Peptides

Chemicals for peptide synthesis were purchased from Sigma-Aldrich, Iris Biotech GmbH, or Rapp Polymere GmbH, and used without further purification. Fmoc-L-4-guanidino-phenylalanine(N, N′-di-Boc)-OH and Fmoc-L-Ala-4-piperidyl(Alloc)-OH were commercially available. Analytical HPLC was performed on a Dionex UltiMate 3000, using a Phenomenex Gemini 110 Å C18 column (3 µm, 4.6×50 mm) with a flow rate of 1.0 ml per min and a linear gradient going from 95% H₂O, 5% acetonitrile with 0.1% HCOOH to 100% acetonitrile with 0.1% HCOOH over 10 min. Preparative HPLC was performed using a Dionex UltiMate 3000, equipped with a Phenomenex Gemini-NX C18 110 Å column, running at a flow rate of 10.0 ml/min and a linear gradient going from 95% H₂O/5% acetonitrile with 0.1% TFA to 100% acetonitrile with 0.1% TFA over 30 min. High resolution mass spectra were obtained on a Micromass LCT high resolution time-of-flight instrument by direct injection. Ionization was performed in positive electrospray mode.

Solid-phase peptide synthesis was performed using N^α-Fmoc-protected amino acids, a HBTU-HOBt activation protocol, and a Tentagel resin with Rink amide linker (0.23 mmol/g); HBTU (3.8 eq.), HOBt-HOAt (4∶1, 4 eq.), Fmoc-AA-OH (4 eq.), DIPEA (7.2 eq.) in NMP. Manual peptide synthesiswas performed with preactivation for 5 min and single couplings for 90 min. Fmoc deprotections were performed using piperidine/NMP (1∶4) for 2+15 min.

Automated peptide synthesis was performed on a Biotage SyroWave. Standard Fmoc-amino acids were coupled in parallel mode 120 min: Fmoc-AA-OH (s5.2 eq.), HBTU (5 eq.), HOBt/HOAt (4∶1, 5 eq.), DIPEA (9.8 eq.). Arginine mimics were coupled at 75°C for 10 min: Fmoc-AA-OH (2 eq.), HBTU (1.9 eq.), HOBt/HOAt (4∶1, 2 eq.), DIPEA (3.6 eq.). Fmoc deprotections were performed using piperidine/NMP (2∶3) for 3 followed by piperidine/NMP (1∶4) for 15 min.

Peptides with Alloc protected amino acids were deprotected to a free amine by treating the fully protected and N-acetylated peptides with a mixture of Pd(PPh₃)₄ (0.05 eq.) and Me₂NH⋅BH₃ (0.2 eq.) in degassed CH₂Cl₂ (30 min) and washed with NMP (5 x). The peptides were then treated with N, N'-di-Boc-1H-pyrazole-1-carboxamidine (5 eq.) in NMP overnight. Following peptide assembly, the resins were washed extensively with NMP and CH₂Cl₂, before peptide release with TFA/H₂O/triethylsilane (95∶2.5∶2.5). Peptide release proceeded for 2 h before the TFA-peptide mixture was collected by filtration. The resin was additionally washed with TFA (2x) and the TFA mixtures were pooled. TFA was removed under a stream of nitrogen and the peptide was precipitated with diethyl ether. The peptides were dissolved in a minimum amount of H₂O/acetonitrile (2∶1) before being purified by preparative HPLC. The purified peptides were dissolved in H₂O/acetonitrile (2∶1) to a final concentration of 1 mM. The solution was brought to pH 7.5–8 with NH₃ in methanol. The peptides were oxidized to form disulfide bridges by addition of 1.2 eq. of H₂O₂ (30–60 min). The oxidization was stopped with the addition of acetic acid (0.1 ml) followed by evaporation and HPLC purification. Mass spectrometry: Mupain-1-12 D9A [M+H]⁺ 1224,4 [M+2H]²⁺ 613,1; mupain-1-16 D9A [M+H]⁺ 1234,5 [M+2H]²⁺ 617,7.

The concentrations of the peptide variants were determined by measurements of OD₂₈₀ and the use of sequence-derived extinction coefficients provided by the Protparam tool on the Expasy server (located at http://www.expasy.org).

Proteases

cDNA encoding full length muPA, full length huPA and site directed mutants were cloned into the pTT5 or pCDNA3.1 vectors. All variants contained a C-terminal hexa-His tag. The cDNAs were transfected into human embryonic kidney 293 (HEK293) 6E suspension cells, which were cultured in a humidified 5% CO₂ incubator at 37°C. The medium used was F17 medium (Invitrogen) supplemented with 0.1% Pluronic F-68, a nonionic detergent (Invitrogen), 4 mM L-Gln (Lonza), and 25 µg/ml of the selective agent for eukaryotic cells G418 (Invitrogen). M_r∼25,000 linear polyethylenimine (400 µg) (Polysciences) was preincubated with cDNA (200 µg) for 15 min and added to 200 mL cells with a density of 1×10⁶ cells/mL. Twenty-four hours post-transfection, Tryptone N1 (Organotechnie SAS) was added to a final concentration of 0.5% (w/v). Conditioned medium was collected 96 h post-transfection, and the recombinant proteins were purified using immobilised metal ion affinity chromatography followed by benzamidine-Sepharose affinity chromatography. The purified proteins were at least 95% pure, as judged by Coomassie Blue-stained SDS-PAGE gels. To ensure that the uPAs purified from the conditioned media were completely in the two-chain form, they were treated with plasmin for 2 hours in a 1∶100 ratio.

