Design and Characterization of a Hybrid Miniprotein That Specifically Inhibits Porcine Pancreatic Elastase*,

Kai Hilpert {ddagger}, Helga Wessner {ddagger}, Jens Schneider-Mergener §, Karin Welfle ¶, Rolf Misselwitz ¶, Heinz Welfle ¶, Andreas C. Hocke ||, Stefan Hippenstiel || and Wolfgang Höhne {ddagger} **

From the {ddagger}Humboldt University of Berlin, Medical Faculty Charité, Department of Biochemistry, Monbijoustrasse 2, 10117 Berlin, Germany, the §Humboldt University of Berlin, Medical Faculty Charité, Department of Medical Immunology, Schumannstrasse 20-21, 10117 Berlin, Germany, the Max-Delbrück-Center of Molecular Medicine, Robert-Rössle-Strasse 10, 13122 Berlin, Germany, and the ||Humboldt University of Berlin, Medical Faculty Charité, Department of Internal Medicine, Augustenburger Platz 1, 13353 Berlin, Germany

Received for publication, December 1, 2002 , and in revised form, April 7, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Studying protease/peptide inhibitor interactions is a useful tool for understanding molecular recognition in general and is particularly relevant for the rational design of inhibitors with therapeutic potential. An inhibitory peptide (PMTLEYR) derived from the third domain of turkey ovomucoid inhibitor and optimized for specific porcine pancreatic elastase inhibition was introduced into an inhibitor scaffold to increase the proteolytic stability of the peptide. The trypsin-specific squash inhibitor EETI II from Ecballium elaterium was chosen as the scaffold. The resulting hybrid inhibitor HEI-TOE I (hybrid inhibitor from E. elaterium and the optimized binding loop of the third domain of turkey ovomucoid inhibitor) shows a specificity and affinity to porcine pancreatic elastase similar to the free inhibitory peptide but with significantly higher proteolytic stability. Isothermal titration calorimetry revealed that elastase binding of HEI-TOE I occurs with a small unfavorable positive enthalpy contribution, a large favorable positive entropy change, and a large negative heat capacity change. In addition, the inhibitory peptide and the hybrid inhibitor HEI-TOE I protected endothelial cells against degradation following treatment with porcine pancreatic elastase.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Proteases can play a decisive role as indirect virulence factors promoting infection by viruses, bacteria, or parasites. Moreover, inappropriate or altered host protease activities are involved in many diseases, such as cancer, blood clotting, Alzheimer's disease, rheumatoid arthritis, arteriosclerosis, and pulmonary emphysema. Consequently protease inhibitors have a huge potential as therapeutic tools for treating any number of diverse diseases (1). A well known example is the generation of protease inhibitors against human immunodeficiency virus, type 1, protease (1). However, for each individual indication major efforts are required to find the appropriate specific, effective, stable, and non-toxic protease inhibitor. Apart from this, investigating inhibitor/protease interactions leads to a better understanding of protein molecular recognition and specificity in general.

There are different methods and strategies to develop an inhibitor for a given protease. Previously, we described a method for characterizing and optimizing peptide inhibitor/protease interactions using cellulose-bound peptides (2). We demonstrated an optimization process for a 9-mer peptide originating from the third domain of the turkey ovomucoid inhibitor, OMTKY3.1 This 56-amino acid protein strongly inhibits a broad spectrum of serine proteases, e.g. bovine {alpha}-chymotrypsin with a Ki = 5.5 x 1012 M (3). The peptide from the binding loop of OMTKY3 was optimized against porcine pancreatic elastase (PPE, EC 3.4.21.36 [EC] ). This 240-amino acid enzyme is a member of the S1 class of serine proteases. Its active site comprises eight subsites (S5–S3'; according to the nomenclature of Schechter and Berger (4)) and cleaves the amide bond of amino acids with small hydrophobic side chains (5). Our optimization procedure led to a 7-mer peptide (PMTLEYR), which although showing a high specificity and affinity toward PPE, turned out to be unstable against proteolytic degradation by PPE. For more detailed investigation of the inhibitor/PPE interaction, e.g. by x-ray structural and biophysical analysis of corresponding complexes, as well as for medical applications, high proteolytic stability is an important prerequisite. One possible way to create a more stable inhibitor molecule is to introduce the inhibitory peptide sequence into a stable scaffold. Such scaffolds are provided by the squash-type inhibitors.

