Structural Determinants of the Calpain Inhibitory Activity of Calpastatin Peptide B27-WT*

Russell BettsDagger , Shantel WeinsheimerDagger , Grant E. BlouseDagger , and John AnagliDagger §||

From the Dagger  Division of Biochemical Research, Department of Pathology, Henry Ford Health Sciences Center, § Protease Program, Karmanos Cancer Institute, and  Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48202

Received for publication, August 15, 2002, and in revised form, December 12, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Calpastatin is the natural specific inhibitor of calpain. Recent research has linked uncontrolled calpain activation to tissue damage after neuronal and cardiac ischemias, traumatic spine and brain injuries, as well as Alzheimer's disease and cataract formation. An imbalance between the activities of calpain and calpastatin is believed to be responsible for the pathological role of calpain. An important key to understanding calpain regulation by calpastatin is to determine, at the molecular level, how calpastatin interacts with calpain to inhibit its enzymatic activity. A 27-residue peptide (DPMSSTYIEELGKREVTIPPKYRELLA) derived from subdomain 1B of the repetitive domains of calpain, named peptide B27-WT, was previously shown to be a potent inhibitor of µ- and m-calpain. In this report, a combination of beta -alanine scanning mutagenesis and kinetic measurements was used to probe, in a quantitative, systematic, and simultaneous fashion, the relative contribution of the amino acid side chain and backbone functionalities to the overall calpain-inhibitory activity of B27-WT. The study identified two "hot spots," Leu11-Gly12 and Thr17-Ile18-Pro19, in B27-WT within which the residues critical for inhibitory function are clustered. Mutation of any one of the key residues in either of the two hot spots resulted in a dramatic loss of inhibitory activity. Furthermore, it was shown that a restricted conformation of the Leu11-Gly12 and Thr17-Ile18-Pro19 backbones is required for the peptide inhibitory function. These results suggest a plausible model in which the two hot spots are situated at or near the interface(s) of the calpain-calpastatin complex and act in a concerted fashion to inhibit calpain. The information on the specific contribution of the amide bond and side chain of each key residue to the bioactivity of B27-WT will contribute to a better understanding of the mechanism of calpain inhibition and lead to novel and effective therapies based on the specific inhibition of dysregulated or overactivated calpain.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Calpain (EC 3.4.22.17), a nonlysosomal, intracellular calcium-activated neutral cysteine proteinase, has been implicated in a variety of important physiological processes, including signal transduction, cell proliferation and differentiation, and apoptosis (1-4). The consequences of uncontrolled calpain activation have been witnessed in certain pathological conditions associated with excessive increases in intracellular Ca2+ levels, namely, in tissue destruction after spine and brain injuries, neuronal injury after cardiac and brain ischemia, demyelination diseases (multiple sclerosis), muscular dystrophy, Alzheimer's disease, arthritis, and cataract formation (5, 6). There are two well established calpain isoforms, µ- and m-calpain,1 which are ubiquitously expressed and require micro- and millimolar concentrations of Ca2+ to reach maximal activity, respectively, in vitro. The classical µ- and m-calpains exist as heterodimers consisting of a large (80-kDa) catalytic subunit and a small (28-kDa) regulatory subunit that dissociates in the presence of calcium (7-9). Tissue-specific calpains and other calpain-related genes have now been identified (10).

The activity of calpain is tightly regulated through Ca2+-induced conformational changes in the proteinase and by interaction with its specific endogenous inhibitor calpastatin (11-13). Modulation of calpain activity by phospholipids and protein activators has also been reported (14). Calpain and calpastatin are widely distributed in all animal cells, with calpastatin in excess of calpain in erythrocytes and heart muscle (1, 14). It has recently been reported that calpastatin resides predominantly in phosphorylated-aggregated bodies, with the inhibitor protein being dephosphorylated and distributed in a Ca2+-dependent manner (11, 12). This observation demonstrates the vigorous regulation placed on the calpain-calpastatin system whereby both enzyme and inhibitor are under strict Ca2+ control.

The primary structure of calpastatin is divided into four repeating inhibitory domains (domains 1-4) and an NH2-terminal domain that lacks inhibitory function (domain L) (6, 15-19; see Fig. 1). The function of domain L is not clear, although it has been suggested that this domain may play a role in regulating calcium ion channels (16). Inside each repetitive domain are three conserved subdomains referred to as A, B, and C. Subdomains A and C are rich in acidic residues that are scattered among hydrophobic residues and are predicted to form amphiphilic alpha -helices. It has been established, using the BIAcoreTM surface plasmon resonance technology, that subdomain A binds to domain IV of the catalytic subunit of calpain, whereas subdomain C binds to domain VI in the regulatory subunit (17-19). Although neither subdomain A nor C inhibits calpain activity directly, these subdomains do act to potentiate the inhibitory ability of calpastatin by promoting its tight binding to calpain (14, 17-19). Previous studies using bacterially expressed calpastatin fragments have established that the inhibitory activity of calpastatin resides in subdomain B of the repeating structures (domains 1-4) (15, 20-23). Moreover, a chemically synthesized 27-residue peptide corresponding to subdomain 1B of human calpastatin showed strong inhibition against µ- and m-calpain, although to a slightly lesser extent than a whole functional domain 1 of calpastatin (18). The subdomain 1B peptide, which we have named peptide B27-WT (see Fig. 2A), bears no regular secondary structures such as alpha -helix and beta -sheets; however, 1H NMR studies of B27-WT have shown the presence of a well defined type I beta -turn in the Pro20-Lys21-Tyr22-Arg23 region and probably another turn in the Glu10-Leu11-Gly12-Lys13 region (21). It has been proposed that these beta -turns could play an important role in mediating the biological activity of the peptide.


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Fig. 1.   Schematic representation of human calpastatin domains. The repetitive domains (1-4) are illustrated with the L domain at the NH2 terminus. The amino acid sequences of the binding subdomains A (gray) and C (white), and the inhibitory subdomain B (black) are shown.

An important key to understanding calpain function and regulation is to determine, at the molecular level, how calpastatin interacts with calpain to inhibit its enzymatic activity. Recent x-ray crystallographic studies on Ca2+-free µ- and m-calpain, as well as the Ca2+-bound protease core (domains I and II) of µ-calpain have provided very important insights into possible mechanisms by which calpain could be activated by Ca2+ (9, 13, 24). However, no crystal structures or molecular modeling data are available for the calpain-calpastatin complex. Even though calpastatin subdomain B or peptide B27-WT was shown to be responsible for calpain inhibition, no rigorous structure-function analysis has been performed to examine the individual contributions of each amino acid residue to the overall inhibitory activity. Because this type of study is absolutely critical to establish a detailed understanding of the mechanism employed by B27-WT to inhibit calpain, we embarked on a study that would identify the structural elements in the peptide which are important for calpain inhibition. This was accomplished by using "beta -alanine scanning" mutagenesis to determine the effect of site-specific amide bond and/or side chain modifications on the biological activity of B27-WT.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Porcine erythrocyte calpain I was purchased from Calbiochem. Suc-Leu-Tyr-AMC was obtained from Sigma. Fmoc-protected amino acids, Fmoc-Ala-Wang resin, piperidine, DIPEA, HBTU, and trifluoroacetic acid were purchased from Advanced ChemTech (Louisville, KY). Dimethylformamide and tetrahydrofuran were purchased from Burdick & Jackson (Muskegon, MI) and used without further purification. All other reagents were of analytical grade.