The cloning, production, and purification of recombinant uPA catalytic domain (residues 159–411), harbouring a H99Y mutation, to be used for crystallisation and isothermal titration calorimetry (ITC), was largely as described previously [16]. Basically, the recombinant catalytic domain of huPA-H99Y was secreted from a stable Pichia pastoris strain (X-33) after induction by methanol and captured by a cation exchange column. The protein was further purified on a gel filtration column (Superdex 75 HR 10/30 column from GE Health Care) equilibrated with 20 mM sodium phosphate, pH 6.5, 150 mM NaCl. The protein was eluted as a single peak under these conditions, with a retention time of approximately 13.6 ml. The recombinant uPA catalytic domain expressed in this way is an active protease with an activity comparable to full-length two-chain uPA [16]. The protein was dialysed in 20 mM potassium phosphate, pH 6.5 overnight and concentrated to 10 mg/ml, using stirred ultrafiltration cells (Millipore and Amicon Bioseparations, Model-5124), prior to protein crystallization. The recombinant catalytic domain of huPA-H99Y to be used for ITC assays was further purified with benzamidine-Sepharose affinity chromatography.

Crystallization and data collection of uPA or uPA H99Y in complex with mupain-1 variants

The crystallization trials were carried out with the sitting-drop vapour-diffusion method. The crystals of the catalytic domain of huPA-H99Y were obtained by equilibrating huPA-H99Y protein against a reservoir solution containing 2.0 M ammonium sulfate, 50 mM sodium citrate, pH 4.6, and 5% polyethylene glycol (PEG) 400 at room temperature. The crystals appeared in about 3 days. The crystals of huPA-H99Y were then soaked for 2 weeks in new soaking buffer (40% PEG 4000, 0.1 M Tris-HCl, pH 7.4), containing 1 mM mupain-1 variants. A solution of 20% PEG 4000, 0.1 M Tris-HCl, pH 7.4 and 20% (v/v) glycerol was used as cryoprotectant for X-ray diffraction data of the crystals at the BL17U beamline, Shanghai Synchrotron Radiation Facility and 3W1A beamline, Beijing Synchrotron Radiation Facility (BSRF). The diffraction data was indexed and integrated by HKL2000 program package [17].

Crystal structure determination and refinement

The crystal structures of the different complexes were solved by molecular replacement [18], using the uPA structure (PDB code: 2NWN) [16] as the search model. The electron density for the peptide was clearly visible in the uPA active sites and was modelled based on the F_o–F_c difference map. The structures was refined (ccp4 program package)[18] and manually adjusted (by the molecular graphics program COOT) [19] iteratively until the convergence of the refinement. Solvent molecules were added using a F_o–F_c Fourier difference map at 2.5 σ in the final refinement step. Statistics of data collection and final model refinement are summarized in Supporting Table S1. The final structure was analysed by software Pymol [20].

Model of mouse uPA

The sequence of the catalytic domain of muPA (positions 16–243) (Uniprot P06869, EC: 3.4.21.73) was homology modeled onto the X-ray crystal structure of huPA-H99Y, using Molecular Operating Environment [21]. The sequence identity is 71% between muPA and huPA-H99Y (S1 Fig.). The generated molecular model was refined by CNS program package [22].

Determination of K_M values

To determine the K_M values for hydrolysis of S-2444 (pyro-Glu-Gly-Arg-p-nitroanilide) by the different uPA variants used in the present study, a 200 µL 2-fold dilution series of the substrate (4 - 0 mM for huPA variants, 24 – 0 mM for muPA variants) in a buffer of 10 mM HEPES, pH 7.4, 140 mM NaCl (HEPES-buffered saline, HBS), with 0.1% bovine serum albumin (BSA), was incubated 2 min at 37°C, prior to the addition of a fixed concentration of each protease (approximaterly 2 nM final concentration). The initial reaction velocities (V_i), monitored as the changes in absorbance at 405 nm, were plotted against the initial substrate concentration ([S]) and non-linear regression analysis was used to determine the K_M according to equation 1:

(1)

The K_M values for hydrolysis of S-2444 by the uPA variants employed in the present study are listed in S2 Table.

