From the 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
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ABSTRACT |
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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 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 -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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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
-helix and
-sheets; however, 1H NMR studies of B27-WT
have shown the presence of a well defined type I
-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
-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 "-alanine scanning" mutagenesis to determine the effect of site-specific amide bond and/or
side chain modifications on the biological activity of B27-WT.
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EXPERIMENTAL PROCEDURES |
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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 -Ala-containing
analogs (B27-
-Ala-X) were synthesized using a multiple
peptide synthesizer (Advanced ChemTech, model 348
) 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-
-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),
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(Eq. 1) |
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(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 -Ala-containing
Analogs of B27-WT--
The effects of single amino acid modifications
on the calpain-inhibitory activity of the library of
-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--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
-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).
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RESULTS |
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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 -Ala substitution (
-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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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,
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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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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
M1 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 -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
-turn
regions are shown in Fig. 5.
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The inhibitory activities of the -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
-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
-Ala had a moderate
effect (IC50 = 50-85 nM) on the inhibitory potential of the peptide. B27-
-Ala mutations at positions 1, 7, 9, 13, 16, 20, and 22 were weak inhibitors (IC50 = 100-130 nM). Mutants B27-
-Ala10 and
Ala14 showed very weak inhibitory activities
(IC50 = 180-280 nM), whereas a group of
mutants comprising B27-
-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 -Ala replacement were observed with
peptide B27-
-Ala11 and B27-
-Ala18 (Fig.
5, A and B). These Leu11
-Ala11 and Ile18
-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-
-Ala12 (IC50 > 750 nM), in
which a -CH2- group was inserted between the N
and C
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
-Ala produced a relatively small effect
(IC50 = 99 nM) on the potency of the peptide.
Because the Gly residue has no side chain, this
-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
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 -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
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-
-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 -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
Ala17 mutant (IC50 = 61 nM) was about 10-fold more potent than the Thr17
-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-
-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 -turns,
respectively (21). Interestingly, the
-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
-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 -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.
|
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 -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).
|
With the exception of two mutants, B27--Ala6
(IC50 = 59 nM) and B27-
-Ala8
(IC50 = 64 nM), which were not as effective in
inhibiting autolysis as suggested by their IC50 values, the
B27-
-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-
-Ala20
(IC50 = 129 nM) inhibited both autolytic steps.
The IC50 values for B27-
-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-
-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.
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DISCUSSION |
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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 -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
-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 -helix and
-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
-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 -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 +/+
,
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
-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
-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
Ala11 or
Ile18
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 -system has a
pseudo-aromatic character that can take part in aromatic and
-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
-turn in region
Pro20-Lys21-Tyr22-Arg23
(21), are less important for bioactivity.
|
All protein protease inhibitors for which three-dimensional structures
are available have revealed an intricate combination of secondary
structure motifs such as -helices,
-sheets,
-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
-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
-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 -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.
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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 |
9. |
Hosfield, C. M.,
Elce, J. S.,
Davies, P. L.,
and Jia, Z.
(1999)
EMBO J.
18,
6880-6889 |
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 |
18. |
Ma, H.,
Yang, H. Q.,
Takano, E.,
Hatanaka, M.,
and Maki, M.
(1994)
J. Biol. Chem.
269,
24430-24436 |
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.,
Ba![]() |
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 |
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] |