Peptide Synthesis-- The 27-residue calpastatin- (162-188)-peptide (B27-WT, DPMSSTYIEELGKREVTIPPKYRELLA) encoded by exon 1B of the human calpastatin gene (25) and its beta -Ala-containing analogs (B27-beta -Ala-X) were synthesized using a multiple peptide synthesizer (Advanced ChemTech, model 348Omega ) using Fmoc/t-butyl chemistry. All peptides were synthesized on 150 mg (0.12 mmol) of preloaded Fmoc-Ala-Wang resin (0.8 mmol/g; 100-200 mesh). N-alpha -Fmoc deprotection was performed with 25% piperidine in dimethylformamide followed by washing with tetrahydrofuran (4 × 3 ml) and dimethylformamide (2 × 3 ml). The Fmoc-amino acids were activated with 0.9 eq of HBTU and 2 eq of DIPEA and coupled for 45 min using a 5-fold excess of activated amino acids. Side chain protecting groups used were (t-butyl) for Asp, Glu, Ser, Thr, and Tyr; (Pbf) for Arg; (t-butoxycarbonyl) for Lys. Side chain deprotection and cleavage of peptides from the resin were performed with 3 ml of trifluoroacetic acid in the presence of phenol/ethanedithiol/thioanisole/water (82.5:5:5:2.5:5, v/v). The crude peptides were purified by reverse phase HPLC on a Vydac C18 column (10 µm, 250 × 50 mm; Grace Vydac, Hisperia, CA) using a linear gradient of 0.1% trifluoroacetic acid in water (buffer A) to 0.1% trifluoroacetic acid in acetonitrile (buffer B) over 100 min. Analytical reverse phase HPLC was performed on a Jupiter C5 (10 µm, 250 × 4.6 mm; Phenomenex, Torrance, CA) using a 60-min gradient or a Discovery series C18 (5 µm, 150 × 4.6 mm; Supelco, Inc., Bellefonte, PA) using a 40-min gradient of 100% buffer A to 100% buffer B. Beckman System Gold equipment (Beckman-Coulter, Fullerton, CA) was used for preparative HPLC, and analytical HPLC was performed with a Waters model 600E apparatus (Waters, Inc., Milford, MA). The purity of the peptides was confirmed by electrospray ionization and/or matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The concentrations of the peptide solutions used for the calpain inhibition studies were determined by amino acid analysis performed at the University of Michigan Protein Structure Facility (Ann Arbor, MI).

Stopped-flow Measurements of Calpain Inhibition by Calpastatin Peptide B27-WT-- Calpain activity was monitored using two different types of substrates: Suc-Leu-Tyr-AMC and fluorescein thiocarbamoyl-casein (FTC-casein). The AMC fluorophore moiety of Suc-Leu-Tyr-AMC has negligible fluorescence when its amino group is involved in an amide bond with the peptidyl portion (Suc-Leu-Tyr) of the substrate. Upon cleavage of the Tyr-AMC amide bond by calpain, the released AMC product, with a free amino functionality, emits a strong fluorescence signal at 460 nm when excited at 370 nm. Fluorescence emitted by the fluorescein groups on FTC-casein (~ 3:1, number of probes/molecule of casein) is quenched through fluorescence resonance energy transfer because of the close proximity of the probes (10-100Å) on the intact substrate. An increase in fluorescence intensity is detected when FTC-casein is digested by calpain to produce fluorescein-labeled polypeptides that are distant from each other in solution.

Rapid kinetic measurements of calpain inhibition by B27-WT in the presence of either Suc-Leu-Tyr-AMC or FTC-casein were performed by stopped-flow fluorometry on an SX.18MV Stopped-Flow Reaction Analyzer (Applied Photophysics Ltd., Surrey, UK). The stopped-flow experiments were carried out under pseudo-first order conditions with B27-WT in at least 5-fold excess over calpain. Buffer conditions were as follows. Buffer C contained 20 mM HEPES, 130 mM KCl, 5 mM dithiothreitol, and 0.1 mM EDTA, pH 7.4. Buffer D contained 20 mM HEPES, 130 mM KCl, 5 mM dithiothreitol, and 2 mM CaCl2, pH 7.4. Two separate experiments were carried out to determine the dependence of the pseudo-first order rate constant, kobs, on inhibitor concentration using either Suc-Leu-Tyr-AMC or FTC-casein. A third experiment examined the dependence of kobs on Suc-Leu-Tyr-AMC concentration.

In the first set of experiments, the rate of calpain-mediated hydrolysis of Suc-Leu-Tyr-AMC was monitored at various concentrations of inhibitor. Excitation was at 370 nm, and a filter with a cutoff below 405 nm was used to monitor the emission of liberated AMC. Calpain was prepared at twice the final reaction concentration (50 nM) in buffer C, and twice the final reaction concentration of Suc-Leu-Tyr-AMC (0.2 mM) was prepared in buffer D. Various concentrations of inhibitor were premixed with substrate in buffer D to achieve final inhibitor concentrations between 0.1 and 1.1 µM.

A second set of experiments was carried out in which FTC-casein was used as substrate to determine the dependence of kobs on inhibitor concentration. Fluorescein was excited at 490 nm, and a filter with a cutoff below 505 nm was used to observe fluorescence emission. 20 nM calpain and 1.0 µM FTC-casein in buffer C (syringe 1) were rapidly reacted with 1.0 µM FTC-casein and various concentrations of B27-WT in buffer D (syringe 2) to final concentrations of 10 nM calpain, 1 µM FTC-casein, and peptide B27-WT (0.05-2.5 µM). The final concentration of substrate (1.0 µM) was prepared in both buffer C and D to prevent any change in fluorescence resonance energy transfer caused by dilution in the reaction cell.

For the third set of experiments, 1 µM B27-WT and 50 nM calpain concentrations were fixed, and the concentration of Suc-Leu-Tyr-AMC was varied between 0.05 and 1.0 mM in the final reaction mixture. The filter settings were the same as in the first set of experiments.

Analysis of Stopped-flow Kinetic Data-- The second order rate constant, ka, of B27-WT-µ-calpain complex formation was determined under pseudo-first order conditions as described previously and could be best fit to Equation 1 (26),


[P]=v<SUB><UP>s</UP></SUB>t+(v<SUB><UP>0</UP></SUB>−v<SUB><UP>s</UP></SUB>)(1−e<SUP>−k<SUB><UP>obs</UP></SUB>t</SUP>)/k<SUB><UP>obs</UP></SUB> (Eq. 1)
where [P] is the product concentration, and v0 and vs are the initial and steady-state velocities, respectively. kobs is the pseudo-first order rate constant for the approach to steady state and is described by Equation 2 (26).
k<SUB><UP>obs</UP></SUB>=k<SUB>a</SUB>[I]/1+[S<SUB><UP>0</UP></SUB>])/K<SUB>m</SUB>+k<SUB>d</SUB> (Eq. 2)

The Michaelis constants (Km) for calpain-catalyzed hydrolysis of Suc-Leu-Tyr-AMC (3.29 ± 0.18 mM) and FTC-casein (13.12 ± 5.55 µM) were determined experimentally using standard Lineweaver-Burk plots with substrate concentrations of 0.1-4 mM and 0.5-8 µM for Suc-Leu-Tyr-AMC and FTC-casein, respectively (data not shown). All stopped-flow traces were best fit by a single exponential function with a linear component to obtain kobs and are reported as the averages of 6-10 replicate traces. The functional inhibitor concentration was determined by dividing experimental inhibitor concentration by the factor 1 + [S0]/Km to correct for the competitive effect of the substrate. The second order association rate constant (ka) was calculated from the slope of the linear regression fit to the dependence of kobs on the corrected inhibitor concentration, and the ordinate intercept gave the first order dissociation rate constant (kd). For the substrate dependence reaction, ka was calculated from the slope of the linear regression fit to the dependence of kobs on 1/(1 + [So]/Km) divided by the concentration of B27-WT (1.0 µM), and the ordinate intercept gave kd. The equilibrium constant Ki (calculated) was calculated as kd/ka.

