Biochem. J. (2010) 429, 379–389 (Printed in Great Britain)
379
doi:10.1042/BJ20100337
Conformational flexibility and allosteric regulation of cathepsin K
Marko NOVINEC*†, Lidija KOVAČIȇ, Brigita LENARČIȆ§ and Antonio BAICI*1
*Department of Biochemistry, University of Zürich, 8057 Zürich, Switzerland, †Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of
Ljubljana, 1000 Ljubljana, Slovenia, ‡Department of Molecular and Biomedical Sciences, Jožef Stefan Institute, 1000 Ljubljana, Slovenia, and §Department of Biochemistry and
Molecular and Structural Biology, Jožef Stefan Institute, 1000 Ljubljana, Slovenia
The human cysteine peptidase cathepsin K is a key enzyme
in bone homoeostasis and other physiological functions. In
the present study we investigate the mechanism of cathepsin
K action at physiological plasma pH and its regulation by
modifiers that bind outside of the active site. We show that at
physiological plasma pH the enzyme fluctuates between multiple
conformations that are differently susceptible to macromolecular
inhibitors and can be manipulated by varying the ionic strength
of the medium. The behaviour of the enzyme in vitro can
be described by the presence of two discrete conformations
with distinctive kinetic properties and different susceptibility
to inhibition by the substrate benzyloxycarbonyl-Phe-Arg-7amino-4-methylcoumarin. We identify and characterize sulfated
glycosaminoglycans as natural allosteric modifiers of cathepsin
K that exploit the conformational flexibility of the enzyme to
regulate its activity and stability against autoproteolysis. All
sulfated glycosaminoglycans act as non-essential activators in
assays using low-molecular-mass substrates. Chondroitin sulfate
and dermatan sulfate bind at one site on the enzyme, whereas
heparin binds at an additional site and has a strongly stabilizing
effect that is unique among human glycosaminoglycans.
All glycosaminoglycans stimulate the elastinolytic activity of
cathepsin K at physiological plasma pH, but only heparin also
increases the collagenolytic activity of the enzyme under these
conditions. Altogether these results provide novel insight into the
mechanism of cathepsin K function at the molecular level and its
regulation in the extracellular space.
INTRODUCTION
as a target for osteoporosis therapy and at least two cathepsin K
inhibitors are currently in clinical trials [10].
Cathepsin K can cleave most extracellular substrates, including
the collagen triple helix and it has been reported that its
collagenolytic activity depends on complex formation with CS
(chondroitin sulfate), which increases the activity and stability of
the enzyme [11]. The crystal structure of cathepsin K in complex
with a chondroitin-4-sulfate hexasaccharide, however, shows that
the CS-binding site is distant from the active site and no effect on
enzyme activity was observed [12].
There have been several other reports about the influence
of GAGs (glycosaminoglycans) on the activity of cysteine
cathepsins. HP (heparin) and HS (heparan sulfate) were found
to increase the activity and stability of papain and cathepsin
B [13,14], whereas, and in contrast, intralysosomal membranebound GAGs act as inhibitors of lysosomal enzymes [15–17].
Furthermore, GAGs facilitate the autocatalytic conversion of procathepsins B and S into their mature forms [18,19].
Bone resorption by osteoclasts occurs at acidic pH, which has
also been found to be optimal for the stability of cathepsin K [20].
Other extracellular actions of cathepsin K, such as degradation
of elastic fibres in the walls of blood vessels, occur however
at pH values equal or close to the physiological plasma pH.
This indicates that cathepsin K retains significant activity under
such suboptimal conditions. In the present study we investigate
the activity of cathepsin K and its regulation at physiological
plasma pH, as is found in the extracellular matrix. We report that
under these conditions the enzyme exists in multiple functionally
distinct conformational states in vitro. We identify sulfated GAGs
as allosteric modifiers of cathepsin K and investigated their effect
on the activity and stability of cathepsin K under these conditions.
Allostery is the coupling of conformational changes between two
separated sites. Initially described in oligomeric systems [1–3],
allostery is known today as a widely used mechanism in
the regulation of monomeric proteins. The ability to undergo
conformational changes in response to ligand binding is an
intrinsic property of many, if not all, non-fibrous proteins.
Conformational changes triggered by an allosteric modifier can
range from major structural movements to subtle, even virtually
unrecognizable, rearrangements within the protein [4].
Although long believed to be relatively unstable in the extracellular milieu, it has become clear in recent years that cysteine
cathepsins are major players in extracellular proteolysis. Much
work has been dedicated in investigating the regulation of these
enzymes by proteinaceous competitive inhibitors [5], but little is
known about other mechanisms involved in the regulation of their
activity.