Squash-type inhibitors are natural serine protease inhibitors occurring in plant seeds of Curcubitaceae. They consist of only ~30 amino acids and have a rigid and stable structure cross-linked by three disulfide bridges. They are resistant to proteolytic cleavage and stable at elevated temperatures (for review, see Ref. 6). EETI II from the squirting cucumber Ecballium elaterium is a well characterized squash-type inhibitor with a high affinity and specificity for trypsin (Kd of 1.2 x 1012 M) (7). It was shown to fold into its native structure even if considerable amino acid exchanges and length variations are incorporated within its binding loop (8). Natural squash inhibitors that inhibit porcine pancreatic elastase are also known from the literature. The inhibitors MCEI I (9) and MCEI II, III, and IV (10) were isolated from bitter gourd (Momordica charantia) and inhibit PPE with Ki values between 9.7 x 107 and 4.7 x 109 M (9, 10).

We describe here the introduction of sequences, derived from the inhibitory peptide PMTLEYR, into the squash inhibitor EETI II, and the characterization of several hybrid squash inhibitor variants with respect to inhibition strength, specificity, and stability. The energetics of the inhibitor-elastase complex formation at different temperatures and in various buffer systems was studied using isothermal titration calorimetry (ITC). Furthermore, the effects of the optimized inhibitory peptide PMTLEYR and of one of the hybrid squash inhibitors (HEI-TOE I) on elastase-treated human endothelial cells were compared.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Peptide Synthesis—The peptides and squash inhibitors were synthesized in reduced form on solid phase according to standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) machine protocols using a multiple peptide synthesizer (Abimed, Langenfeld, Germany). After a purification step by reverse phase HPLC, the disulfide bridges were formed using charcoal according to a recently described cyclization protocol (11). The degree of disulfide formation was monitored by measuring the decrease of free cysteine content with 5,5'-dithiobis(2-nitrobenzoic acid). The cyclization procedure was applied until no free cysteine was detectable. After disulfide formation another reverse phase HPLC purification step was performed. The purity of the final products was greater than 98%.

Ki and Proteolytic Stability Determination—All enzymes and substrates were purchased from Serva (Heidelberg, Germany). The activity of the enzymes was measured by monitoring hydrolysis of the corresponding substrates on a recording spectrophotometer model UV-160A (Shimadzu, Duisburg, Germany). Varying the substrate (63 µM to 2.5 mM) and inhibitor concentrations (55 nM to 1 mM) the Ki values were determined in a 200-µl assay at 25 °C. For PPE, human leukocyte elastase (HLE), and trypsin a 0.1 M Tris buffer, pH 8.5, was used; in the case of chymotrypsin, a 0.1 M Tris buffer, pH 7.5, was used. The activity of chymotrypsin (Serva, Heidelberg, Germany) was determined using Suc-Ala-Ala-Phe-pNA (Serva, Heidelberg, Germany); for PPE, Suc-Ala-Ala-Ala-pNA; for HLE, Suc-Ala-Ala-Val-pNA; and for trypsin, N{alpha}-benzoyl-DL-arginine-pNA, all solubilized in Me2SO.

The proteolytic stability of the peptides was measured (determined via PPE activity) by preincubation of different inhibitor concentrations (10–500 µM) with PPE (15–150 units in 150-µl assay volume) for between 2 min and 8 days in 0.1 M Tris buffer, pH 8.5, at 25 °C.

Isothermal Titration Experiments—Isothermal titrations were performed at 6.8, 11.5, 16.0 and 25 °C using a MicroCal MCS isothermal titration calorimeter (MicroCal Inc., Northampton, MA) as described recently (12, 13). PPE and HEI-TOE I were dialyzed against the following buffers at pH 6.0 containing 0.15 M NaCl, 50 mM PIPES, 50 mM MES, and 50 mM MOPS, which have ionization enthalpies at 7.0 °C of 11.1 kJ·mol1, 15.2 kJ·mol1, and 21.1 kJ·mol1, respectively (14). The ionization enthalpies at different temperatures were calculated using a heat capacity change, , of 0.019 kJ·mol1·K1 for PIPES (14). The concentrations of PPE and HEI-TOE I were determined spectrophotometrically at 280 and 276 nm, respectively, using extinction coefficients of 5.2 x 104 M–1·cm1 (15) and 1.885 x 103 M–1·cm1 (calculated from the amino acid composition according to Gill and von Hippel (16)). In a typical titration experiment 5 µl of HEI-TOE I solution (around 560 µM) were titrated into 1.4 ml of PPE solution (around 12 µM). The experimental raw data were corrected by subtraction of the heat of dilution of the peptide, transformed into kJ·mol1 of injectant, then fitted to a model of identical and independent binding sites using the ORIGIN software package provided with the calorimeter (MicroCal Inc.). Enthalpy changes, , of complex formation were determined by correction of experimentally estimated enthalpy changes, , for of the respective buffer.