Determination of K_i values

For routine determination of K_i values for the inhibition of the various enzymes under steady state inhibition conditions, a fixed concentration of purified enzyme or conditioned media from transfected cells (approximately 2 nM enzyme as the final concentration) was pre-incubated in a total volume of 200 µL HBS with 0.1% BSA at 37°C, with various concentrations of mupain-1 variants for 15 min prior to the addition of the chromogenic substrate in concentrations approximately equal to the K_M value for each particular variant. The initial reaction velocities were monitored at an absorbance of 405 nm. The inhibition constants (K_i) were subsequently determined from the non-linear regression analyses of plots for V_i/V_o versus [I]₀, using Equation 2, derived under assumption of competitive inhibition:

(2)where V_i and V₀ are the reaction velocities in the presence and absence of inhibitor, respectively; [S]₀ and [I]₀ are the substrate and inhibitor concentrations, respectively; K_M is K_M for substrate hydrolysis by each protease. In Equation 2, it is assumed that [S]_free ≈ [S]₀ and [I]_free ≈ [I]₀. These conditions were fulfilled, as less than 10% of the substrate was converted to product in the assays and as the assay typically contained a final concentration of each protease of 2 nM and inhibitor concentrations in the µM range.

In cases, in which we observed no measurable inhibition (i.e., <10%) at the maximal inhibitor concentration used, i.e., 400 µM, the accuracy of the assay allowed us to conclude that the K_i value was more than 1000 µM (indicated as “>1000 µM” in the tables).

The validity of performing the K_i determinations with uPA-containing conditioned media from transfected cells was verified by controls in which the determinations were performed with conditioned media as well as with purified preparations. These controls were performed with murine uPA wt and human uPA wt, obtaining indistinguishable values with the two types of samples [13].

Surface plasmon resonance (SPR) analysis

To determine the equilibrium binding constants (K_D), the association rate constants (k_on) and dissociation rate constants (k_off) for peptide binding to uPA, surface plasmon resonance analysis was performed on a Biacore T200 instrument (Biacore, Uppsala, Sweden). A CM5 chip was coupled with the uPA variant (muPA or huPAH99Y) to be analysed, by injecting a concentration of 30 µg/mL uPA in immobilization buffer (10 mM sodium acetate, pH 5.0), aiming for an immobilised level of approximately 500 response units (RU). Immobilisation was followed by surface blocking with ethanolamine. A reference cell was prepared in the same way, without coupling of uPA. Mupain-1 variants in HBS with 0.1% BSA, in a dilution series, were injected at a flow rate of 30 µL per min during 60 s at 25°C. Subsequently, the dissociation was monitored during 600 s. Kinetic constants (k_on and k_off) were calculated with the Biacore Evaluation Software, using the 1∶1 kinetic fit. The K_D values were calculated as k_off/k_on.

ITC

For these experiments, we used the catalytic domain of huPA-H99Y expressed in and purified from Pichia pastoris strain X-33 (see above). The protein was dissolved in and dialysed against a buffer of 20 mM sodium phosphate, pH 7.4, 140 mM NaCl. The protein concentration was determined by absorbance at 280 nm, using an extinction coefficient of 43810 M⁻¹cm⁻¹. The peptides were dissolved in the above-mentioned buffer. All isothermal titration calorimetry experiments were performed with a MicroCal^TM ITC200 instrument equilibrated to a temperature of 25°C (298oK). The concentration of uPA-H99Y catalytic domain used in the 200 µl sample cell was 5–50 µM, depending on the affinity of the ligand. Titrations were performed by injecting 2 µl of the ligand until the total syringe volume of 40 µl was spent. Titration of ligand into buffer was performed to obtain buffer correction. The equilibrium association constant KA and the reaction enthalpy ΔH were calculated by fitting the integrated titration peaks using a one-binding-site model in the ITC ORIGIN7 programme package. The following formulas for Gibbs free energy ΔG were used to analyse the measured energies

(3)

(4)where R is the gas constant and T the absolute temperature. ΔS, the entropic change during the reaction, was calculated using equations 3 and 4 and the measured K_A and ΔH values.

NMR spectroscopy

Peptide samples were dissolved in a buffer of 10 mM sodium phosphate, 140 mM NaCl in D₂O/H₂O (7∶93, v/v). The pH was adjusted to 7.4. For chemical-shift reference and to increase the long-term stability of the samples, 2,2-dimethyl-2-silapentane-5-sulfonic acid (10 µM) and NaN₃ (150 µM) were added. The peptide concentrations were 5.0 and 6.7 mM for mupain-1 and mupain-1-16, respectively. NMR experiments were acquired with a Bruker Avance III 500 spectrometer (500.13 MHz; Bruker Biospin, Rheinstetten) equipped with a standard inverse triple-resonance TXI 5 mm probe. Two-dimensional TOCSY data (80 ms mixing time), NOESY data (200 ms mixing time) and natural abundant ¹³C HSQC data were acquired for both peptides. The experiments were acquired at 5°C to slow down peptide tumbling, favour lowest-energy conformations, and obtain the highest signal in the NOESY spectra for assignment. Assignments were obtained by standard methods with CCPN software [23]. Visualisation of spectra and integration of NOE peaks were performed in SPARKY [24]. Random coil shifts were calculated by using values provided by Kjærgaard et al. [25] and corrected by subtraction of correction values from Schwarzinger [26] which contains values for oxidised Cys and for cis-Pro. These correction values were obtained by subtracting Schwartzinger's values for Cys_red from Cys_ox and trans-Pro from cis-Pro for all the different proton types in these residues. The order parameter (S²) was calculated according to the method of Berjanskii and Wishart [27] as implemented within TALOS+ [28].