Cathepsin K is one of the most potent mammalian peptidases. It
is the major proteolytic enzyme involved in bone metabolism and
its deficiency causes pycnodysostosis, a rare disease characterized
by bone abnormalities [6]. It is also expressed in several other cell
types of the fibroblast and haemopoietic lineages, as well as some
epithelioid cells and aortic smooth muscle cells. Besides bone
resorption, it plays important roles in embryonic development,
spermatogenesis and thyroid hormone release [7]. Previous
findings also suggest roles for cathepsin K in schizophrenia [8] and
obesity [9]. Excessive cathepsin K activity has also been reported
in association with cardiovascular and pulmonary diseases, as
well as arthritis and cancer [7]. Because of its role in bone matrix
degradation it has received considerable attention in recent years
Key words: chondroitin sulfate, conformational change, dermatan
sulfate, heparin, non-essential activation, substrate inhibition.
Abbreviations used: AMC, 7-amino-4-methylcoumarin; CS, chondroitin sulfate; DS, dermatan sulfate; DTT, dithiothreitol; DxS, dextran sulfate;
GAG, glycosaminoglycan; HP, heparin; HS, heparan sulfate; N2TY, thyroglobulin type 1 domain 1 of human nidogen-2; R state, relaxed state;
T state, tense state; Z-, benzyloxycarbonyl-.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2010 Biochemical Society
380
M. Novinec and others
EXPERIMENTAL
Intrinsic fluorescence spectroscopy
Materials
Intrinsic fluorescence spectra of cathepsin K were recorded
in 50 mM Hepes, pH 7.40, containing from 0 to 300 mM
NaCl in single-use acrylic cuvettes (1 cm × 1 cm) at 25 ◦C
with magnetic stirring. Samples were excited at 295 nm and
emitted fluorescence spectra were either recorded from 310
to 400 nm or the fluorescence was monitored continuously at
340 nm (5 nm bandwidth). The final enzyme concentration in
all experiments was 0.2 μM. When recording time-dependent
changes in fluorescence, recording was started immediately upon
diluting the enzyme from a stock solution (1 mg/ml cathepsin K
in 50 mM Hepes, pH 5.0, containing 500 mM NaCl, 1 mM DTT,
25 μg/ml DxS) into the reaction mixture.
Recombinant human cathepsin K was produced according to
the procedure described by D’Alessio et al. [21]. Enzyme
concentration was determined by active-site titration with
the irreversible inhibitor E-64 (Bachem). The fluorogenic
substrates Z-FR-AMC (benzyloxycarbonyl-Phe-Arg-7-amino4-methylcoumarin) and Z-VVR-AMC (benzyloxycarbonyl-ValVal-Arg-7-amino-4-methylcoumarin) were from Bachem. DS
(dermatan sulfate) from porcine intestinal mucosa was from
Calbiochem. Heparin sodium salt, DxS (dextran sulfate) sodium
salt, leupeptin hydrochloride and chondroitin-4-sulfate
sodium salt from bovine trachea were from Sigma–Aldrich.
Although labelled ‘chondroitin-4-sulfate’ this sample was a
co-polymer of the 4- and 6-isomers along the same chain and
contained 69 % 4-sulfate and 25 % 6-sulfate; DS contained 98 %
4-sulfate and the balance to 100 % was non-sulfated material
for both CS and DS. The uronic acid moieties were consistently
glucuronic acid and iduronic acid in CS and DS, respectively, as
measured by HPLC analysis of the unsaturated disaccharides as
described previously [22]. The weight-average molecular masses,
Mw , of CS and DS were 23299 and 26488 Da, respectively [23].
All concentrations of GAGs are given as molar concentrations
of disaccharide units. Bovine neck ligament elastin and soluble
ETNA-elastin were from Elastin Products Company. Soluble
calf-skin collagen was from the Worthington Biochemical
Corporation. The cross-linking reagent sulfo-SBED {sulfoN-hydroxysuccinimidyl-2-[6-(biotinamido)-2-(p-azido benzamido)-hexanoamido] ethyl-1,3 -dithioproprionate} was from
Thermo Scientific. Recombinant human stefin A and recombinant human N2TY (thyroglobulin type 1 domain 1 of human
nidogen-2) were produced according to published procedures
[24,25].
