reflected insignificantly stronger inhibition of trypsin by cospin, when comparedwith cnispin. Changes of P1 residue to Asn did not have such a markedeffect on the inhibition of chymotrypsin, where Asn can still bind tolarger chymotrypsin's S1 pocket, optimized for accepting large hydro-phobic residues, when compared with narrow S1 pocket of trypsin,optimized for lysine or arginine residues.

In conclusion, yet another loop of the β-trefoil protease inhibitorswas identified in cnispin as a trypsin inhibitory loop, showing the

) (PDB ID: 3N0K) and trypsin (gray). Details of the β2–β3 (B) and β11–β12 (C) loops ofnegatively charged oxyanion hole (S1 pocket) shown in red. Stabilization of the β2–β3volved in hydrogen bonds, are marked.

Fig. 8. Schematic representation of the β-trefoil fold loops involved in protease inhibition.The protease family inhibited by individual loops is indicated in the adjacent gray circlesand the involvement of different β-trefoil fold loops is indicated by numbers: 1 (β2–β3)inhibition of trypsin by papaya protease inhibitor and cospin; 2 (β4–β5) inhibition oftrypsin/chymotrypsin by most members of family I3 protease inhibitors, including STI; 3(β5–β6) inhibition of trypsin by API-A and macrocypin 4 and inhibition of legumain bymycocypins, and inhibition of subtilisin by BASI; 4 (β9–β10) inhibition of trypsin byAPI-A; 5 (β11–β12) inhibition of trypsin by cnispin; and 6 (β1–β2 and β3–β4) inhibitionof papain-like cysteine proteases by mycocypins.

1756 P. Avanzo Caglič et al. / Biochimica et Biophysica Acta 1844 (2014) 1749–1756

great plasticity of the β-trefoil fold conferring inhibition of the sameprotease through different loops and inhibition of different proteasesthrough the same loop.

Acknowledgements

We are grateful to Dr Jože Brzin for valuable discussions and to DrRoger Pain for critical reading of the manuscript. This work wassupported by the Research Agency of the Republic of Slovenia [GrantsP4-0127 (to J.K.) and P1-0048 (to D.T.)]. The funding source had norole in study design, data collection and analysis, decision to publish,or preparation of the manuscript.

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.bbapap.2014.07.004.

References

[1] N.D. Rawlings, A.J. Barrett, A. Bateman, MEROPS: the database of proteolyticenzymes, their substrates and inhibitors, Nucleic Acids Res. 40 (2012) D343–D350.

[2] W. Bode, R. Huber, Natural protein proteinase inhibitors and their interaction withproteinases, Eur. J. Biochem. 204 (1992) 433–451.

[3] M. Laskowski, M.A. Qasim, What can the structures of enzyme–inhibitor complexestell us about the structures of enzyme substrate complexes? Biochim. Biophys. Acta1477 (2000) 324–337.

[4] D. Krowarsch, T. Cierpicki, F. Jelen, J. Otlewski, Canonical protein inhibitors of serineproteases, Cell. Mol. Life Sci. 60 (2003) 2427–2444.

[5] S. Khamrui, J. Dasgupta, J.K. Dattagupta, U. Sen, Single mutation at P1 of achymotrypsin inhibitor changes it to a trypsin inhibitor: X-ray structural (2.15A) and biochemical basis, Biochim. Biophys. Acta 1752 (2005) 65–72.

[6] Z. Malik, S. Amir, G. Pal, Z. Buzas, E. Varallyay, J. Antal, Z. Szilagyi, K. Vekey, B. Asboth,A. Patthy, L. Graf, Proteinase inhibitors from desert locust, Schistocerca gregaria:engineering of both P(1) and P(1)′ residues converts a potent chymotrypsininhibitor to a potent trypsin inhibitor, Biochim. Biophys. Acta 1434 (1999) 143–150.

[7] R. Helland, H. Czapinska, I. Leiros, M. Olufsen, J. Otlewski, A.O. Smalas, Structuralconsequences of accommodation of four non-cognate amino acid residues in theS1 pocket of bovine trypsin and chymotrypsin, J. Mol. Biol. 333 (2003) 845–861.

[8] H. Jering, H. Tschesche, Replacement of lysine by arginine, phenylalanine andtryptophan in the reactive site of the bovine trypsin–kallikrein inhibitor (Kunitz)and change of the inhibitory properties, Eur. J. Biochem. 61 (1976) 453–463.

[9] S. Kojima, N. Fushimi, A. Ikeda, I. Kumagai, K. Miura, Secretory production of chickenovomucoid domain 3 by Escherichia coli and alteration of inhibitory specificitytoward proteases by substitution of the P1 site residue, Gene 143 (1994) 239–243.

[10] L. Wen, I. Lee, G. Chen, J.K. Huang, Y. Gong, R. Krishnamoorthi, Changing theinhibitory specificity and function of Cucurbita maxima trypsin inhibitor-V bysite-directed mutagenesis, Biochem. Biophys. Res. Commun. 207 (1995) 897–902.

[11] M. Renko, J. Sabotič, D. Turk, Beta-trefoil inhibitors — from the work of Kunitzonward, Biol. Chem. 393 (2012) 1043–1054.

[12] A.A. Kortt, A.A. Kortt, A.A. Kortt, Isolation and properties of a chymotrypsin inhibitorfrom winged bean seed (Psophocarpus tetragonolobus (L) Dc.), Biochim. Biophys.Acta Protein Struct. 624 (1980) 237–248.