The total entropy can be dissected according to Equation 1,

(Eq. 1)
in terms of the main contributions of solvation entropy, , conformational entropy, , and translational and rotational entropy, . The value of is debated in the literature and here J·mol1·K1 was taken, which is close to the cratic entropy (17). Change of the solvation entropy, , was calculated according to Equation 2,

(Eq. 2)
where T is the temperature in Kelvin, K is the reference temperature where is zero (18) and {Delta}Cp is the heat capacity change.

Entropic contributions of the hydrophobic effect () to HEI-TOE I-PPE binding were calculated according to Equation 3 (19),

(Eq. 3)
where {Delta}Anp is in Å2, T is in degrees Kelvin, and is in entropy units (e.u. or cal·mol1·K1). Ts is the temperature where {Delta}S0 is zero and was calculated according to Equation 4,

(Eq. 4)
with the experimental values J·mol1·K1 at 298 K and {Delta}Cp,exp = –1.275 kJ·mol1·K1. At Ts the entropy changes from the hydrophobic effect and from any other entropic contribution despite add to zero.

(Eq. 5)
Polar ({Delta}ASAp) and nonpolar ({Delta}ASAnp) buried surface areas were calculated according to Refs. 20 and 21 by means of empirical Equations 6, 7, 8,

(Eq. 6)

(Eq. 7)

(Eq. 8)
where T is in °C, a = 1.88 J·K1·mol1·Å2, b = –1.09 J·K1·mol1·Å2, c = –35.3 J·mol1·Å2, and d = 131 J·mol1·Å2.

Buried surface areas used for thermodynamic calculations were obtained from atomic coordinates of elastase-HEI-TOE I complex (Protein Data Bank code 1mcv [PDB] , adapted for the calculations) with program CALCASA of the program suite STC (structure-based thermodynamic calculations) (17). In CALCASA is implemented the Lee and Richards algorithm (22); probe radius and slice widths are 1.4 and 0.25 Å, respectively. Identical structures for both PPE and HEI-TOE I were assumed in the complex and in free form. The program suite STC sums up to the individual contributions of all residues buried in the complex. Program THERMO of the STC suite calculates thermodynamic parameters {Delta}Cp,calc, , , and dissociation constant Kd (17). THERMO provides the possibility to vary arbitrarily the value for and thus to achieve apparent but meaningless agreement of experimental and calculated {Delta}G0.

Preparation of Human Endothelial Cells and F-actin Staining— Human umbilical cord vein endothelial cells (HUVEC) were isolated from umbilical cord veins and identified as described previously (23). Briefly, cells obtained from collagenase digestion were washed, resuspended, cultivated in MCDB 131, 10% fetal calf serum, and seeded onto 24-well Thermanox® slides (Nunc, Wiesbaden, Germany) (23, 24). Studies were performed using confluent endothelial cell monolayers in their second passage. For the investigations with the protease inhibitors human umbilical cord vein endothelial cells were incubated with 0.5 unit of PPE for 1 h. HUVEC were grown on Thermanox® slides. After stimulation, slides were fixed for 1 h in 3% paraformaldehyde at room temperature and rinsed three times in phosphate-buffered saline. Permeabilization of cell membranes was performed using 0.1% Triton X-100 for 5 min followed by three wash steps with phosphate-buffered saline. F-actin was stained with phalloidin Alexa 488 (Molecular Probes, Eugene, OR) (1:400) as described previously (24). Thermanox® slides were placed on glass slides and embedded with Gelmount mounting media (Biomeda). Slides were analyzed using a Pascal 5 confocal scanning laser microscope (Zeiss, Jena, Germany) equipped with an air-cooled argon laser (Axioskop 2 Mot microscope, Zeiss). Alexa 488 fluorescence was excited with a 488-nm argon-ion laser beam and imaged using a NT80/20/488 beamsplitter and a 505-nm longpass emission filter. All images were recorded with a 40 x 1.3 NA Plan-Apochromat III DIC objective with image resolution 1024 x 1024 pixels.


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Rational Design of Hybrid Squash Inhibitors—The binding loop of squash inhibitor EETI II is located at the N terminus of the miniprotein and is stabilized by two cysteine bonds (see Fig. 1 for the corresponding sequences). Two different strategies were used to insert the inhibitory peptide into this binding loop. First, the inhibitory peptide was introduced in such a way that its P1 position (according to the nomenclature of Schechter and Berger (4), the main specificity-determining amino acid residue adjacent to the scissile peptide bond of a peptide or polypeptide substrate) Leu-4 resembled that of the original squash inhibitor EETI II. Also, to maintain the important disulfide bridges, the sequence of the inhibitory peptide PMTLEYR was modified in position 2 to PCTLEYR. Furthermore, to avoid problems with an adjacent positive charge, and since a substitution for methionine is possible without decreasing inhibition (2, 8), the Arg-7 was changed to methionine, a conserved position in most squash inhibitors. This inhibitor with the N-terminal sequence now reading PCTLEYM (see Fig. 1) was called HEI-TOE I: hybrid elastase inhibitor-turkey ovomucoid E. elaterium.