Kinetic measurements
Prior to the reactions the enzyme was kept on ice either in
low-salt buffer [50 mM Hepes, pH 7.40, containing 1 mM EDTA
and 2.5 mM DTT (dithiothreitol)] or in high-salt buffer (50 mM
Hepes, pH 7.40, containing 300 mM NaCl, 1 mM EDTA and
2.5 mM DTT), as described in the text. Buffers were prepared
and used at 25 ◦C. All measurements were performed in lowsalt buffer in single-use acrylic cuvettes (1 cm × 1 cm) that were
◦
kept at a constant temperature of 25 +
− 1 C and subject to
magnetic stirring. Z-FR-AMC was used as the substrate, except
where indicated otherwise. Reactions were started by adding
enzyme to the reaction-mixture-containing buffer, substrate and
modifier, where appropriate. Reaction progress was monitored
fluorimetrically at an excitation wavelength (λex ) of 383 nm and
an emission wavelength (λem ) of 455 nm. The final enzyme active
site concentration in the assays was 0.1 nM. Blanks containing
standard concentrations of AMC (7-amino-4-methylcoumarin)
were recorded under reaction conditions to assure that total
substrate consumption in the experiments was less than 10 %.
Stability of the cathepsin K activity
Cathepsin K was incubated at 37 ◦C at a final enzyme
concentration of 0.2 μM in 50 mM Hepes, pH 7.40, containing
1 mM EDTA and 2.5 mM DTT, in the presence or absence of
0.2 mM GAGs and 300 mM NaCl, or 5 mg/ml soluble ETNAelastin. Residual enzyme activity was determined by removing
aliquots from the reaction mixture at regular time intervals and
measuring their activity using the substrate Z-FR-AMC (10 μM
final concentration).
Elastinolytic assays
Suspensions of bovine neck ligament elastin (5 mg/ml) were
prepared in 50 mM Hepes, pH 7.40, containing 1 mM EDTA
and 2.5 mM DTT. GAGs (0.2 mM final concentration) were
added to the suspensions just prior to addition of the enzyme
(final concentration 0.1 μM). The same experiments where
also performed in two other buffers, 50 mM Hepes, pH 7.40,
containing 300 mM NaCl, 1 mM EDTA and 2.5 mM DTT and
50 mM Mes, pH 6.20, containing 1 mM EDTA and 2.5 mM
DTT. All mixtures were incubated in an Eppendorf Thermomixer
Compact at 37 ◦C with shaking (1200 rev./min) and reactions
were stopped after various incubation times by addition of
trichloroacetic acid to a final concentration of 5 % (w/v). After
centrifugation (14 000 g for 10 min), clear supernatants (200 μl)
were diluted to 3.0 ml with 0.2 M borate buffer, pH 8.50, and
reacted with 1.0 ml of a fluorescamine solution (0.15 mg/ml in
acetone). The fluorescence of the samples was measured at λex
390 nm and λem 480 nm. Peptide concentrations were determined
from a standard curve produced in the same manner using a
standardized concentration of L-alanine.
Collagen digestion
Soluble calf-skin collagen was diluted in 50 mM Hepes, pH 7.40,
containing 1 mM EDTA to a final concentration of 0.5 mg/ml.
The solutions were supplemented with 2.5 mM DTT and 0.2 mM
GAGs and digestion started by addition of cathepsin K (final
concentration 0.25 μM). All reactions were incubated for 16 h at
25 ◦C and then stopped by addition of SDS/PAGE sample buffer.
Polypeptides were separated by SDS/PAGE (8 % gels) and stained
with Coomassie Brillant Blue R-250.
Docking of GAG octasaccharides to cathepsin K
Kinetic models and data analysis
The detailed descriptions of kinetic models used in this work
are available in the Supplementary Theoretical background
section (at http://www.BiochemJ.org/bj/429/bj4290379add.htm).
All mathematical analyses and graphical manipulations were
performed with GraphPad Prism 5.0 software.
c The Authors Journal compilation c 2010 Biochemical Society
Octasaccharide models of DS (consisting of four 4sulfo-N-acetylgalactosamine-iduronic acid disaccharides) were
constructed with the CambridgeSoft ChemOffice 11 Suite.
Torsion angles between monosaccharides were adjusted manually
according to published data [26] and the structures then optimized
by energy minimization using the MMFF94 force-field. Heparin
Allosteric regulation of cathepsin K
381
Intrinsic tryptophan fluorescence
Figure 1 Dependence of the inhibitory efficiency of macromolecular
inhibitors of cathepsin K on ionic strength
Reactions were performed by adding 0.1 nM cathepsin K to a reaction mixture containing the
substrate10 μM Z-FR-AMC and inhibitor at 25 ◦C. (A) Examples of progress curves of substrate
hydrolysis in the presence of stefin A (20 nM) recorded in 50 mM Hepes, pH 7.40, containing
the indicated concentrations of NaCl. (B) Steady-state reaction rates v s (left-hand panel) and
values of the first-order rate constant λ (right-hand panel) for the interaction between enzyme
and inhibitor. The inhibitors used were 20 nM stefin A (stfA) or 50 nM N2TY. The values of
v s and λ were determined by non-linear regression using eqn (1) (Supplementary material at
http://www.BiochemJ.org/bj/429/bj4290379add.htm).