Steady-state Kinetics of Calpain Inhibition by beta -Ala-containing Analogs of B27-WT-- The effects of single amino acid modifications on the calpain-inhibitory activity of the library of beta -Ala-containing B27-WT analogs were evaluated using steady-state fluorescence measurements. Various concentrations of the inhibitor peptides in 20 µl of assay buffer (20 mM HEPES buffer, pH 7.4, containing 130 mM KCl) and 0.76 µg of calpain in 10 µl of assay buffer were added to microtiter wells. After the solutions were mixed for 5 min, 65 µl of assay buffer containing 1.5 mM CaCl2 and 5 mM dithiothreitol was added, yielding a final concentration of 1.0 mM Ca2+, 0.05 mg/ml calpain, and 3.4 mM dithiothreitol. The enzyme-inhibitor complex was allowed to form while shaking the reaction mixture in the microtiter plate over a 15-min period, at which time 5.0 µl of a 6 mM Suc-Leu-Tyr-AMC solution was added to reach a final substrate concentration of 300 µM. Residual calpain activity in the enzyme-inhibitor complex was determined by measuring the fluorescence increase over time resulting from the Ca2+-dependent hydrolysis of Suc-Leu-Tyr-AMC, using a Fluoroscan Ascent FL microtiter plate reader (Labsystems, Inc.). The percent enzyme inhibition was determined by comparing the activity of the inhibitor-treated enzyme with that of a control reaction without inhibitor. The residual activities reported are the average of six to eight individual reactions for each inhibitor concentration tested. IC50 values (inhibitor concentration required for half-maximal inhibitory activity) were determined after dose-response curve analysis. Data were analyzed with the Ascent software version 2.4.1 provided with the instrument and a SigmaPlot 2001 software package (SPSS, Inc., Chicago).

Gel Electrophoresis-- The Ca2+/calpastatin peptide-treated calpain samples were also analyzed by gel electrophoresis. 2 µM calpain was incubated with various concentrations of B27-WT or 50 µM B27-beta -Ala-X mutant in the presence or absence of Ca2+ for 15 min. The reactions were terminated by adding equal volumes of 2 × sample buffer (150 mM Tris-HCl, 20% glycerol, 4% w/v SDS, 10% v/v beta -mercaptoethanol, and 0.04% bromphenol blue, pH 6.8), followed by heating the samples at 95 °C for 5 min. Samples were stored at -20 °C until ready to be analyzed. SDS-PAGE was performed using the buffer system described by Laemmli (27). Gels were stained with GelCode® Blue Stain Reagent (Pierce), and band intensities were analyzed by densitometry (Bio-Rad GelDoc model 2000).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Synthesis and Characterization of the Calpastatin Peptides-- A 27-residue human calpastatin subdomain 1B (162-188) peptide (named B27-WT, DPMSSTYIEELGKREVTIPPKYRELLA) was selected for this study. B27-WT and a library of B27-WT analogs containing specific positional modifications, a beta -Ala substitution (beta -alanine scan), as shown in Fig. 2A, were synthesized. A group of NH2-terminally truncated fragments of B27-WT was also generated (Fig. 2B). The synthetic peptides were purified to near homogeneity by HPLC, and their molecular weights were confirmed by electrospray ionization and/or MALDI mass spectrometry (Table I). No contamination by modified or incompletely deprotected peptides was detected after final purification.


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Fig. 2.   A, alignment of amino acid sequences of B27-WT and its beta -Ala analogs. A library consisting of peptide B27-WT (derived from subdomain 1B of human calpastatin) and 27 beta -Ala mutants was synthesized; only selected examples of the mutant peptides are shown in this figure. The nomenclature for the library is specified on the right of the figure. The mutant peptides are numbered beta -Ala1-beta -Ala27 starting at the NH2 terminus. The peptide with no structural modification was named B27-WT and, for example, the peptide with Pro19 replaced by beta -Ala was named B27-beta -Ala19. B, NH2-terminally truncated fragments of B27-WT. Truncated peptides were generated starting with an 11-mer (CLP-1) that contains only the type I beta -turn. The residues linking the type I and type II beta -turns were added two at a time (CLP-2 and -3) until Gly-12 (CLP-4).

                              
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Table I
Analysis of the synthetic peptides for purity and for calpain inhibitory activity
Peptide B27-WT: D - P - M - S - S - T - Y - I - E - E - L - G - K - R - E - V - T - I - P - P - K - Y - R - E - L - L - A                          1    2    3     4    5    6    7    8   9   10   11  12  13   14  15  16  17  18  19  20   21  22  23   24  25  26  27                                                                Type II beta -turn region                         Type I beta -turn region

Calpain Inhibition by Calpastatin Peptide B27-WT-- Stopped-flow kinetic measurements were used to investigate the effectiveness and the mechanism of calpain inhibition by B27-WT. A simple, competitive inhibition was assumed, as shown in Scheme I,



<UP><SC>Scheme</SC> 1</UP>
where ka is the second order rate constant of complex formation and kd is the rate constant of its decomposition.

Stopped-flow traces of the fluorescence changes observed after rapid mixing of calpain with various concentrations of inhibitor preincubated with fixed concentrations of fluorogenic peptide (200 µM) or fluorescently labeled casein (1 µM) substrate are shown in Fig. 3, A and B, respectively. The dependence of the pseudo-first order rate constant (kobs) on the concentration of B27-WT was also examined. The experiments were performed under pseudo-first order conditions ([B27-WT] [calpain I]), which were verified by the independence of kobs on the concentration of calpain. The combination of a decrease in the steady-state rate, i.e. inhibition (Fig. 3, A and B) and an increase in kobs as the concentration of B27-WT increases (Fig. 4, A and B) provides evidence for a competitive component to the inhibitory mechanism. As shown in Fig. 4, kobs increased linearly with increasing concentrations of B27-WT up to the highest concentration investigated, when either Suc-Leu-Tyr-AMC (Fig. 4A) or FTC-casein (Fig. 4B) was used as substrate, confirming a classical competitive inhibition in both cases. For the experiments performed with Suc-Leu-Tyr-AMC, a second order association rate constant (ka) of (8.14 ± 0.21) × 105 M-1 s-1 and a first order dissociation rate constant (kd) of 0.035 ± 0.010 s-1 were obtained. The equilibrium constant, Ki (= kd/ka), of 42.6 ± 11.9 nM was calculated from the association and dissociation rate constants. The ka and kd values obtained for the studies with FTC-casein as substrate were (4.28 ± 0.01) × 105 M-1 s-1 and 0.0587 ± 0.0162s-1, respectively, giving a Ki value of 137.4 ± 38.13 nM. The Ki value determined for the calpain-peptide B27 interaction was about 3-fold larger with casein compared with Suc-Leu-Tyr-AMC as substrate.


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Fig. 3.   Stopped-flow traces for the inhibition of calpain by B27-WT. The progress of µ-calpain inhibition by B27-WT was followed by measuring the fluorescence emitted by AMC following rapid mixing of calpain and B27-WT in the presence of CaCl2 and substrate as described under "Experimental Procedures." A, progress curves with 50 nM µ-calpain, 1 mM CaCl2, 200 µM Suc-Leu-Tyr-AMC, and 0-0.3 µM B27-WT. B, progress curves with 10 nM µ-calpain, 1 mM CaCl2, 1 µM FTC-casein, and 0-0.6 µM B27-WT. No cleavage of the substrate was detected in the absence of calcium (B).