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FIG. 1.
Schematic representation of the three-dimensional structure of hybrid squash inhibitor HEI-TOE I with the binding loop in red (derived from the complex with PPE, Protein Data Bank code 1mcv [PDB] ) and sequences of different natural squash inhibitors from E. elaterium (EE) and M. charantia (MC), and three hybrid inhibitor variants (HEI). The amino acids of the binding loops are printed bold and the P1 positions underlined. In the case of HEI-TOE II the position P1 is not known and therefore not labeled.

 

In the second strategy, the optimized peptide was inserted between the two cysteine residues of the squash inhibitor N-terminal binding loop. Christmann et al. (25) showed that it is possible to replace the six residues of this loop by a 17-residue epitope from human bone Gla protein. This EETI II variant was fully oxidized and correctly folded, and the epitope inserted into the binding loop was readily recognized by a monoclonal antibody (25). We inserted the optimized peptide into the binding loop in the same way, creating the variant HEI-TOE II (Fig. 1).

Both hybrid squash inhibitors were synthesized, purified, and oxidized as described under "Experimental Procedures." Inhibition of PPE activity was measured during the course of disulfide bridge formation. Interestingly, HEI-TOE I and II showed opposing behaviors. Whereas PPE inhibition by HEI-TOE I increases during disulfide formation it decreases for HEI-TOE II (Fig. 2A). To verify these data, the Ki values for HEI-TOE I and II were determined before disulfide formation, after disulfide formation and after a subsequent second purification step to eliminate improperly folded or cross-reacting species. For comparison, the Ki values for the free peptides corresponding to the binding loop of the two hybrid squash variants were included (Fig. 2B and Table I). The data confirmed that only the inhibitor HEI-TOE I, with the P1 site position conserved within the structure, inhibits PPE strongly after folding and disulfide formation is finished. We cannot exclude that non-native disulfide bonds are predominantly formed with HEI-TOE II, resulting in the decreased inhibition strength of this inhibitor variant, but more likely, the decreasing inhibitory strength during the formation of disulfide bridges reflects structural restrictions imposed on the PPE-interacting loop region.



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FIG. 2.
A, determination of the inhibition of PPE activity at different times during the cyclization process of the inhibitors. {circ} is the symbol of HEI-TOE I, • represents HEI-TOE II, and {blacksquare} is the symbol of the peptide PMTLEYR. The cyclization was performed in 0.1 M Tris buffer, pH 8.5, at room temperature with stirring. The peptide concentration of each peptide in the cyclization assay was 3 x 105 M. B, determination of the Ki value of the HEI-TOE I and II in different purification steps in comparison with free peptides equivalent to the binding loop. The given deviations are ranges.

 

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TABLE 1
Specificity and pH dependence of ovomucoid derived and squash-type inhibitors

Comparison of the specificity and pH dependence of inhibition for the following inhibitors: ovomucoid inhibitor (third domain) from turkey (OMTKY3), the derived peptide PACTLEYRC, the peptide PMTLEYR optimized against PPE, the squash-type trypsin inhibitor EET1 II from E. elaterium, and the hybrid inhibitors HEI-TOE I, II, and III. ND, not determined; NI, no inhibition; PPE, porcine pancreas elastase; HLE, human leucocyte elastase; BPC, bovine pancreas chymotrypsin; BPT, bovine pacreas trypsin.

 

The natural elastase-specific squash inhibitors MCEI I–IV differ only in N-terminal additions of glutamic acid residues: MCEI I has no Glu at the N terminus, but MCEI IV has three (see Fig. 1). This correlates directly with considerable differences in their affinity, where the inhibition strength of MCEI III is 240-fold higher than that of MCEI I (10). To investigate the influence of those Glu residues on our hybrid squash inhibitor, we added the three N-terminal residues of MCEI III (EER) to our sequence (Fig. 1, HEI-TOE III). This variant indeed showed increased inhibition of PPE, but only by a factor of 1.8 at pH 8.5 (Table I). Thus, the mode of interaction with PPE is apparently different for HEI-TOE III and MCEI III.