Inhibitory mechanism and binding mode studied by X-ray crystal structure analysis

While being unable to generate crystals of muPA, we did manage to crystallise huPA-H99Y, a murinised version of human uPA which, in contrast to human uPA wt, is able to bind mupain-1, although with a somewhat lower affinity than muPA [12], [13]. We determined the structures huPA-H99Y in complex with mupain-1 (CPAYSRYLDC) itself as well as with either of two other inhibitory peptides, namely mupain-1-12 (CPAYS[4-guanidinophenylalanine]YLDC) and mupain-1-16 (CPAYS[L-3-(N-amidino-4-piperidyl)alanine]YLDC). Mupain-1-12 and mupain-1-16 have around 10-fold higher affinities to huPA-H99Y than mupain-1 [13]. X-ray data collection and model refinement statistics are shown in S1 Table. Important features of the structures are illustrated in Fig. 2. The contact distances between the residues of the peptides and residues of huPA-H99Y are shown in S3-S5 Tables. The B-factors are listed in S7 Table.

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Figure 2. X-ray crystal structure analysis of huPA-H99Y in complex with peptidic inhibitors.

(A) Overall structure of the complexes between huPA-H99Y and mupain-1 (cyan), mupain-1-12 (salmon), mupain-1-16 (grey), and mupain-1-16-D9A (red). (B) A zoom on interactions of mupain-1's Arg⁶ in the S1 pocket; polar interactions are indicated by stippled lines. (C) A zoom on the polar interactions (stippled lines) between huPA-H99Y residue Arg³⁵ and mupain-1 residues Tyr⁷ and Asp⁹. (D) A zoom on the polar interaction (stippled lines) between huPA-H99Y residue Tyr⁹⁹ and mupain-1 residue Ser⁵. (E) An overlay of the active site areas of the huPA-H99Y – mupain-1 complex and the huPA S195A – PAI-1 Michaëlis complex (pdb entry 3pb1; [29]); the P2, P1, and P1′ residues are indicated, those of PAI-1 in salmon. (F) A zoom on the Lys¹⁴³ – Gln¹⁹² area of the huPA-H99Y – mupain-1 complex; distances, in Å, between different residues are indicated. In all parts of the figure, huPA-H99Y are shown in wheat cartoon presentation. In Fig. 1E, huPA S195A is shown in grey cartoon presentation. The peptides are shown in stick representation. huPA-H99Y residues are labelled with black letters, peptide residues with dark red letters.

https://doi.org/10.1371/journal.pone.0115872.g002

The analysis showed very similar conformations of the three peptides when bound to huPA-H99Y (Fig. 2A). The RMSD values among these peptides are quite small (0.32–0.35 Å). In the complexes, the inhibitory peptides adopt cyclic conformations with an overall Ω shape. The disulfide bonds are the main structural restraint responsible for this conformation. Beginning from the N-terminus, the cyclic peptides approach the active site of huPA-H99Y from the 99-loop, insert residue 6 into the S1 pocket, and exit the active site towards the 37-loop (Fig. 2A). In each of the structures, the amino acid in position 6 of the peptide, i.e., Arg, 4-guanidinophenylalanine, or L-3-(N-amidino-4-piperidyl)alanine, forms polar interactions to Asp¹⁸⁹, Ser¹⁹⁰, and Gly²¹⁸ in the S1 pocket (Fig. 1B). In addition, the X-ray crystal structure analysis showed that the huPA-H99Y residues Arg³⁵, Val⁴¹, Leu^97b, Tyr⁹⁹, Gln¹⁹², Trp²¹⁵, and Arg²¹⁷ have the largest contact surface area to the peptides (S3-S5 Tables). In particular, Arg³⁵ forms polar interactions with peptide residues Tyr⁷ and Asp⁹ (Fig. 2C) and Tyr⁹⁹ forms polar interactions with peptide residue Ser⁴ (Fig. 2D). In the enzyme-peptide structures, the peptides are constrained by two type I tight β-turns (Pro²-Ala³-Tyr⁴-Ser⁵ and Tyr⁷-Leu⁸-Asp⁹-Cys¹⁰) and three intra-peptide H-bonds (Pro² O – Ser⁵ N; Serⁱ⁵ Oγ - Tyr⁷ N; Ser⁵ Oγ - Arg⁶ N; S2 Fig.). The tight β-turns are likely to play an important role in maintaining the conformation and stability of the bound peptide. On the enzyme side, there are no major changes in the conformation of surface loops of the enzyme following peptide binding based on the comparison of the structure of the enzyme in the absence or the presence of peptide.