models were retrieved from the PDB (PDB code 1HPN). Coordinates of cathepsin K were extracted from the crystal structure
of the cathepsin K–CS complex (PDB code 3C9E). All docking
calculations were performed with AutoDock 4 [27] using the
Lamarckian genetic docking algorithm. A detailed description of
the procedure is available in the Supplementary section. Surface
potentials were calculated with the Adaptive Poisson–Boltzman
Solver [28]. All images were created with PyMOL (DeLano
Scientific; http://www.pymol.org).
RESULTS
Interaction of cathepsin K with macromolecular inhibitors
The initial observation that led us to investigate the phenomena
presented in this paper was that macromolecular inhibitors fail
to inhibit cathepsin K under certain experimental conditions at
pH 7.40. In the present study we show results obtained with
the slow-binding inhibitors of cathepsin K, cystatin stefin A and
N2TY. Figure 1(A) shows several examples of progress curves
of the hydrolysis of a fluorogenic substrate by cathepsin K in
the presence of a large excess of stefin A (20 nM inhibitor
and 0.2 nM enzyme) measured in buffers with increasing ionic
strength. The ionic strength had a large impact on the efficiency
of the inhibitor and similar progress curves were obtained with
N2TY. Figure 1(B) shows that both the residual enzyme activity
and the value of the apparent first-order rate constant for the
binding of inhibitor (λ) were affected. Only 50 % inhibition was
achieved at the lowest ionic strength (50 mM Hepes, pH 7.40,
I 19 mM), and over 90% inhibition, comparable with that
observed at acidic pH values, was achieved when the ionic strength
was more than 0.15 M and reached the maximum at I 0.3 M.
To investigate whether the different susceptibility towards
macromolecular inhibitors results from a conformational change,
intrinsic fluorescence spectra of cathepsin K were recorded.
Cathepsin K contains four tryptophan residues, three of which
are located in or near the active centre (Figure 2A). It is therefore
reasonable to assume that conformational changes of this part of
the molecule will be reflected in a change of intrinsic fluorescence.
Initially, near-UV emission spectra were recorded in two different
environments: a low-salt buffer (50 mM Hepes, pH 7.40), where
the effect of macromolecular inhibitors was minimal, and a highsalt buffer (50 mM Hepes, pH 7.40, containing 300 mM NaCl),
where maximal efficiency of inhibitors was achieved. The spectra
(Figure 2B) showed an approx. 20 % decrease in signal intensity
and a red-shift of 3 nm upon increasing the ionic strength.
Time-dependent measurements were performed to investigate
further the effect of ionic strength on the intrinsic fluorescence of
cathepsin K. These showed a slow decrease of fluorescence
emission intensity at 340 nm upon dilution of the enzyme
with a stock solution (1 mg/ml enzyme concentration, pH 5.00,
500 mM NaCl and 25 μg/ml DxS) into the reaction mixtures
containing increasing concentrations of NaCl (Figure 2C).
Concentrations of up to 300 mM showed increasing effects
on the decrease of fluorescence intensity, whereas increasing
further the concentration of NaCl had no additional effect. In these
experiments the reaction mixtures were supplemented with the
reversible inhibitor leupeptin. Even though its presence may have
contributed to the overall effect it was necessary to minimize the
probability of enzyme autodegradation during the measurements.
A possible interpretation of the results presented thus far
is the existence of multiple conformations of cathepsin K;
the slow decay of intrinsic fluorescence can be interpreted as
a slow transition between these conformational states. This
behaviour was originally described by Frieden [29] as a regulatory
mechanism in metabolic processes. A minimal mechanism
consisting of a population of enzyme molecules that can exist in
two conformational states is sufficient to describe the experiments
in the present study, even though the treatment of cathepsin K as
a fluctuating enzyme would probably be more appropriate at the
single-molecule level [30]. In analogy with other allosterically
regulated proteins we termed these states the T (tense) and R
(relaxed) states, which predominate at low and high ionic strength
respectively. At the same time this nomenclature reflects the
different susceptibility of the two states towards macromolecular
inhibitors. This hypothesis is supported by the plot of the values
of the apparent rate constant (k) for the exponential decay of
fluorescence as a function of ionic strength shown in Figure 2(D).