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Fig. 4.   Dependence of the pseudo-first order rate constant of inhibition, kobs, on B27-WT concentration. kobs was determined using 200 µM Suc-Leu-Tyr-AMC (A) or 1 µM FTC-casein (B) as substrate. The rate constants reported are from the averages of 6-10 replicate traces. The experiments were carried out as described under "Experimental Procedures," and the values for kobs were obtained from the best fit of the single exponential function with a linear component of the stopped-flow traces. The functional inhibitor concentration reported was determined by dividing experimental inhibitor concentration by the factor 1 + [S0]/Km to correct for the competitive effect of the substrate, using the following constants: Km = 3.29 ± 0.18 mM (Suc-Leu-Tyr-AMC), and 13.12 ± 5.55 µM (FTC-casein). A second order rate constant (ka) was obtained from the slope of kobs versus the functional inhibitor concentration ([B27-WT]/(1 + [S0]/Km)). A, dependence of kobs on the concentration of B27-WT (0.1-1.1 µM) at 200 µM Suc-Leu-Tyr-AMC () or Suc-Leu-Tyr-AMC (0.05-1 mM) at 1 µM B27-WT (open circle ). B, dependence of kobs on the concentration of B27-WT (0.1-2.5 µM) at 1 µM FTC-casein ().

The competitive nature of calpain inhibition by B27-WT was examined further at different concentrations of Suc-Leu-Tyr-AMC using fixed inhibitor and enzyme concentrations ([I0] = 1.0 µM and [E0] = 50 nM), and kobs was plotted as a function of 1/(1 + [S0]/Km) as shown in Fig. 4A. The linear decrease of kobs with increasing concentrations of Suc-Leu-Tyr-AMC confirms the competitive nature of the inhibition as assumed initially. Also, the ka and kd values of (4.58 ± 0.044) × 105 M-1 s-1 and 0.0234 ± 0.0377 s-1 obtained from the substrate dependence studies were in good agreement with those determined from the inhibitor dependence studies. The rapid kinetic measurements demonstrate that B27-WT exhibits a tight, reversible, and competitive inhibition of calpain in the presence of either a protein (casein) or a small molecular weight (Suc-Leu-Tyr-AMC) substrate.

Evaluation of the Contribution of Individual Amino Acid Residues to the Overall Bioactivity of B27-WT-- To determine the individual contribution of each residue in B27-WT to the overall inhibitory function, we measured the effect of the native peptide and its beta -Ala mutants (Fig. 2A) on porcine erythrocyte calpain activity. The IC50 values for B27-WT and the mutant peptides are tabulated in Table I, and the dose-response curves of calpain inhibition by peptides mutated in the hot spots and putative beta -turn regions are shown in Fig. 5.


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Fig. 5.   Inhibition of calpain by beta -Ala-containing analogs of B27-WT. Various concentrations (0-1 µM) of the inhibitor peptides were preincubated with 0.76 µg of calpain in a Ca2+-containing assay buffer, followed by the addition of 300 µM Suc-Leu-Tyr-AMC as described under "Experimental Procedures." Residual calpain activity in the enzyme-inhibitor complex was measured in a fluorescence microtiter plate reader, and the percent enzyme inhibition was determined by comparing with activity measured in the absence of inhibitor. Residual activities measured between 0 and 300 nM are shown in the graphs. The dose-response curves of peptides with mutations in the hot spots and beta -turns have been grouped together (A-D). A, dose-response curve analysis of calpain inhibition by calpastatin peptides with beta -Ala and L-Ala mutations in the first hot spot, Leu11-Gly12. open circle , B27-WT; , B27-beta -Ala11; black-square, B27-Ala11; and triangle , B27-beta -Ala12. B, dose-response curve analysis of calpain inhibition by calpastatin peptides with beta -Ala mutations in the second hot spot, Tyr17-Ile18-Pro19. open circle , B27-WT; black-square, B27-beta -Ala17; black-triangle, B27-beta -Ala18; and black-down-triangle , B27-beta -Ala19. C, dose-response curve analysis of calpain inhibition by calpastatin peptides with L-Ala mutations in the second hot spot, Tyr17-Ile18-Pro19. open circle , B27-WT; , B27-Ala17; triangle , B27-Ala18; and down-triangle, B27-Ala19. D, dose-response curve analysis of calpain inhibition by calpastatin peptides with beta -Ala mutations in the putative type I beta -turn region. open circle , B27-WT; , B27-beta -Ala20; triangle , B27-beta -Ala21; down-triangle, B27-beta -Ala22; black-square, B27-beta -Ala23; and black-triangle, B27-beta -Ala24.

The inhibitory activities of the beta -Ala mutant peptides have been grouped into four main categories: strong (IC50 < 50 nM), moderate (IC50 = 50-100 nM), weak (IC50 = 100-300 nM), or no inhibition against calpain (IC50 > 750 nM). As shown in Table I, mutation of Pro2, Met3, Ser4, Ser5, Arg23, or Glu24 to beta -Ala had practically no effect at all on the ability of the peptides to inhibit calpain. These mutants exhibited activities (IC50 < 40 nM) that were comparable with that of the wild type peptide, suggesting that neither the side chains nor the amide backbones of the mutated residues are important for calpain-calpastatin interaction or the bioactive conformation of B27-WT. Replacement of Thr6, Ile8, Glu15, Lys21, Leu25, Leu26, or Ala27 with beta -Ala had a moderate effect (IC50 = 50-85 nM) on the inhibitory potential of the peptide. B27-beta -Ala mutations at positions 1, 7, 9, 13, 16, 20, and 22 were weak inhibitors (IC50 = 100-130 nM). Mutants B27-beta -Ala10 and Ala14 showed very weak inhibitory activities (IC50 = 180-280 nM), whereas a group of mutants comprising B27-beta -Ala at positions 11, 12, 17, 18, and 19 exhibited practically no inhibition (IC50 > 750 nM) against calpain (see Fig. 5, A and B). Leu11, Gly12, Thr17, Ile18, and Pro19 are, therefore, the key amino acid residues that are essential for the inhibitory activity of B27-WT. The individual contributions of the amino acid residues to the overall inhibitory activity can be ranked in the following order: Ile18 > Leu11 > Gly12 > Pro19 > Thr17 > Arg14 > Glu10 > Glu9 >=  Pro20 > Tyr7 > Tyr22 > Asp1 >=  Lys13 > Val16 > Leu26 > Leu25 > Ile8 > Ala27 > Thr6 > Lys21 >=  Glu15 > Arg23 > Met3 > Pro2 >=  Ser5 > Glu24 > Ser4.

The most dramatic effects of a beta -Ala replacement were observed with peptide B27-beta -Ala11 and B27-beta -Ala18 (Fig. 5, A and B). These Leu11 right-arrow beta -Ala11 and Ile18 right-arrow beta -Ala18 mutants had no inhibitory activity even at concentrations in the micromolar range. The results suggest that the side chains of Leu11 and Ile18 and/or the backbones of the Leu11-Gly12 and Ile18-Pro19 regions play a critical role in mediating the inhibitory activity of the peptide. Furthermore, B27-beta -Ala12 (IC50 > 750 nM), in which a -CH2- group was inserted between the Nalpha and Calpha of Gly12, exhibited more than a 15-fold decrease in inhibitory activity relative to the native peptide, B27-WT (IC50 = 33 nM). However, replacing Lys13 with beta -Ala produced a relatively small effect (IC50 = 99 nM) on the potency of the peptide. Because the Gly residue has no side chain, this beta -Ala-based mutagenesis study clearly revealed the importance of the Gly12-Lys13 amide bond for the overall inhibitory effect of peptide B27 on calpain. To probe the contribution of the Leu11-Gly12 amide bond, we carried out a classical "L-Ala substitution" which conserved the amide bond but modified the side chain functionality of Leu11. The Leu11 right-arrow Ala11 mutant was found to exhibit an IC50 of 113 nM (Fig. 5A). The fact that a moderately effective inhibitor was obtained by restoring the Leu11-Gly12 amide bond to its original position while shortening the branched -CH2-CH(CH3)CH3 side chain of Leu to a -CH3 in mutant B27-Ala11 strongly suggests that the Leu11-Gly12 backbone contributes a considerable part to the biological function of calpastatin.