The inhibitory mechanism of these peptides and the reason that they are inhibitors and not substrates readily become evident from the structural analysis. The overall conformation of the peptide backbone on the enzyme surface is quite similar to that of the reactive centre loop of plasminogen activator inhibitor-1 (PAI-1) in its Michaëlis complex with uPA S195A, aligning into the active site in a substrate-like manner (Fig. 2E; [29]). However, compared to PAI-1 in its Michaëlis complex with uPA S195A, the scissile bond of mupain-1 in its complex with uPA-H99Y is shifted approximately 0.5 Å away from residue 195. The distance from the Ser¹⁹⁵ Oγ to the carbonyl group of the P1 residue of mupain-1 and derivatives is too large (approximately 3.9 Å) to allow the nucleophilic attack associated with catalysis (Fig. 2E). Moreover, the distance from the oxygen atom of the carbonyl group of the P1 residue to the amido group of the Ser¹⁹⁵ is too large (approximately 4 Å) for formation of a polar interaction, implying that oxyanion stabilisation cannot take place (Fig. 2E).

In spite of the X-ray crystal structure analysis having a good resolution, no differences could be detected between the complexes of huPA-H99Y with each of the peptides mupain-1, mupain-1-12, and mupain-1-16, the K_i values of which differ around 10-fold. A 10-fold difference in K_i corresponds to a Δ(ΔG) for binding of approximately 6 kJ/mol, about the same energy as that of an average hydrogen bond. In this case, it is therefore possible that small differences in hydrophobic interactions and in the length and angles of polar interactions, at the detection limit by the structural analysis, may account for the differences in K_i values. Alternatively, and more likely, the peptide-enzyme complexes, in solution, may sample a number of similar conformations of which only the most stable one is selected during crystallisation.

Analysis of peptide-huPA-H99Y exosite interactions by site-directed mutagenesis

We determined the K_i values for inhibition of variants of huPA-H99Y by variants of mupain-1, mupain-1-12, and mupain-1-16. The Ala-substituted residues of the peptide and of huPA-H99Y are those deemed to be important for binding from the X-ray crystal structure analysis (Table 1). In good agreement with the structural analysis, Ala-substitution of mupain-1 residues Pro², Tyr⁴, Ser⁵, and Arg⁶ led to strong reductions in affinity, Ala-substitution of Tyr⁷ to a moderate reduction in affinity, and Ala-substitution of Leu⁸ to a very small change in affinity. The observed effect of Ala-substituting the peptide's Ser⁵ is in good agreement with the fact that the binding of mupain-1 to huPA is dependent on the substitution of its His⁹⁹ with Tyr [12], shown here to be able to form a hydrogen bond with Ser⁵ (Fig. 2D). Surprisingly, Ala-substitution of Asp⁹ of mupain-1, mupain-1-12, and mupain-1-16 led to a 3–10-fold increased affinity to huPA-H99Y. This observation is in contrast to the expectancies from the X-ray crystal structure analysis, which predicts a polar interaction between Asp⁹ of mupain-1 and Arg³⁵ of huPA-H99Y (Fig. 2C; S3-S5 Tables).

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Table 1. Inhibition of huPA-H99Y and huPA-H99Y exosite mutants by mupain-1 variants.

https://doi.org/10.1371/journal.pone.0115872.t001

Ala-substitution of huPA-H99Y residues Arg³⁵, Val⁴¹, Lys¹⁴³, and Gln¹⁹² led to 2–4 fold reductions in affinity to mupain-1, but smaller if any reductions in the affinity to mupain-1-D9A, mupain-1-12, mupain-1-16, mupain-1-12-D9A, and mupain-1-16-D9A. The effects of the R35A and Q192A mutations are in good agreement with the predictions of polar interactions from the X-ray crystal structure analysis (Fig. 2C, Fig. 2F). The V41A mutation may result in loss of hydrophobic interactions. From the X-ray crystal structure analysis, Lys143 is not predicted to make any direct contacts to the peptides, but the observed effect of the K143A mutation may be caused by an indirect effect through a polar interaction between Lys 143 and Gln192 (Fig. 2F). The observed changes following the Ala substitutions in the enzyme were in all cases small, in agreement with the fact that the predicted polar interactions are surface exposed. In general, there was less dependence on the exosite interactions with the peptides with the unnatural P1 residues and the peptides with an D9A substitution. This observation suggests that the effects of the exosite mutations and the D9A substitution is influenced by interactions in the S1 pocket.