[13] J.K. Dattagupta, A. Podder, C. Chakrabarti, U. Sen, D. Mukhopadhyay, S.K. Dutta, M.Singh, Refined crystal structure (2.3 A) of a double-headed winged beanalpha-chymotrypsin inhibitor and location of its second reactive site, Proteins 35(1999) 321–331.

[14] M. Azarkan, S. Martinez-Rodriguez, L. Buts, D. Baeyens-Volant, A. Garcia-Pino,The plasticity of the beta-trefoil fold constitutes an evolutionary platform for prote-ase inhibition, J. Biol. Chem. 286 (2011) 43726–43734.

[15] R. Bao, C.Z. Zhou, C. Jiang, S.X. Lin, C.W. Chi, Y. Chen, The ternary structure of thedouble-headed arrowhead protease inhibitor API-A complexed with two trypsinsreveals a novel reactive site conformation, J. Biol. Chem. 284 (2009) 26676–26684.

[16] J. Sabotič, T. Popovič, V. Puizdar, J. Brzin, Macrocypins, a family of cysteine proteaseinhibitors from the basidiomycete Macrolepiota procera, FEBS J. 276 (2009)4334–4345.

[17] M. Renko, J. Sabotič, M.Mihelič, J. Brzin, J. Kos, D. Turk, Versatile loops inmycocypinsinhibit three protease families, J. Biol. Chem. 285 (2010) 308–316.

[18] S. Odani, K. Tominaga, S. Kondou, H. Hori, T. Koide, S. Hara, M. Isemura, S.Tsunasawa, The inhibitory properties and primary structure of a novel serineproteinase inhibitor from the fruiting body of the basidiomycete, Lentinus edodes,Eur. J. Biochem. 262 (1999) 915–923.

[19] J. Sabotič, S. Bleuler-Martinez, M. Renko, P. Avanzo Caglič, S. Kallert, B. Štrukelj, D.Turk, M. Aebi, J. Kos, M. Künzler, Structural basis of trypsin inhibition andentomotoxicity of cospin, serine protease inhibitor involved in defense ofCoprinopsis cinerea fruiting bodies, J. Biol. Chem. 287 (2012) 3898–3907.

[20] P. Avanzo, J. Sabotič, S. Anžlovar, T. Popovič, A. Leonardi, R.H. Pain, J. Kos, J. Brzin,Trypsin-specific inhibitors from the basidiomycete Clitocybe nebularis withregulatory and defensive functions, Microbiology 155 (2009) 3971–3981.

[21] J.F. Morrison, The slow-binding and slow, tight-binding inhibition of enzyme-catalyzed reactions, Trends Biochem. Sci. 7 (1982) 102–105.

[22] P.J.F. Henderson, Linear equation that describes steady-state kinetics of enzymesand subcellular particles interacting with tightly bound inhibitors, Biochem. J. 127(1972) 321–333.

[23] D. Turk, MAIN software for density averaging, model building, structure refinementand validation, Acta Crystallogr. Sect. D: Biol. Crystallogr. 69 (2013) 1342–1357.

[24] H.K. Song, S.W. Suh, Kunitz-type soybean trypsin inhibitor revisited: refinedstructure of its complex with porcine trypsin reveals an insight into the interactionbetween a homologous inhibitor from Erythrina caffra and tissue-type plasminogenactivator, J. Mol. Biol. 275 (1998) 347–363.

[25] S. Kojima, N. Takagi, T. Minagawa, N. Fushimi, K.I. Miura, Effects of amino acidreplacements around the reactive site of chicken ovomucoid domain 3 on theinhibitory activity toward chymotrypsin and trypsin, Protein Eng. 12 (1999) 857–862.

[26] W. Apostoluk, J. Otlewski, Variability of the canonical loop conformations in serineproteinases inhibitors and other proteins, Proteins 32 (1998) 459–474.

[27] J.E. Swedberg, S.J. de Veer, K.C. Sit, C.F. Reboul, A.M. Buckle, J.M. Harris, Mastering thecanonical loop of serine protease inhibitors: enhancing potency by optimising theinternal hydrogen bond network, PLoS One 6 (2011) e19302.

[28] D.N. Patil, A. Chaudhary, A.K. Sharma, S. Tomar, P. Kumar, Structural basis for dualinhibitory role of tamarind Kunitz inhibitor (TKI) against factor Xa and trypsin,FEBS J. 279 (2012) 4547–4564.

[29] M. Alvarez-Fernandez, A.J. Barrett, B. Gerhartz, P.M. Dando, J. Ni, M. Abrahamson,Inhibition of mammalian legumain by some cystatins is due to a novel secondreactive site, J. Biol. Chem. 274 (1999) 19195–19203.

[30] N. Eswar, B. Webb, M.A. Marti-Renom, M. Madhusudhan, D. Eramian, M.-y. Shen, U.Pieper, A. Sali, Comparative protein structure modeling using MODELLER, CurrentProtocols in Protein Science. 50 (2.9) (2007) 2.9.1–2.9.31 (http://onlinelibrary.wiley.com/doi/10.1002/0471140864.ps0209s50/abstract).

[31] S.N. Yang, M.M. Wolska-Klis, J.R. Cann, Gel electrophoresis of reactingmacromolecules. Rate-limited self-association, Anal. Biochem. 196 (1991) 192–198.