Based on the results of the beta -Ala scan, Ile18 was ranked as the most critical residue in peptide B27-WT. The contributions of the amide and side chain functionalities of Ile18 were investigated further by evaluating an Ile18 right-arrow Ala18 mutant. A very weak inhibitor, exhibiting an IC50 > 300 nM, was achieved when the Ile18-Pro19 peptide bond was restored, while the Ile18 side chain was replaced by a -CH3 group in mutant B27-Ala18 (see Table I and Fig. 5C). As can be deduced easily from a comparison of the IC50 values of 33, >300, and >1,000 nM for B27-WT, B27-Ala18, and B27-beta -Ala18, respectively, both the Ile18-Pro19 backbone and the side chain of Ile18 contribute to the B27-WT bioactivity. Thus, in addition to participating with its carbonyl group to the bioactive conformation of B27-WT, Ile18 also provides a hydrophobic side chain that appears to be a key structural element for the inhibitory function of peptide B27. This finding suggests that the Ile18 side chain is potentially involved in protein-protein interaction with calpain.

The other key residues in B27-WT are Thr17 and Pro19. Replacement of either Thr17 or Pro19 with beta -Ala resulted in a significant loss of inhibitory function (Fig. 5B). The peptide backbone of the Thr17-Ile18-Pro19-Pro20 region was therefore examined further for its contribution to the calpain inhibitory function of B27-WT. As shown in Fig. 5C, the Thr17 right-arrow Ala17 mutant (IC50 = 61 nM) was about 10-fold more potent than the Thr17 right-arrow beta -Ala17 mutant (IC50 > 500 nM, Fig. 5B) and only about 2-fold less potent than the native peptide B27 (IC50 = 33 nM). This suggests the importance of the Thr17-Ile18 backbone and, to a lesser extent, the branched -CH(CH3)-OH side chain of Thr17 in the calpain inhibitory activity of the peptide. Peptide B27-Ala19 (Fig. 5C, IC50 = 44 nM), with a typical amide bond instead of the imide bond between Ile18 and Pro19 in the native peptide, exhibited close to a full inhibitory activity compared with the native peptide B27-WT (IC50 = 33 nM) and the noninhibitory B27-beta -Ala19 mutant (Fig. 5B). Therefore, even though Pro19 is a highly conserved residue across three different species (pig, rabbit, and human) and repetitive domains (domains 1-4) of calpastatin, a Pro residue does not seem to be absolutely required in position 19 to maintain the calpain-inhibitory function of the inhibitor. However, a normal peptide bond between the 19th and 20th residues is necessary to retain the bioactive conformation of B27-WT.

Two regions in calpastatin subdomain 1B, Glu10-Leu11-Gly12-Lys13 and Pro20-Lys21-Tyr22-Arg23, have been predicted to possess type II and type I beta -turns, respectively (21). Interestingly, the beta -Ala mutations that resulted in a severe loss of inhibitory activity were carried out on residues located within (Leu11 and Gly12) or adjacent (Thr17, Ile18, and Pro19) to the beta -turns. Taken together, our structure-activity analysis has identified important contributions of the amide backbones as well as the side chains of some key amino acid residues, clustered at two hot spots, Leu11-Gly12 and Thr17-Ile18-Pro19, within B27-WT which are absolutely critical for the overall activity of the inhibitor. Furthermore, the results of this study demonstrate that the simultaneous or concerted interaction of the two hot spots is required for inhibitory activity because mutation of either one of these key residues resulted in the complete loss of activity. This observation is supported further by the fact that NH2-terminally truncated fragments of B27-WT (Fig. 2B) lacking any one of these key residues failed to inhibit calpain.

Effect of B27-WT and Its beta -Ala Mutants on Calpain Autolysis-- The activation of calpain involves more than a simple Ca2+-induced conformational change. On incubation with Ca2+, the NH2 termini of the catalytic and regulatory subunits of the enzyme are rapidly autolyzed from 80 to 76 kDa, and 28 to 18 kDa, respectively (28). Careful examination of bovine erythrocyte calpain I autolysis in vitro, under conditions that slowed down the process, revealed a two-stage limited cleavage of a 14-amino acid segment from the NH2 terminus of the catalytic subunit to yield a 78-kDa intermediate form, followed by cleavage of an additional 12-amino acid segment to yield the 76-kDa active enzyme form. Cleavage of the 28-kDa subunit was subsequent to generation of the 76-kDa catalytic form (28). Because autolysis of the classical µ- and m-calpains appears to be an intermolecular event that could be inhibited by a calpain inhibitor, we decided to examine the ability of B27-WT to block the autoproteolytic processing of the catalytic subunit of porcine erythrocyte µ-calpain. The native 80-kDa subunit was converted to a 76-kDa form within 1 min when 2 µM calpain was incubated with 1 mM Ca2+ in the absence of inhibitor (Fig. 6, -control). It must be noted here that the commercial preparation of the enzyme, which we used for the autolysis experiment, contained both the 80- and 76-kDa forms of the catalytic subunit (Fig. 6, +control). When calpain was incubated with calcium and B27-WT at inhibitor concentrations that did not completely inactivate the enzyme, the 78-kDa intermediate form of the catalytic subunit was "trapped" during the autoproteolytic process. The extent of autolytic conversion of the intact large subunit from an 80-kDa protein to 78- and 76-kDa fragments depended on the concentration of inhibitor in the preincubation mixture (Fig. 6). Above 110 µM inhibitor, only the intact 80- and the contaminant 76-kDa bands could be seen, whereas at inhibitor concentrations between 10 and 100 µM, the 78-kDa form was also present. Considering the fact that the original enzyme preparation contained some of the 76-kDa form as a contaminant (Fig. 6, +control), it appears that a 50-fold molar excess of B27-WT effectively inhibited the initial stage (80 to 78 kDa) of autolysis.


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Fig. 6.   Inhibition of calpain autolysis by B27-WT. The degradation of the large subunit of µ-calpain from 80 right-arrow 78 right-arrow 76 kDa was monitored by SDS-PAGE after incubation with increasing amounts of B27-WT. The + control lane (+C) includes calpain in the absence of Ca2+ and in the absence of B27-WT. The - control lane (-C) includes calpain after a 15-min incubation in the presence of Ca2+ without B27-WT. 23 µg of µ-calpain was incubated for 15 min with Ca2+ and 0, 14, 28, 42, 56, 85, or 113 µM B27-WT, noted as lane numbers. The reaction was stopped by adding SDS-PAGE reducing buffer (2×) and heating at 95 °C for 5 min. 4-µg samples were analyzed by 7.0% SDS-PAGE, and the gel was stained with GelCode® Blue Stain Reagent.

We next examined the contribution of each amino acid residue in B27-WT to the overall effectiveness of the peptide in blocking autolysis. 2 µM µ-calpain was preincubated with a 25-fold molar excess of B27-WT or one of the 27 beta -Ala mutants in the presence of 1 mM Ca2+ for 15 min, followed by SDS-PAGE of the reaction mixture. At this inhibitor concentration (50 µM), B27-WT slowed the two-step conversion of the 80-kDa subunit to the 76-kDa form, generating a mixture of 80-, 78-, and 76-kDa forms (Fig. 7, WT lane).