The K_i determinations were supported by determinations of K_D values with SPR (Table 2; S3 Fig.) and ITC (Table 3). The K_D values determined by SPR and ITC agreed well with the K_i values, in particular when considering that K_D values were routinely determined at 25°C and the K_i values at 37°C; separate control experiments showed that the K_i values were 2–3 fold lower at 25°C than at 37°C (data not shown). The SPR measurements showed that the 9–12-fold increased affinities to huPA-H99Y following the D9A substitution were associated with 3–4-fold increased k_on values as well as 2–3-fold decreased k_off values (Table 2). The ITC measurements showed that the increased affinity of the Ala⁹ peptides to huPA-H99Y, as compared to the original Asp⁹ peptides, was associated with a binding entropy penalty but mainly accounted for by a more favourable binding enthalpy (except with mupain-1 and mupain-1 D9A, in which case the differences were not statistically significant; Table 3). The most ready molecular interpretation of the SPR and ITC data, taken together, is that the D9A substitution renders the peptide more flexible in solution, thereby making the binding entropy less favourable, the association activation energy lower, and the k_on higher, and allows a more favorable binding enthalpy and a more stable bound state, thereby increasing the dissociation activation energy and decreasing the k_off.

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Table 2. Surface plasmon resonance analysis of the binding of peptides to muPA or huPA-H99Y.

https://doi.org/10.1371/journal.pone.0115872.t002

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Table 3. Isothermal titration calorimetry for binding of peptides to huPA-H99Y.

https://doi.org/10.1371/journal.pone.0115872.t003

Considering the unexpected effect of the D9A substitution, we also crystallised mupain-1-16 D9A in complex with huPA-H99Y (S1 and S6 Tables), but in spite of the much higher affinity of the D9A peptides, the structures of the mupain-1-16 and the mupain-1-16-D9A complex were indistinguishable (Fig. 2A). Noticeably, however, the relative B-factor for mupain-1-16 D9A was higher than that for mupain-1-16 (1.39 versus 1.24; S7 Table).

In summary, while most interactions between the peptides and huPA-H99Y predicted by X-ray crystal structure analysis were largely in agreement with the results of the site-directed mutagenesis, the increased affinity following the D9A substitution in mupain-1 was unexpected and could not be correlated with any structural differences observable by X-ray rystal structuyre analysis. However, the SPR and ITC analyses indicated a more stable complex following the D9A substitution, in spite of a binding entropy penalty.

Analysis of peptide-muPA exosite interactions by site-directed mutagenesis

Next, we analysed the K_i values for inhibition of several muPA variants by mupain-1, mupain-1-12, and mupain-1-16, Ala substituting residues of the peptide and the enzyme in positions implicated in peptide-enzyme interaction by the X-ray crystal structure analysis of the huPA-H99Y-peptide complex (see S1 Fig. for alignment of the amino acid sequences of the catalytic domains of huPA and muPA). Previously, we showed that Ala-substitution of mupain-1 residues Pro², Tyr⁴, Ser⁵, Arg⁶, and Tyr⁷ leads to substantial loss of affinity to muPA, while Leu⁸ and Asp⁹ could be Ala-substituted with minimal consequences [12]. Thus, the effect of the D9A substitution is different with muPA and huPA-H99Y. Now, we found that Ala-substitutions of muPA residues Lys⁴¹, Tyr⁹⁹ and Lys¹⁴³ increase the K_i values 5–50-fold (Table 4). However, in contrast to observations with huPA-H99Y, there were no effects of Ala substitutions of muPA residues in the 37-loop. From a model of muPA in complex with mupain-1, it seems that Lys⁴¹ is able to make polar interactions with Tyr⁷ (2.3 Å) and Asp⁹ (2.8 Å) of mupain-1 (Fig. 3C), explaining the effect of substitution of this muPA residue. From the model, Lys¹⁴³ is predicted to be more than 6 Å away from the closest part of the peptide and more than 6 Å away from Lys¹⁹² (Fig. 3B), leading to the notion that the peptide and/or residue 192 have different conformations in the huPA-H99Y-mupain-1 complex and the muPA-mupain-1 complex. Thus, the observed importance of mupain-1 residues Pro², Tyr⁴, Ser⁵ and Arg⁶ and muPA residue Tyr⁹⁹ suggests that the CCPAYSR stretch of peptides occupies a position on muPA similar to that inferred from the X-ray crystal structure analysis of the peptide-huPA-H99Y complexes, while the stretch YLD of mupain-1 makes different interactions in the two complexes.

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Figure 3. Structural model of mupain-1 in complex with muPA.

(A) Overall model of the complex between muPA (teal ribbon presentation) and mupain-1 (red stick representation). The enzyme is shown in teal cartoon presentation. (B) A zoom on the Lys¹⁴³ area of the model, showing distances from Lys¹⁴³ to the closest atoms in the peptide. (C) A zoom on the Lys⁴¹ area of the model, showing the distances between Lys41 and peptide residues Tyr⁷and Asp⁹. (D) A zoom on the entrance to the S1 pocket of the model, with Val²¹³ at the entrance indicated; the enzyme is represented as teal surface. huPA-H99Y residues are labelled with black letters, peptide residues with dark red letters.

https://doi.org/10.1371/journal.pone.0115872.g003

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Table 4. Inhibition of muPA wt and muPA exosite mutants by mupain-1 variants.