Specificity and Stability of Native and Hybrid Squash Inhibitors—For comparison, Ki values for the two hybrid miniproteins HEI-TOE I and HEI-TOE II were determined with PPE and three other proteases: HLE, chymotrypsin (BPC), and trypsin (BPT) from bovine pancreas (Table I). A specificity similar to PPE has been reported for HLE (26). BPT was chosen because EETI II, the scaffold donor, shows a high specificity toward trypsin. BPC is included because of its deviating specificity and strong inhibition by OMTKY3. HEI-TOE II did not significantly distinguish between the tested serine proteases, in contrast to HEI-TOE I, which showed high specificity for PPE. The observed PPE specificity was similar to that of the optimized peptide PMTLEYR. In fact HEI-TOE I affinity was about seven times higher than that of PMTLEYR, but 3–4 orders of magnitude lower than that of OMTKY3, which in turn showed little difference between PPE, HLE, and BPC but did not inhibit BPT at all (27). The natural squash inhibitor EETI II is highly specific for trypsin due to the Arg in position P1 of the binding loop, but, at a low affinity level, it does not distinguish between the elastases PPE and HLE. The natural inhibitor MCEI III described as an inhibitor for PPE has a low affinity against trypsin and, at high affinity level, does not distinguish between both elastases (PPE and HLE) and also chymotrypsin (see Table I). In contrast to the free peptide, the hybrid inhibitor variant HEI-TOE II containing the optimized peptide PMTLEYR directly inserted into the EETI II binding loop shows no much difference in affinity toward all proteases tested (Table I). Obviously, the distortion of peptide conformation imposed by the framework constraint occurs in such a way that important contacts, especially to PPE, cannot be established. The data also show that the substitutions leading from EETI II to the hybrid variant HEI-TOE I transform a squash inhibitor previously highly specific for trypsin into an specific inhibitor for porcine pancreatic elastase.

Another feature of inhibitors is important: their stability against proteolytic cleavage, here either at the P1 position or somewhere else within structurally exposed sequence parts. OMTKY3 itself, the starting protein for our previous optimization experiments, is very stable against proteolytic attack (28). The peptide PACTLEYRP representing the binding loop of OMTKY3 was synthesized, and its stability against proteolytic cleavage was measured as a change in the degree of PPE inhibition (data not shown). The peptide undergoes rapid degradation by PPE with a half-life ranging below 1 h. This suggests that the binding sequence itself is not responsible for the high hydrolytic stability of OMTKY3 but rather the rigid structure imposed by the scaffold. The time scale for degradation of the optimized peptide PMTLEYR enters the same range as for PACTLEYRP if 10-fold more enzyme is used (data not shown). The hybrid miniprotein HEI-TOE I is clearly more stable against proteolytic attack than the free inhibiting peptides (24% residual inhibitory activity after 145 h of incubation with PPE). The time-dependent reduction of the inhibitor peptides was also ascertained by HPLC measurements (data not shown). In the case of the natural squash inhibitor MCEI III no cleavage is detectable even over 8 days. The difference between the peptides and the squash inhibitors could be explained by a lower efficiency of the acyl enzym formation for the complete miniproteins because of the rigid structure of the binding loop. In addition, it was reported that protein inhibitors with the scissile bond already cleaved can bind to the enzyme as well and thus maintain inhibition or even resynthesize the cleaved peptide bond (29). HEI-TOE I binding to PPE causes small shifts of loops in the vicinity of the binding region of the protease (30). It is possible that these shifts impose a higher degree of flexibility on the binding loop, thus resulting in a faster cleavage of the hybrid inhibitor compared with the natural squash inhibitor MCEI III. The free peptides are even more flexible, and the acyl enzyme may form faster.

The pH dependence of Ki of the hybrid squash inhibitors HEI-TOE I and III is not very pronounced (Table I) and similar to that of the inhibitory peptide PMTLEYR. At pH 6.0, where the proteolytic activity of PPE is diminished, the affinity of HEI-TOE I for PPE is large enough for the co-crystallization of the complex (31). The complex structure shows a complete HEI-TOE I inhibitor with clear electron density for the scissile Leu-Glu bond in the active site of PPE (30).

Comparison of the structure of HEI-TOE I in a complex with PPE and a structure of EETI II variant (for sequence see Fig. 1) in a complex with trypsin2 showed a good match in the peptide backbone (data not shown). This means that the overall structure of HEI-TOE I is similar to the EETI II donor framework. There are only small deviations in the binding loop and also in two other loop regions. Interestingly, the exchange of two amino acids strictly conserved in the binding loop of all squash family inhibitors (proline at position 4 and isoleucine at position 6, nomenclature by Otlewski and Krowarsch (6)) has no influence on the structure. Thus there is no indication why these two positions are conserved in the squash-type inhibitors. Possibly, there is an influence on the folding process within the cellular milieu of the plant seed, or the residues have some influence on the interaction with target proteases that so far are unknown, a description that applies in general to the function of squash-type inhibitors in the seeds of Curcubitacea.