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Fig. 7.   Calpain autolysis in the presence of B27-WT or B27-beta -Ala-X mutants. The µ-calpain (2 µM) large subunit degradation from 80 right-arrow 78 right-arrow 76 kDa followed a 15-min incubation with 50 µM B27-WT or B27-beta -Ala-X mutant peptide. The reaction was stopped by adding SDS-PAGE reducing buffer (2×) and heating at 95 °C for 5 min. 4-µg samples of peptide B27-treated calpain were analyzed by 7.0% SDS-PAGE, and the gels were stained with GelCode® Blue Stain Reagent. The lane numbers represent the position of beta -Ala in the B27 mutant peptides. Control lanes include calpain in the absence of Ca2+ and without B27-WT (+C) or calpain in the presence of Ca2+ and without B27-WT (-C). 50 µM B27-WT was also incubated with calpain and run alongside samples of B27-beta -Ala-X peptide-inhibited reactions to compare the extent of calpain inhibition.

With the exception of two mutants, B27-beta -Ala6 (IC50 = 59 nM) and B27-beta -Ala8 (IC50 = 64 nM), which were not as effective in inhibiting autolysis as suggested by their IC50 values, the B27-beta -Ala-X mutants with moderate to strong inhibitory activities (IC50 <=  100 nM) slowed both steps of autolysis (80 to 78 kDa and 78 to 76 kDa) with an efficacy comparable with what was obtained with 50 µM B27-WT. On the other hand, 50 µM B27-beta -Ala20 (IC50 = 129 nM) inhibited both autolytic steps. The IC50 values for B27-beta -Ala at positions 6, 8, and 20 are close to the borderline (100 nM) between the moderate and weak categories of inhibitors. Thus, the apparent discrepancies between the qualitative autolysis data and quantitative substrate kinetic results for these three mutants seem to be within an allowable margin of experimental error. As a general trend, B27-beta -Ala-X mutants that were weakly effective (IC50 = 100-300 nM) in inhibiting the Suc-Leu-Tyr-AMC hydrolytic activity of porcine µ-calpain (see Table I) blocked only the second step (78 to 76 kDa) of the autolytic process, resulting in the accumulation of the 78-kDa form of the catalytic subunit and a loss of the 80-kDa subunit in the presence of Ca2+. Mutants that exhibited practically no inhibitory activities (IC50 > 1000 nM) against the substrate cleaving ability of calpain were unable to inhibit any of the early steps of autolysis (Fig. 7). This study suggests that in the presence of nonphysiological levels of Ca2+, the calpastatin peptide could inhibit the ability of calpain to proteolyze its substrates by blocking the formation of the 76-kDa form of the catalytic subunit.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Peptide deletion/truncation experiments have been used previously to localize the inhibitory activity of calpastatin to regions around a well conserved sequence, TIPPXYR, in subdomain B of each of the four repeating domains of the protein (15, 23). Despite its usefulness in determining the minimum active sequence necessary for bioactivity, the peptide truncation approach provides few details about the contributions of each residue to the overall biological function of a protein. Classical L-amino acid residue replacement approaches, such as alanine scanning, made possible using the tools of molecular biology, allow systematic structural modifications at the resolution of individual residues and are good for monitoring side chain interactions. However, these point mutations do little to probe the conformational properties of the peptide backbone. Furthermore, alanine, proline, glycine and cysteine ("nonentropic" amino acids) are not usually mutated in alanine scans (29).

The work presented in this report combined beta -alanine scanning mutagenesis with kinetic measurements to probe, in a quantitative, systematic, and simultaneous fashion, the relative contribution of each amino acid side chain and peptide backbone (main chain) in the functional region of calpastatin to the overall calpain-inhibitory activity. The beta -alanine substitution modifies both the amino acid side chain and the COOH-terminal amide bond of the residue being probed. This strategy enables "structure-conformation-activity relations" (SCARs) to be delineated if further analyses are carried out on key residues to differentiate an effect because of a side chain modification from one that is the result of a change in peptide (amide) bond conformation. The SCARs approach is, therefore, capable of providing insights into the importance of different side chain groups and/or specific amide bonds for ligand-receptor interactions, which normal L-Ala scanning cannot provide.

Earlier studies by Cottin et al. (30) and by Shiba et al. (31) on the mechanism of calpain inhibition by calpastatin indicated a noncompetitive type of inhibition. However, more recent studies, performed in two independent laboratories, on calpain inhibition by calpastatin-derived peptides showed a competitive type of inhibition (15, 23). Graphical methods (Dixon plots) were used to examine the kinetics of inhibition in all of the above mentioned studies. As pointed out by Laskowski and Sealock (32), graphical methods are not suitable for the determination of the mechanism of tight binding inhibitors. To circumvent the problems associated with the analyses of tight binding inhibition, calpastatin peptides that were derived from the most weakly inhibiting domain of the four repetitive domains of calpastatin were used in the more recent studies (15, 23).

In the first part of our study, stopped-flow kinetic measurements were used to determine the ka, kd, and Ki values and the mechanism of inhibition of calpain by B27-WT, derived from the most potent inhibitory subdomain of calpastatin. A competitive type of inhibition was observed when either FTC-casein or a small molecular weight dipeptide fluorogenic substrate (Suc-Leu-Tyr-AMC) was used for the assay. Our results suggest that B27-WT competes with either of the substrates for interaction with calpain. The binding of B27-WT to calpain is not expected, however, to proceed through direct interaction with the catalytic center cysteine of the proteinase. B27-WT probably functions like an exosite binding inhibitor that interacts with surface residues adjacent to the active center of calpain, preventing access of substrates to the active center, without direct blockage of the catalytic residues. This mechanism is supported by the observation that calpain, with the active center cysteine protected with either a reversible or irreversible inhibitor, still retains the ability to efficiently bind a calpastatin subdomain B peptide (23, 33). This experimental observation has been exploited in our laboratory to purify calpain from bovine kidney and heart (33). Purification was accomplished by passing the active site inhibitor-treated crude tissue extract through a column charged with B27-WT immobilized on CNBr-activated Sepharose 4B (Amersham Biosciences). Further support is provided by a study described by Croall and McGrody in 1994 (34), in which a cysteine-specific cross-linking of an inhibitory calpastatin peptide to calpain did not result in the labeling of the active site cysteine of the enzyme. The study also suggested that the binding site for the calpastatin peptide was close to domain III of the catalytic subunit of calpain.

The main goal of our study was to determine the structural elements in B27-WT that are critical for its biological activity. Previous studies have shown that the calpastatin polypeptide is largely a random coil in solution (22, 35, 36). This earlier observation was supported further by evidence from 1H NMR studies (21) and circular dichroism spectroscopy experiments performed by our group,2 demonstrating the absence of regular secondary structures such as alpha -helix and beta -sheets in B27-WT. Based upon these findings, we hypothesized that the specific and high affinity binding of B27-WT to calpain is most likely the result of multisite protein-peptide interactions mediated by small and discrete structural elements present in key residues in the peptide. Therefore, by modifying the side chain and/or peptide backbone of the key B27-WT residues, the ability of the peptide to inhibit calpain would be altered. Through beta -Ala scanning and conventional Ala mutations on selected amino acids, we identified, for the first time, two hot spots, Leu11-Gly12 and Thr17-Ile18-Pro19, in calpastatin subdomain 1B within which the key amide backbones as well as amino acid side chains that are most critical for the overall activity of the inhibitor are clustered. Mutation of any one of the key residues in either of the two hot spots resulted in a dramatic loss of inhibitory activity, suggesting that a simultaneous or concerted interaction involving both hot spots is required for inactivation of calpain by the calpastatin inhibitor peptide. Because hot spots tend to cluster near the center of protein-protein interfaces (29), we predict that the key contact residues of the calpain-calpastatin complex are located in this region of the peptide.