https://doi.org/10.1371/journal.pone.0115872.t004

Interestingly, the D9A substitution, while having no effects on the affinities of the peptides to muPA wt, muPA-Y99A, and muPA-K143A, strongly increased the affinity to muPA-K41A (Table 2). Thus, the K_i values for inhibition of muPA by the Ala⁹ peptides depend differently on exosite mutations than those for the original Asp⁹ peptides. This observation shows than the interactions between the Ala⁹ peptides and muPA differ from the interactions between the Asp⁹ peptides and muPA. Importantly, the D9A substitution in all cases reduced the difference in K_i between huPA-H99Y and muPA (Tables 1 and 4): For the Asp⁹ peptides, the K_i value for inhibition of huPA-H99Y were 28, 7, and 55-fold higher than those for inhibition of muPA, while the corresponding values for the Ala⁹ peptides were only 7, 1, and 4-fold higher.

The K_i values were in good agreement with the K_D values determined by SPR (Table 2).

Analysis of S1-P1 interactions by site-directed mutagenesis

In order to characterise the mechanism of the increase in affinity following substitution of the P1 Arg with either of the two unnatural P1 residues, we introduced mutations in the S1 pocket of muPA, i.e., S190A and V213T (Table 5). The X-ray crystal structure analysis of the peptide-huPA-H99Y complexes implicated Ser¹⁹⁰ in hydrogen bonding to the P1 residues (Fig. 2B; S3–S5 Tables); many serine proteases, including tissue-type plasminogen activator (tPA), have an Ala in this position. Val²¹³ forms a hydrophobic patch at the entrance to the S1 pocket (Fig. 3D); we substituted the Val with a Thr, as some serine proteases, including plasma kallikrein, has a Thr in this position. The S190A substitution resulted in a decreased affinity for all the peptides, while the V213T mutation resulted in an increased affinity for all the peptides (Table 5).

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Table 5. Inhibition of muPA wt and muPA S1 pocket mutants by mupain-1 variants.

https://doi.org/10.1371/journal.pone.0115872.t005

In order to visualise the effects of the S1 pocket mutations, the K_i values for the inhibition of the muPA mutants by each of the 6 peptides were plotted logarithmically versus the K_i values for inhibition of muPA wt by the same peptides (Fig. 4). For data points above the y = x line, the mutations increase the K_i value or decrease the affinity; for points above the y = x line, the mutations decrease the K_i value or increase the affinity. The slopes of the lines defined by the data points (which may be referred to as an “interdependence factor”) were 0.68 in the case of the S190A mutation and 1.27 in the case of the V213T mutation. The slopes being different from 1 shows that the effects of the S1 pocket mutations vary with the P1 residue: Following the S190A mutation, the K_i value increases more with mupain-1-12 and mupain-1-16 variants than with the Arg variants, while following the V213T mutation, the K_i value decreases more with the mupain-1-12 and mupain-1-16 variants than with the mupain-1 Arg variants. For instance, the V213T mutation increases the affinity for the mupain-1 Arg variants around 2.5-fold, but increases the affinity for the L-3-(N-amidino-4-piperidyl)alanine] variants around 5-fold. These findings are compatible with the notion that each of the three P1 residues fit differently into the S1 pocket.

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Figure 4. The relationship between the K_i values for inhibition of muPA S1 mutants and the K_i values for inhibition of muPA wt.

The figure is based on the K_i values presented in Table 6. The x-axis shows the K_i values for inhibition of muPA wt by the indicated peptides. The y-axis shows the corresponding K_i values for inhibition of muPA S190A (filled dots) or muPA V213T (open dots). The lines resulted from simple linear regression analysis. The slopes of the lines are 0.68 (muPA-S190A) and 1.27 (V123T). The stippled line is the one which would have resulted if the K_i values for inhibition of the muPA S1 mutants had been identical to those for inhibition of muPA wt (y = x).

https://doi.org/10.1371/journal.pone.0115872.g004

To further visualise the importance of the P1 residues of mupain-1, the K_i values for inhibition of the muPA exosite mutants K41A, Y99A, and K143A by mupain-1, mupain-1-12, and mupain-1-16 and their D9A variants were plotted logarithmically versus the corresponding K_i values for the wt enzymes (Fig. 5). The above-mentioned difference between the Asp⁹ and the Ala⁹ peptides with respect to inhibition of muPA K41A was obvious in the plot, the data points in an Ala⁹ group falling below the y = x line and the data points for the Aap⁹ group falling above the y = x line. There was no such split with the other muPA mutants. With the K41A and the Y99A mutants, the slopes of the lines defined by the data pairs were relatively close to 1. However, with the K143A mutant, the slopes were around 1.4. This slope corresponds to a 9-fold increase in K_i for mupain-1 following the K143A mutation and an only 2.9-fold increase in K_i for mupain-1-16 following the K143A mutation and similar differences when comparing the other peptides. This observation shows that the identity of the amino acid in position 143 influences the effect of a P1 substitution and vice versa.