Isothermal Titration Calorimetry of the HEI-TOE I Binding to PPE—The binding of HEI-TOE I to PPE was measured by ITC in three different buffers at pH 6.0 and in the temperature range between 6.8 and 25 °C. The advantage of ITC over other techniques is that the enthalpy change of the reaction is measured directly, allowing a complete thermodynamic description of the binding reaction (42). The experimental raw data were corrected and fitted to a model with identical and independent binding sites. As an example the titration of PPE with HEI-TOE I at 6.8 °C in 50 mM PIPES, pH 6.0, and 0.15 M NaCl is shown (Fig. 3A). The binding of HEI-TOE I to PPE is stoichiometric under all conditions examined in this study and is characterized by a very small endothermic heat effect. The thermodynamic data calculated for the binding of HEI-TOE I to PPE are summarized in Table II. The dissociation constant for the inhibitor-elastase complex determined in the temperature range between 6.8 and 25 °C is ~1–5 x 106 M. For comparison, the inhibition constant Ki = 1.3 x 107 M of HEI-TOE I measured at pH 6.5 and 25 °C is about 1 order of magnitude lower (see Table I). This deviation may be due to different experimental conditions, especially in the enzyme concentrations used or the Me2SO content of the kinetic assay necessary because of problems with the substrate solubility.



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FIG. 3.
Isothermal titration of PPE with HEI-TOE I at 6.8 °C in 50 mM PIPES, pH 6.0, 0.15 M NaCl. A, plot of integrated heat (black squares) from each injection, corrected for the heat of dilution of the inhibitor, versus the molar ratio of HEI-TOE I to PPE. The data were fitted (solid line) to a model assuming identical and independent binding sites. B, enthalpy-entropy compensation shown by the plot of versus T{Delta}S0. {circ}, 50 mM PIPES, pH 6.0, 0.15 M NaCl at 6.8, 11.5, 16.0, and 25.0 °C; *, 50 mM MOPS, pH 6.0, 0.15 M NaCl at 7.2 °C; and {triangledown}, 50 mM MES, pH 6.0, 0.15 M NaCl at 7.1 °C. C, temperature dependence of {Delta}H0, T{Delta}S0, and {Delta}G0. The curves were calculated with {Delta}Cp,exp = –1.275 kJ·mol1·K1 obtained from the slope of the fit curve through the experimental values (shown as asterisks).

 

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TABLE II
Thermodynamic parameters of the complex formation between HEI-TOE I and PPE

T is in °C, Kd is in M, , , , {Delta}G0, and T{Delta}S0 are in kJ·mol1, and {Delta}S0 is in kJ·mol1·K1. is the experimental enthalpy change, is the enthalpy corrected for the ionization enthalpies of the buffers. T{Delta}S0 was calculated from and .

 

The binding of HEI-TOE I to PPE is entropy driven with unfavorable positive enthalpy below 37 °C and favorable positive entropy contributions below 62 °C to the Gibbs free energy, {Delta}G0 (Fig. 3C). With increasing temperature, both (the enthalpy corrected for the ionization enthalpy of the buffer) and T{Delta}S0 become smaller (Table II). Plotting of versus T{Delta}S0 gives an almost linear correlation between these parameters with a slope of 0.95 and a correlation coefficient of 0.98 (Fig. 3B). Changes in {Delta}G0 in the temperature range 6.8–25 °C are much smaller than changes in and T{Delta}S0 (Fig. 3C). This is caused by enthalpy-entropy compensation, which is expected for processes where and T{Delta}S0 show similar temperature dependences. We recently observed further examples of such processes with other protein-peptide interactions (12, 13).

The heat capacity change, {Delta}Cp, is an important thermodynamic quantity of any association reaction. {Delta}Cp,exp was determined from the slope of versus temperature to {Delta}Cp,exp = –1.275 kJ· mol1·K1. Knowledge of {Delta}Cp,exp permits the calculation of the solvation entropy (Equation 2), and with we obtained the contribution of the conformational entropy, , to the total entropy change of the reaction, (Equation 1). The calculations provide of 406, 385, 365, and 326 J·mol1·K1 and unfavorable of –125, –127, –126, and –143 J·mol1·K1 at the temperatures 6.8, 11.5, 16.0, and 25.0 °C. Thus, according to these calculations the total favorable entropy contribution to the binding energy of complex formation between HEI-TOE I and PPE results from a large favorable solvation entropy, which is partially compensated by negative terms of conformational, translational, and rotational entropy contributions. This favorable entropy contribution is due to the release of solvent from the binding interfaces mainly induced by hydrophobic effects and corresponds to the displacement of the considerable amount of 21 water molecules from the elastase binding site during binding of HEI-TOE I (30). A further, although unknown, amount of water molecules is displaced from the surface of HEI-TOE I.