Of 26 amide bonds and 25 side chains that are present in B27-WT, we have been able to pinpoint the key structural elements that are crucial for the biological activity of the peptide to five specific main chain (backbone) and two side chain functionalities. The main contributions of the backbone to the B27-WT inhibitory activity are from the Leu11-Gly12-Lys13 and Thr17-Ile18-Pro19 segments. The Leu11-Gly12-Lys13 segment is located within a region of B27-WT which is probably a type II beta -turn, in which case Leu11 and Gly12 would be the i + 1 and i + 2 residues, respectively (21). The type II turn is relatively flat, and the central peptide lies in a plane perpendicular to the plane of the turn. Also, the i + 2 position in type II turns is dominated by Gly, which most readily adopts the +/+ phi ,psi conformation found in this structure (37). Turns have been suggested as the bioactive conformations involved in many recognitional processes, with the side chain groups in corner positions pointing outward and serving as a site for molecular recognition (38, 39). Thus, a compact and stable local turn conformation of the Leu11-Gly12 main chain would be crucial for a strong interaction between the Leu11 side chain and a calpain site and/or effective hydrogen bonding between its amide backbone and specific residues in calpain. Our beta -Ala scan also tested for the flexibility of the peptide backbone and has shown that the structural integrity of the three amide bonds in the Thr17-Ile18-Pro19-Pro20 backbone is crucial for the inhibitory activity of B27-WT. In addition to the Leu11 side chain, the Ile18 side chain situated NH2-terminally to a type I beta -turn (Pro20-Lys21-Tyr22-Arg23) was revealed to be a key structural element that is critical for the bioactivity of B27-WT. We predict that the Leu11 and Ile18 side chains are involved in hydrophobic interactions with complementary binding sites in Ca2+-bound calpain because a conventional Leu11 right-arrow Ala11 or Ile18 right-arrow Ala18 mutation significantly reduced the inhibitory potential of B27-WT. In addition to identifying the particular amide bonds and side chains that constitute the structural elements contributing to biological activity, our studies indicate that the Leu11-Gly12 and Thr17-Ile18-Pro19-Pro20 regions have restricted local structures that maintain the inhibitory function of the peptide.

In general, bioactive peptides that have no secondary structure constraints usually must be relatively large to interact effectively with a protein because the peptide must provide interactions that compensate for the entropic penalty of docking a flexible structure in a relatively ordered and static orientation. The results from our study indicate that other residues such as Glu9, Glu10, and Arg14, which flank the first hot spot region, could play an important role in maintaining a functional local conformation or serve as sites for calpain interaction.

An amino acid sequence alignment of the 27-residue repetitive inhibitory subdomains of human, pig, and rabbit calpastatins shows only five residues that are identical and two that are conservatively substituted in all 12 subdomains, i.e. 4 repetitive subdomains from three different species (Fig. 8). Except for Leu11 and Arg14, which are replaced by Cys11 and Asp14, respectively, in subdomain 2B, the calpastatin residues that are important for inhibitory activity are either identical or conservatively substituted in the 12 subdomains covering three species. Much to our surprise, however, Arg23, one of the five residues that are identical across the 12 inhibitory subdomains, does not seem to be important for bioactivity (Table I). Arg is one of the amino acid residues that appears in hot spots with high frequency (29). Arginine can interact at protein-protein interfaces by utilizing a hydrogen-bonding network with up to five hydrogen bonds and a salt bridge with its positively charged guanidine motif. The electron delocalization of the guanidinium pi -system has a pseudo-aromatic character that can take part in aromatic and pi -interactions. Another interesting observation was that the side chains of Lys21 and Tyr22, two residues that have been predicted to be in corner positions i + 1 and i + 2 of a type I beta -turn in region Pro20-Lys21-Tyr22-Arg23 (21), are less important for bioactivity.


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Fig. 8.   Amino acid sequence alignment to give maximum homology among repetitive subdomains B of human (H), pig (P), and rabbit (R) calpastatins. Residues identical (#) or conservatively substituted (*) in all 12 subdomains covering three species are indicated.

All protein protease inhibitors for which three-dimensional structures are available have revealed an intricate combination of secondary structure motifs such as alpha -helices, beta -sheets, beta -barrels, and loops, designed to interact with and inactivate their cognate proteases with a high selectivity (40). Calpastatin is mostly in a random coil conformation as determined by circular dichroism and NMR spectroscopy studies (22). Apart from the possibility of local turn structures in B27-WT, none of the biophysical studies done so far on the peptide indicates the presence of even loosely preformed secondary structure motifs. It is most likely that the specific and high affinity binding between B27-WT and calpain is mediated by several discrete and localized structural elements rather than a global well structured conformation of the peptide. Therefore, our structure-function analysis has been based mainly on effects of the beta -Ala mutagenesis which could lead to a change in the local conformation of the peptide and/or loss of a specific interaction with calpain. However, one cannot completely rule out possible effects of a beta -Ala mutation on the overall structure of B27-WT required for calpain inhibition. It is conceivable that the calpastatin polypeptide assumes a more structured conformation when it interacts with its target proteinase calpain.

The functional groups that are critical for the inhibitory activity of B27-WT have been revealed in this report. Whether the positional modifications introduced in the beta -Ala library of the peptide perturbed the bioactive conformation and/or the fully productive interaction of the bioactive conformation(s) with calpain is not completely clear at this stage of our study. NMR studies have been scheduled to investigate whether B27-WT assumes an overall better structured conformation upon binding to calpain in the presence of calcium and whether mutation of a key residue could disrupt the overall structural integrity of the calpain-bound peptide. It must be noted, however, that even x-ray crystallography and NMR studies do not easily resolve the fundamental issue of distinguishing between direct effects of a substitution on inhibition and indirect effects caused by structural changes. Nevertheless, the present study integrated the functional importance of individual residues with the structural and dynamic aspects of inhibition. We propose a structural model for the competitive inhibition of calpain by B27-WT in which the two hot spots in the inhibitor interact with two separate sites in the extended substrate binding region of the proteinase in a manner akin to the closing of a "gate" leading to the active site of the enzyme. This model is being tested by using photoaffinity labeling techniques to map the topology of the inhibitor binding region.

The number of human diseases linked to uncontrolled calpain activation continues to increase (41). The pathological role of calpain has been attributed to an imbalance between calpain and calpastatin activities, underscoring the importance of elucidating the structural basis of the inhibitory activity of calpastatin. Although the exact mechanism by which calpastatin inhibits calpain remains unclear, the present study has provided very useful insights into the possible mode of action of B27-WT by pinpointing the structural determinants of the biological activity of the inhibitor. It is expected that the information derived from this study will contribute an important part to the overall understanding of calpain inhibition and possibly lead to novel and effective therapies based on the specific inhibition of dysregulated or overactivated calpain.

    ACKNOWLEDGEMENT

We are grateful to Dr. Herbert Halvorsen for reading the manuscript and providing useful comments.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants R01 NS39075 (to J. A.,) and P60 AR20557 (to the University of Michigan Multipurpose Arthritis and Musculoskeletal Diseases Center).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Division of Biochemical Research, Dept. of Pathology, Henry Ford Health Sciences Center, One Ford Place, 5D, Detroit, MI 48202. Tel.: 313-876-7460; Fax: 313-876-2380; E-mail: janagli1@hfhs.org.