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Figure 5. The relationship between the K_i values for inhibition of muPA exosite mutants and the K_i value for inhibition of muPA wt.

The figure is based on the K_i values presented in Table 5. The x-axes show the K_i values for inhibition of muPA wt by the indicated peptides. The y-axes shows the corresponding K_i values for inhibition of muPA K41A, Y99A, or K143A. The lines resulted from simple linear regression analysis. The slope of the lines are 1.04 (muPA K41A with Asp⁹ peptides); 1.15 (muPA K41A with Ala⁹ peptides); 1.15 (muPA Y99A); 1.40 (muPA K143A). The stippled line shows the line which would have resulted if the K_i values for exosite mutants and muPA wt had been identical (y = x).

https://doi.org/10.1371/journal.pone.0115872.g005

The effects of substituting the P1 residue were also analysed by SPR (Table 2). The increased affinities associated with the unnatural P1 residues were found to be accounted for almost exclusively by lower off-rates.

Taken together, these observations are in agreement with the conclusion that the increased affinity with the two unnatural P1 residues is caused by changes in the binding in the S1 pocket. Interestingly, there seems to be a cross-talk between interactions of the peptide in the S1 pocket and interactions with Lys143.

Analysis of the importance of the P1 residue by NMR

For a further characterization of the importance of the P1 residue, mupain-1 and mupain-1-16 were subjected to ¹H liquid-state NMR analysis. Analysis of the backbone amide and α-proton region of a TOCSY spectrum revealed a doubling of the expected number of peaks of mupain-1 and mupain-1-16. Following standard procedures for analysis of TOCSY and NOESY spectra [30], the NMR data could be assigned to two markedly different resonance forms, as illustrated by two parallel “backbone walks” connecting sequential residues (Fig. 6). Based on analysis of ¹³C chemical shifts assigned by natural abundance ¹³C-HSQC (difference between C_β and C_γ chemical shifts) (S8 Table) [31], the two sets of resonances were inferred to be due to cis-trans isomerization around the Cys¹-Pro² peptide bond. By integration of isolated peaks in the TOCSY spectrum, the steady state ratio between the two forms was found to be 1∶3 in favor of the trans conformation. Since no exchange cross peaks were observed between the two distinct resonance sets corresponding to the two conformers in the time frame of a 200 ms NOESY experiment, the interconversion between the two conformations was estimated to be slower than 1 s⁻¹ for both mupain-1 and mupain-1-16. The assigned chemical shifts were close to the random coil values, indicating that the peptides are flexible in solution. Both the proton and carbon chemical shifts for the two isomeric forms were substantially different for most of the residues, suggesting that the average conformations for the cis and trans isomers are different. It is notable that the spectral differences due to the cis-trans isomerism propagated throughout the entire peptide chain and not just to the adjacent residues, indicating that the structures are not completely disordered. Furthermore, the chemical shifts for trans-mupain-1 and cis-mupain-1 are very similar to those of trans-mupain-1-16 and cis-mupain-1-16, respectively (as illustrated, e.g., by H_α in S4 Fig.). This similarity strongly suggests that the structures or average conformations of each of the isomers of each of the two peptides are very similar. Also the similarity in steady state ratio of the cis- and trans-conformations of the two peptides is in agreement with the notion that the two peptides have the same overall structural and dynamic trends.

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Figure 6. TOCSY (orange) and NOESY (green) of mupain-1-16.

Assignments of the two isomeric forms is illustrated by connecting TOCSY H_α(i)-H_N(i) and NOESY H_α(i)-H_N(i+1) cross peaks with lines representing a shared reference commonly referred to as “backbone walks”.

https://doi.org/10.1371/journal.pone.0115872.g006

Importantly, the presence of the peptides in both a trans- and a cis-conformation in solution should be contrasted with the fact that only the trans-conformation was observed in the crystal structures of the peptide-enzyme complexes.

The flexibility of the peptides was probed by deriving a predicted order parameter, S², based on the chemical shifts, using random coil index implemented within TALOS+ (Fig. 7). Depending on movements on the picosecond to nanosecond time scale, an S² value of 0 corresponds to a completely disordered peptide and an S² value of 1 to a completely rigid one. The predicted order parameter was found to be relatively low, as also observed from the TALOS+ classification, in which most of the residues fall in the dynamic category.

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Figure 7. Predicted order parameter S² (top) calculated by TALOS+.

The dots (bottom) represent classification of the residue from TALOS+, as based on the mobility of the backbone, the certainty of the angles of the reference triplets and whether the angles fall into allowed regions in the Ramachandran plot [34]. The colour codes represent good (green), dynamic (yellow), ambiguous (red), and no prediction (blue) for the two different peptides in cis and trans conformations.

https://doi.org/10.1371/journal.pone.0115872.g007

In summary, the NMR analysis of the peptides in solution did not reveal differences in solution structures between mupain-1 and mupain-1-16.