Spolar and Record (19) proposed Equation 3 enabeling calculation of the entropic contributions of the hydrophobic effect () from structural information. We calculated J·mol1·K1 at temperature Ts = 335 K using Equation 3 with the buried non-polar area {Delta}ASAnp = –1134 Å2 known from the structure of the elastase-hybrid squash inhibitor complex (Protein Data Bank code 1mcv [PDB] ). Ts is the temperature where the total entropy sums up to zero and was calculated with Equation 4. At Ts, is equal but of opposite sign to , which is the sum of all other contributions to the total entropy . For the folding of proteins was found within the range –14 to –34 J·mol1·K1 per amino acid residue R with an average value of –23 J·mol1·K1 for the contribution of one amino acid (19). Thus, J·mol1·K1 calculated for the elastase-hybrid squash inhibitor complex accounts for contributions from 9 amino acids. This rough estimate is close to the number of 7 amino acids from the binding loop of the hybrid squash inhibitor interacting with PPE and suggests as main source for the fixation of flexible amino acids from the binding loop of the inhibitor. This consideration is consistent with the fact that assuming no conformational changes upon dissociation yields a J·mol1·K1 (calculated with program STC), which is to small to account for the observed {Delta}G0.

The size of the buried surface areas of polar ({Delta}ASAp) and nonpolar ({Delta}ASAnp) residues was considered a vital feature of the energetics of complex formation (19). Semi-empirical equations were developed to calculate buried surface areas from thermodynamic parameters (Refs. 1820, 32, 33 and references therein), and these equations were also applied to calculate thermodynamic parameters from {Delta}ASA values that were obtained from atomic coordinates.

Calculation of {Delta}Cp according Equation 6 with {Delta}ASAp = –581 Å2 and {Delta}ASAnp = –1134 Å2 known from the crystal structure of the elastase-HEI-TOE complex (Protein Data Bank code 1mcv [PDB] ) results in {Delta}Cp,calc about 18% larger than {Delta}Cp,exp. With Equations 6, 7, 8, {Delta}Cp,exp = –1.275 kJ·mol1·K1 and {Delta}H0 (60 °C) = –36.1 kJ·mol1 result buried polar and nonpolar surface areas that are about 15% smaller than {Delta}ASAp and {Delta}ASAnp. Calculation of reasonable {Delta}G0 from atomic coordinates was not successful for the elastase-squash hybrid inhibitor. Using the program suit STC (17), a much too high and consequently a rather unrealistic of –76 kJ·mol1 were obtained.

The buffer dependence of the experimental enthalpy changes, , points to a proton linkage of complex formation. The data indicate that approximately one proton per binding site (1.1 ± 0.1) is transferred to the buffer at pH 6.0 and 6.8 °C. The most likely candidate for the proton release upon complex formation between HEI-TOE I and PPE is the active site His-57 of PPE. A similar proton linkage was also observed for the binding of OMTKY3 to PPE arising from a shift of the pKa of His-57 in the catalytic triad from 6.7 in the uncomplexed elastase to 5.2 upon complex formation (34). The shift of pKa upon complex formation is important for the mechanism of serine proteases. His-57 is less protonated at lower pKa and more strongly accepts the hydroxyl proton of Ser-195 (34, 35). Similar pKa shifts between ~7 and 5 have been observed for the binding of other protease inhibitors/substrates to different serine proteases (37, 38).

In summary, the binding of the inhibitor HEI-TOE I to PPE is entropy driven, characterized by a small unfavorable enthalpy change, {Delta}H0, a large favorable entropy change, T{Delta}S0, and a large negative experimental heat capacity change, {Delta}Cp. Similar thermodynamic features were recently described for the OMTKY3/PPE interaction (34). With increasing temperature the favorable entropy decreases and becomes compensated by increasing favorable enthalpy contributions. Enthalpy-entropy compensation is known for several protein-ligand complexes. In the past enthalpy-entropy compensation has been regarded as a ubiquitous property of water (39). However, it appears to be a property of all weak intermolecular interactions, of which hydrogen bonding in aqueous solution is merely the most frequently encountered in supramolecular interactions (40). Enthalpy-entropy compensation is expected for any process where {Delta}Cp is much larger than T{Delta}S0. The temperature dependences of {Delta}H0 and T{Delta}S0 may be large, but nevertheless they result in a much smaller temperature dependence of {Delta}G0.