Published, JBC Papers in Press, December 24, 2002, DOI 10.1074/jbc.M208350200

2 J. Anagli, R. Betts, and T. Stemmler, unpublished results.

    ABBREVIATIONS

The abbreviations used are: µ- and m-calpain, the micromolar and millimolar Ca2+-requiring Ca2+-dependent proteinase, respectively; AMC, aminomethylcoumarin; DIPEA, N,N'-diisopropylethylamine; Fmoc, N-(9-fluorenyl)methoxycarbonyl; FTC, fluorescein thiocarbamoyl; HBTU, O-benzotriazole-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate; HPLC, high performance liquid chromatography; MALDI, matrix-assisted laser desorption ionization; Pbf, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Pontremoli, S., and Melloni, E. (1986) Annu. Rev. Biochem. 55, 455-481[CrossRef][Medline] [Order article via Infotrieve]
2. Schollmeyer, J. E. (1988) Science 240, 911-913[Medline] [Order article via Infotrieve]
3. Ono, Y., Sorimachi, H., and Suzuki, K. (1998) Biochem. Biophys. Res. Commun. 245, 289-294[CrossRef][Medline] [Order article via Infotrieve]
4. Pinton, P., Ferrari, D., Di Virgilio, F., Pozzan, T., and Rizzuto, R. (2001) Drug Dis. Res. 52, 558-570[CrossRef]
5. James, T., Matzelle, D., Bartus, R., Hogan, E. L., and Banik, N. L. (1998) J. Neurosci. Res. 51, 218-222[CrossRef][Medline] [Order article via Infotrieve]
6. Wang, K. K., Postmantur, R., Nadimpalli, R., Nath, R., Mohan, P., Nixon, R. A., Talanian, R. V., Keegan, M., Herzog, L., and Allen, H. (1998) Arch. Biochem. Biophys. 356, 187-196[CrossRef][Medline] [Order article via Infotrieve]
7. Yoshizawa, T., Sorimachi, H., Tomioka, S., Ishiura, S., and Suzuki, K. (1995) FEBS Lett. 587, 101-103
8. Pal, G. P., Elce, J. S., and Jia, Z. (2001) J. Biol. Chem. 276, 47233-47238[Abstract/Free Full Text]
9. Hosfield, C. M., Elce, J. S., Davies, P. L., and Jia, Z. (1999) EMBO J. 18, 6880-6889[Abstract/Free Full Text]
10. Sorimachi, H., and Suzuki, K. (2001) J. Biochem. 129, 653-664[Abstract]
11. De Tullio, R., Passalacqua, M., Averna, M., Salamino, F., Melloni, E., and Pontremoli, S. (1999) Biochem. J. 343, 467-472[CrossRef][Medline] [Order article via Infotrieve]
12. Averna, M., De Tullio, R., Passalacqua, M., Salamino, F., Pontremoli, S., and Melloni, E. (2001) Biochem. J. 354, 25-30[CrossRef][Medline] [Order article via Infotrieve]
13. Moldoveanu, T., Hosfield, C. M., Lim, D., Elce, J. S., Jia, Z., and Davies, P. (2002) Cell 108, 649-660[Medline] [Order article via Infotrieve]
14. Molinari, M., and Carafoli, E. (1997) J. Membr. Biol. 156, 1-8[CrossRef][Medline] [Order article via Infotrieve]
15. Maki, M., Takano, E., Osawa, T., Ooi, T., Murachi, T., and Hatanaka, M. (1988) J. Biol. Chem. 263, 1787-1793
16. Hao, L.-Y., Kameyama, A., Kuroki, S., Takano, J., Takano, E., Maki, M., and Kameyama, M. (2000) Biochem. Biophys. Res. Commun. 279, 756-761[CrossRef][Medline] [Order article via Infotrieve]
17. Yang, H. Q., Ma, H., Takano, E., Hatanaka, M., and Maki, M. (1994) J. Biol. Chem. 269, 18977-18984[Abstract/Free Full Text]
18. Ma, H., Yang, H. Q., Takano, E., Hatanaka, M., and Maki, M. (1994) J. Biol. Chem. 269, 24430-24436[Abstract/Free Full Text]
19. Takano, E., Ma, H., Yang, H. Q., Maki, M., and Hatanaka, M. (1995) FEBS Lett. 362, 93-97[CrossRef][Medline] [Order article via Infotrieve]
20. Maki, M., Bagci, H., Hamaguchi, K., Ueda, M., Murachi, T., and Hatanaka, M. (1989) J. Biol. Chem. 264, 18866-18869[Abstract/Free Full Text]
21. Ishima, R., Tamura, A., Akasaka, K., Hamaguchi, K., Makino, K., Murachi, T., Hatanaka, M., and Maki, M. (1991) FEBS Lett. 294, 64-66[CrossRef][Medline] [Order article via Infotrieve]
22. Uemori, T., Shimojo, T., Asada, K., Asano, T., Kimizuka, F., Kato, I., Maki, M., Hatanaka, M., Murachi, T., Hanazawa, H., and Arata, Y. (1990) Biochem. Biophys. Res. Commun. 166, 1485-1493[Medline] [Order article via Infotrieve]
23. Kawasaki, H., Emori, Y., Imajoh-Ohmi, S., Minami, Y., and Suzuki, K. (1989) J. Biochem. 106, 274-281[Abstract]
24. Strobl, S., Fernandez-Catalan, C., Braun, M., Huber, R., Masumoto, H., Nagakawa, K., Irie, A., Sorimachi, H., Bourenkow, G., Bartunik, H., Suzuki, K., and Bode, W. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 588-592[Abstract/Free Full Text]
25. Asada, K., Ishiro, Y., Shimada, M., Shimojo, T., Endo, M., Kimizuka, F., Kato, I., Maki, M., Hatanaka, M., and Murachi, T. (1989) J. Enzyme Inhib. 3, 49-56[Medline] [Order article via Infotrieve]
26. Williams, J. W., and Morrison, J. F. (1979) Methods Enzymol. 63, 437-467[Medline] [Order article via Infotrieve]
27. Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve]
28. Zimmerman, U.-J. P., and Schlaepfer, W. W. (1991) Biochim. Biophys. Acta 1078, 192-198[Medline] [Order article via Infotrieve]
29. Bogan, A. A., and Thorn, K. S. (1998) J. Mol. Biol. 280, 1-9[CrossRef][Medline] [Order article via Infotrieve]
30. Cotttin, P., Vidalenc, P. L., Merdaci, N., and Ducastaing, A. (1983) Biochim. Biophys. Acta 743, 299-302[Medline] [Order article via Infotrieve]
31. Shiba, E., Tsujinaka, T., Kambayashi, J., and Kosaki, G. (1983) Thromb. Res. 32, 207-214[Medline] [Order article via Infotrieve]
32. Laskowski, M., Jr., and Sealock, R. W. (1971) in The Enzymes (Boyer, P. D., ed), Vol. 3 , pp. 375-473, Academic Press, Orlando, FL
33. Anagli, J., Vilei, E. M., Molinari, M., Calderara, S., and Carafoli, E. (1996) Eur. J. Biochem. 241, 948-954[Abstract]
34. Croall, D. E., and McGrody, K. S. (1994) Biochemistry 33, 13223-13230[Medline] [Order article via Infotrieve]
35. Konno, T., Tanaka, N., Kataoka, M., and Maki, M. (1997) Biochim. Biophys. Acta 1342, 73-82[Medline] [Order article via Infotrieve]
36. Shannon, J. D., and Goll, D. E. (1985) in Intracellular Protein Catabolism (Khairallah, E. A. , Bond, J. S. , and Birds, J. W. C., eds) , pp. 233-239, Alan R. Liss, New York
37. Wilmot, C. M., and Thornton, J. M. (1988) J. Mol. Biol. 203, 221-232[Medline] [Order article via Infotrieve]
38. Perczel, A., and Hollosi, M. (1996) in Circular Dichroism and the Conformational Analysis of Biomolecules (Fasman, G. D., ed) , pp. 286-380, Plenum Publishing Corp., New York
39. Rose, G. D., Gierasch, L. M., and Smith, J. A. (1985) Adv. Prot. Chem. 37, 1-109[Medline] [Order article via Infotrieve]
40. Bode, W., and Huber, R. (2000) Biochim. Biophys. Acta 1477, 241-252[Medline] [Order article via Infotrieve]
41. Huang, Y., and Wang, K. W. W. (2001) Trends Mol. Med. 7, 355-362[CrossRef][Medline] [Order article via Infotrieve]


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