The hyperbolic profile describes the dependency of k upon ionic
strength and shows that k approaches zero at low ionic strength and
tends asymptotically to the value 0.015 s−1 for increasing ionic
strength. This means that a certain ionic strength is necessary
to trigger the change in intrinsic fluorescence associated with a
conformational change of the enzyme, whereas at lower ionic
strength the enzyme will remain in the initial conformation.
Activity measurements
To verify the hypothesis presented above, we sought evidence for
functional differences between the two putative conformations.
Experimentally this was achieved by measuring the activity of
cathepsin K in low-salt buffer following pre-incubation in either
low-salt buffer (enzyme assumed to be in the T state) or highsalt buffer (enzyme in R state). The use of this artificial system
with non-physiological ionic strengths was necessary to keep the
c The Authors Journal compilation c 2010 Biochemical Society
382
Figure 2
M. Novinec and others
Intrinsic fluorescence of cathepsin K
(A) Locations of four tryptophan residues in mature cathepsin K. The protein is shown in cartoon representation. Side chains of tryptophan residues and of the catalytic diad Cys25 –His162 are shown
as black sticks. (B) Intrinsic fluorescence spectra of 0.2 μM cathepsin K in 50 mM Hepes, pH 7.40, without NaCl (low salt) or with 300 mM NaCl (high salt). Samples were excited at 295 nm. First
derivatives of the primary spectra are shown in the inset. (C) Time-dependent change in fluorescence intensity upon dilution of cathepsin K into 50 mM Hepes, pH 7.40, containing the indicated
concentrations of NaCl and 10 μM leupeptin. (D) Plot of the values of the rate constant k for the exponential decay of intrinsic fluorescence as a function of ionic strength of the medium. A rectangular
−1
hyperbola fit gave k = 0.015 +
− 0.010 s as the asymptote of the function for increasing ionic strength. AU, arbitrary units.
enzyme predominantly in a single conformation; at physiological
ionic strengths equilibration of the enzyme between both states
would have made the kinetic analyses unfeasible.
Two different substrates (Z-FR-AMC and Z-VVR-AMC) were
used and the calculated kinetic parameters are shown in Table 1.
The hydrolysis of Z-VVR-AMC exhibited ‘classical’ Michaelis–
Menten kinetics at substrate concentrations up to 5-fold the K m .
The T state had 2-fold higher catalytic efficiency, but also 2fold higher K m than the R state. The behaviour with Z-FR-AMC,
the most frequently used endoproteolytic substrate for cysteine
cathepsins, was more complex. It did, however, provide additional
information about the conformational flexibility of cathepsin K
in that it showed that the conformational equilibrium is regulated
not only by ionic strength, but also by the substrate. The complete
reaction scheme to be considered in these experiments is shown
in Figure 3. The enzyme is in equilibrium between the T and
R states in both the free and substrate-bound forms. As an
additional off-path, substrate inhibition occurs at high substrate
concentrations (above K m ) that locks the enzyme in a nonfunctional conformation. This phenomenon has been observed
previously [31], but remained without interpretation.
To simplify the analysis, we investigated the mechanism in
parts, as indicated by boxes in the outline scheme in Figure 3 (upper panel); the progress curves are shown in Supplementary Figure
S1 (at http://www.BiochemJ.org/bj/429/bj4290379add.htm) and
the concentration dependencies are shown in Figures 3(A)–3(C).
The results shown in Figures 3(A) and 3(B) demonstrate that substrate inhibition occurs in a slow manner and affects the reaction
rates at zero time (vz ) and at steady-state (vs ). The model used
to describe this mechanism is shown in Supplementary Scheme
S1 (see http://www.BiochemJ.org/bj/429/bj4290379add.htm).
c The Authors Journal compilation c 2010 Biochemical Society
Table 1 Kinetic parameters for the hydrolysis of Z-VVR-AMC and Z-FR-AMC
by cathepsin K
Experiments were performed at 25 ◦C in 50 mM Hepes, pH 7.40, containing 1 mM EDTA and
2.5 mM DTT after pre-incubation of the enzyme in the same buffer without salt [enzyme (E) in
the T state] or with 300 mM NaCl (enzyme in the R state). Results are best fits (+
− S.E.M.) from
non-linear regression analysis using the Michaelis–Menten eqn (Z-VVR-AMC) or supplementary
eqn (3) (Z-FR-AMC).