The large negative heat capacity change and the large positive entropy contribution indicate that hydrophobic interaction is a major force for the binding (41, 20). Indeed hydrophobic interactions are observed, particularly for the residues Pro-1, Tyr-6, and Met-7 of the inhibitor binding loop (30). Burial of hydrophobic residues in the interface of PPE and HEI-TOE I thus stabilizes the complex by desolvation and an increase in the solvent entropy. Otherwise, this entropy gain is partly compensated by a loss of enthalpy, caused by weaker hydrogen bonds of the released water molecules in the bulk water. The favorable binding entropy is further reduced by unfavorable conformational entropy contributions resulting from the loss of side chain and main chain flexibility upon complex formation. Ordering of water molecules in the binding region can contribute unfavorably to entropy changes or favorably to enthalpy changes, as described for antigen-antibody interaction (36). In our case, the small positive binding enthalpy, , of 14.3 kJ·mol1 at 25 °C is composed of a large favorable polar contribution of –83.1 kJ·mol1 and a large unfavorable nonpolar contribution of 97.3 kJ·mol1, which are comparable with those of the OMTKY3-PPE complex (34). Hydrogen bonding of polar groups is observed in the HEI-TOE I-PPE complex (30) and is expected to contribute to the favorable polar component of .

Cell Assay with Inhibitors—Elastases play an important role in several inflammatory diseases. For example, the uncontrolled release of elastase, e.g. from granulocytes, may have a strong destructive impact on surrounding tissue. Since the potential medical application of elastase inhibitors is considerable, we investigated the effects of the inhibitors in cell assays.

HUVEC are highly sensitive to PPE. As described before (23), treatment of HUVEC with PPE resulted in a loss of endothelial barrier function followed by tissue edema formation. We observed massive endothelial cell retraction and detachment from culture dishes following incubation with PPE (Fig. 4). Such damage was not observed after the same treatment but in the presence of the optimized inhibitor peptide (PMTLEYR) or the hybrid squash inhibitor HEI-TOE I (Fig. 4). A negative control peptide (PMTfEYR) with a sequence similar to the optimized inhibitory peptide, but showing no PPE inhibition, was also used in the cell experiments. As expected this peptide did not protect the cells against damage caused by PPE activity (Fig. 4). Both the inhibitory peptide and the hybrid squash inhibitor could inhibit damage for at least 4 h (data not shown). After more than 4 h cell damage occurred independently of PPE treatment (data not shown). Based on the in vitro data the inhibitors were apparently not cytotoxic to the cell line nor did they invoke any detectable side effects. Moreover, no rapid degeneration of the inhibitor molecules nor adsorption on the cell surface was observed.



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FIG. 4.
HEI-TOE I and PMTLEYR prevented elastase-induced endothelial cell retraction. HUVEC treated with solvent (A), 10 µM HEI-TOE I (C), 10 µM PMTLEYR (E), or 50 µM PMTfEYR (G) showed no gap formation as visualized by phalloidin Alexa 488. Exposure of endothelial cells for 1 h to 0.5 unit of PPE resulted in massive cell retraction and cell detachment accompanied by cell loss from culture dishes (B). Endothelial cell retraction was completely prevented by preincubation of cells with HEI-TOE I (D) or PMTLEYR (F), while PMTfEYR (H) showed no significant effect. Representative fields of HUVEC monolayers (out of three separate experiments) are shown.

 

In conclusion, the data show that squash-type inhibitors are stable miniproteins that can be used as a scaffold to engineer highly specific serine protease inhibitors, providing that the P1 position in the binding loop is conserved.


    FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

The on-line version of this article (available on http://www.jbc.org) contains Supplemental Figs. S1–S4. Back

** To whom correspondence should be addressed. Tel.: 49-30-450528153; Fax: 49-30-450528909; E-mail: wolfgang.hoehne{at}charite.de.

1 The abbreviations used are: OMTKY3, third domain of turkey ovomucoid inhibitor; EETI, trypsin inhibitor from E. elaterium; HEI-TOE, hybrid inhibitor from E. elaterium and the optimized binding loop of the third domain of turkey ovomucoid inhibitor; PPE, porcine pancreatic elastase; MCEI, elastase inhibitor from M. charantia; ITC, isothermal titration calorimetry; HPLC, high pressure liquid chromatography; HLE, human leucozyte elastase; HUVEC, human umbilical cord vein endothelial cells; BPC, bovine pancreatic chymotrypsin; BPT, bovine pancreatic trypsin; ASA, accessible surface area; Suc, succinyl; pNA, p-nitroanilide; PIPES, 1,4-piperazinediethanesulfonic acid; MES, 4-morpholineethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid. Back

2 I. Uson, personal communication. Back



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