Parameter
Z-VVR-AMC
E in T state
E in R state
Z-FR-AMC
E in T state
E in R state
K m (μM)
k cat (s−1 )
k cat /K m (M−1 ·s−1 )
7.7 +
− 1.3
3.2 +
− 0.5
0.37 +
− 0.02
0.19 +
− 0.01
4
(4.8 +
− 0.8) × 104
(5.9 +
0.9)
×
10
−
21 +
−4
5.1 +
− 1.1
91 +
− 12
39 +
−5
6
(4.3 +
− 1.0) × 106
(7.6 +
1.9)
×
10
−
Comparison between experiments started from the T and R
states shows that the former becomes affected at much higher
substrate concentrations, reflecting the higher K m value of
the T state in comparison with the R state. The plot in
Figure 3(C) shows that at low substrate concentrations a slow
re-equilibration of the enzyme occurs. This is characterized
by ‘concave-up’-shaped progress curves (Supplementary Figure
S1B) and is observed only when experiments are started
from enzyme in the T state. The model used to describe
this mechanism is shown in Supplementary Scheme S2 (see
http://www.BiochemJ.org/bj/429/bj4290379add.htm). Calculation of the kinetic parameters showed that k−3 k3 and k6 k−6 ,
meaning that free enzyme prefers the R state and substrate-bound
enzyme prefers the T state. The rate constant k6 corresponds to
Allosteric regulation of cathepsin K
Figure 3
383
Activity profiles of Z-FR-AMC hydrolysis
Activity profiles of Z-FR-AMC (Z-FR↓AMC) hydrolysis show competing effects of conformational change and substrate inhibition. The complete reaction scheme to be considered is shown in the
top panel. The mechanism was analysed in three parts, as indicated by the boxes and the conditions used for each experimental are given to the right of the mechanism. (A–C) Plots of reaction
rates at zero time (v z ) and at steady state (v s ) (left-hand panels) and plots of the values of the rate constant λ (right-hand panels). The calculated values of kinetic parameters corresponding to each
part of the mechanism are also shown. All reactions were measured in 50 mM Hepes, pH 7.40, containing 1 mM EDTA and 2.5 mM DTT after pre-incubation of the enzyme under low salt (enzyme
initially in the T state) or high-salt conditions (enzyme initially in the R state). The profiles in panels (A) and (B) are described by the model of slow substrate inhibition (Supplementary Scheme
S1 at http://www.BiochemJ.org/bj/429/bj4290379add.htm) and analysed with Supplementary eqns (2–4). The profiles in panel (C) are described with the model for conformational equilibrium
(Supplementary Scheme S2 at http://www.BiochemJ.org/bj/429/bj4290379add.htm) and analysed with Supplementary eqns (7–9).
rate constant k in Figure 2(D), which describes the change in
the intrinsic fluorescence of the enzyme that accompanies the
transition from the T to the R state in the presence of leupeptin,
hence in the bound state. The results in Figure 2(D) confirms that
under the conditions used in the experiments (low ionic strength)
the enzyme remains in the T state, meaning that k6 ≈ 0.
Effect of GAGs on the conformation of cathepsin K
The results presented above demonstrate that the conformational
flexibility of cathepsin K can be readily manipulated in vitro.
In biological systems, however, the activity of cathepsin K is
regulated by interactions with other biological macromolecules.
GAGs are known regulators of cathepsin K at acidic pH [32].
Therefore we have investigated whether these polysaccharides
also influence the conformational flexibility of cathepsin K at
physiological plasma pH. We have experimentally determined
the effects of CS, DS, HP and hyaluronan. Hyaluronan had no
effect, whereas all of the sulfated GAGs stimulated the activity of
cathepsin K.
Binding of CS, DS and HP to cathepsin K resulted in
a decrease of the intrinsic fluorescence intensity at 340 nm
(Supplementary Figure S2 at http://www.BiochemJ.org/bj/429/
bj4290379add.htm), similar to that observed for a conformational
c The Authors Journal compilation c 2010 Biochemical Society
384
Figure 4
M. Novinec and others
Effect of GAGs on the conformation of cathepsin K in vitro
(A) Effect of a saturating concentration of DS on the binding of a macromolecular inhibitor to
cathepsin K. Enzyme was first added to a reaction mixture containing inhibitor (20 nM stefin
A) and10 μM Z-FR-AMC as substrate (E + I). DS was added to the reaction mixture (0.2 mM
final concentration). (B) Effect of DS on the substrate inhibition of cathepsin K by Z-FR-AMC.
Enzyme (E) was added into a reaction mixture containing a high concentration of substrate
(100 μM). When the steady-state was reached, DS was added into the reaction mixture (0.2 mM
final concentration). Both experiments were performed in 50 mM Hepes, pH 7.40, containing
1 mM EDTA and 2.5 mM DTT at 25 ◦C with a final enzyme concentration of 0.2 nM. AU, arbitrary
units.
change of the enzyme (Figure 2). The rate of transition increased
in the order CS < DS < HP. Further experiments that provide
insight into the effect of GAGs on the conformation of cathepsin
K are shown in Figure 4. In the experiment shown in Figure 4(A),
cathepsin K was added to a mixture of substrate and the inhibitor
stefin A. Because the enzyme is in the T state under these
conditions, the inhibitor binds only weakly. Addition of GAG
into the reaction mixture then causes a change in the enzyme
conformation, resulting in rapid binding of the inhibitor. A similar
effect is shown in Figure 4(B) which shows a typical example of
slow substrate inhibition of cathepsin K. Addition of DS into
this mixture again appears to cause a conformational change,
which in this case relieves the enzyme from the effect of substrate
inhibition.
Activation of cathepsin K by GAGs
The conformational change upon binding of GAGs to cathepsin
K was reflected in its activity. CS and DS had a similar effect
on cathepsin K. Progress curves had ‘concave-up’ exponential
profiles (Supplementary Figure S3 at http://www.BiochemJ.
org/bj/429/bj4290379add.htm) consistent with a mechanism of
slow non-essential activation. To describe the mechanism of
CS/DS binding to cathepsin K it was sufficient to analyse
separately the situations at zero time and at steady-state. Specific
velocity plots constructed from steady-state reaction rates had
similar profiles of straight lines with a positive slope and a
c The Authors Journal compilation c 2010 Biochemical Society
trend of intercepting the ordinate axis with value 1 at abscissa
values near 1 (Figures 5A and 5B). By combining results from
the specific velocity plots with conventional plots of reaction
rates in the presence of increasing concentrations of CS/DS
(Figure 5C and 5D), we were able to determine the parameters
α, β and K A at steady-state and zero time. Their values show
that CS/DS act by increasing the affinity of the enzyme for the
substrate (α < 1) without major influence on the catalytic activity
(β ≈ 1) thus promoting an effect similar, but not identical, to
the conformational changes undergone by the enzyme alone in
response to low substrate concentration.
In addition to naturally occurring GAGs, cathepsin K was
also bound by DxS, a semi-synthetic highly sulfated
anhydroglucose polymer. Progress curves in the presence of
DxS were linear and plots of reaction rates against DxS
concentration at different substrate concentrations (Figure 5E)
show that DxS acts as an activator at low substrate
concentrations (below a half of the K m ) and as an inhibitor
at substrate concentrations above K m . This behaviour can be
described as hyperbolic mixed-type inhibition or non-essential
activation with a combination of parameters 0 < α < β < 1
in the general modifier mechanism (Supplementary Scheme
S3 at http://www.BiochemJ.org/bj/429/bj4290379add.htm). The
overall effect of DxS on the activity was small ( +
− 25 % at most).
Altogether this indicates that DxS binds at a different site than
CS/DS. Despite the fact that the interaction of cathepsin K with
DxS is not physiologically relevant, it is instrumental for analysing
and interpreting the effect of HP on the enzyme.
The interaction of cathepsin K with HP is more complex
than that of CS/DS or DxS. At low HP concentrations the
shapes of progress curves and the effect on enzyme activity were
analogous to that of CS/DS, whereas higher concentrations caused
a decrease in cathepsin K activity (Figure 5F). This behaviour
can be interpreted by a simultaneous interaction of HP with two
sites on cathepsin K, yielding a composite effect on enzyme
activity. This mechanism is shown in Supplementary Scheme
S7 (at http://www.BiochemJ.org/bj/429/bj4290379add.htm). It is
reasonable to assume that one binding site is the site bound by
CS/DS, as indicated by the slow activation of the enzyme at low
HP concentrations. The second binding site seems to be identical
to the DxS-binding site, as indicated by the similar effects of HP
and DxS on the stability of cathepsin K (see the Stabilization of
cathepsin K activity section).
Considering the effect of HP as a composite effect of those
observed with CS/DS and DxS, we used the values of coefficients
α and β determined for DxS and for CS/DS as constants
in Supplementary eqn (18) (at http://www.BiochemJ.org/
bj/429/bj4290379add.htm). Thereby eqn (18) could be fitted
to the experimental data at all substrate concentrations used
(Figure 5F). The fitted curves showed no significant systematic
deviation from the experimental points, indicating that the
mathematical model was indeed appropriate. The concentration
dependencies were biphasic and consisted of a hyperbolic
activation phase followed by a phase of declining activity. Both
the maximal vA /v0 ratio and the vA /v0 ratio at saturating HP
concentrations depended on substrate concentration. Moreover,
the latter fell under 1 at substrate concentrations above K m ,
i.e. the net effect of HP became inhibitory above a certain
substrate concentration. The calculated K A values were one
order of magnitude lower than those calculated for CS, DS
and DxS, showing that the effect of HP is much stronger
than that of CS, DS or DxS. Owing to the complexity of the
system and the heterogeneity of HP, experimental data were
relatively disperse and therefore we were unable to calculate
shared values for all parameters in Supplementary eqn (18). The
Allosteric regulation of cathepsin K
Figure 5
385
Activation of cathepsin K by GAGs
(A and B) Specific velocity plots constructed from steady-state reaction rates of Z-FR-AMC hydrolysis by cathepsin K in the presence of (A) CS and (B) DS. (C and D) Plots of reaction rates at
zero time (v z ) and steady-state (v s ) in the presence of increasing concentrations of (C) CS and (D) DS. The values of parameters K A , α and β were determined using Supplementary eqns (14) and
(17) (at http://www.BiochemJ.org/bj/429/bj4290379add.htm) and are given for CS and DS, respectively. (E) Activity profile of cathepsin K in the presence of various DxS and substrate (Z-FR-AMC)
concentrations. The values of parameters K A , α and β were calculated with Supplementary eqn (10) (at http://www.BiochemJ.org/bj/429/bj4290379add.htm) and are given on the right of the plot.
(F) Activity profile of cathepsin K in the presence of HP at various substrate (Z-FR-AMC) concentrations. The profile was constructed from steady-state reaction rates. The values of parameters K A ,
α and β were calculated with Supplementary eqn (18) (at http://www.BiochemJ.org/bj/429/bj4290379add.htm) and are given next to the plot. All experiments were performed at 25 ◦C in 50 mM
Hepes, pH 7.40, containing 1 mM EDTA and 2.5 mM DTT. σ = [S]/K m . All concentrations of GAGs are given as molar concentrations of disaccharide units.
consensus was, however, that the parameter c was greater
than 1, meaning that binding at one site hinders binding
at the other [33]. Furthermore, the general trend observed
in the experiments was unequivocal, confirming that the
proposed model is overall adequate for the description of the
system.
c The Authors Journal compilation c 2010 Biochemical Society
386
Figure 6
M. Novinec and others
Structural models of GAGs binding to cathepsin K
(A) Binding of CS/DS to cathepsin K (in surface representation, coloured according to the
electrostatic potential) illustrated on the example of a DS octasaccharide (consisting of four
4-sulfo-N -acetylgalactosamine-iduronic acid disaccharides; shown as sticks). The binding is
proposed to proceed via a slow conformational adaptation of the GAG to the structure of
cathepsin K. (B) A possible binding mode of HP at the second HP-binding site on the bottom
of cathepsin K. The putative binding site is composed of seven positively charged residues
(coloured in shades of blue). The models were constructed using Autodock 4. The surface
potential of cathepsin K was calculated with APBS software and the structures visualized with
PyMOL (DeLano Scientific; http://www.pymol.org).
Structural interpretation of GAG binding
Taken together, activity and intrinsic fluorescence measurements
demonstrate that GAGs act as allosteric regulators that alter the
conformation and activity of cathepsin K by binding at a site other
than the active centre. The location of this site has been revealed
by the crystal structure of a cathepsin K/CS complex [12].
Comparing the structure of CS in this complex with the calculated
structure of CS in solution [26] shows that a rearrangement of the
CS chain occurs upon binding to the enzyme. This indicates that
the slow activation of cathepsin K observed in our experiments
results from a slow conformational change of CS/DS, as illustrated
in Figure 6(A) for DS, which allows for the formation of a more
tightly bound complex between enzyme and GAG.
Binding kinetics show that HP binds cathepsin K at two
different sites. At very low concentrations, the effect of HP was
analogous to CS/DS. It is therefore logical to assume that under
these conditions HP binds to cathepsin K in a manner analogous to
CS/DS. The location of the second HP-binding site was predicted
to be on the bottom of the molecule and the energetically most
favourable docking pose is shown in Figure 6(B). The central part
of the second binding site is composed of six basic residues (Lys40 ,
Lys41 , Arg108 , Arg111 , Arg127 and Lys214 ) organized in a ring-shaped
structure on the bottom of cathepsin K. In addition to these, the
octasaccharide forms contacts with Lys10 , which is also involved
in binding of GAGs at the primary activation site.
A more detailed description and discussion of docking results
is available in the Supplementary material.
K as a function of incubation time at pH 7.40 and at
37 ◦C and the half lives of the enzyme in the presence
and absence of GAGs are shown in Supplementary Table
S1 (at http://www.BiochemJ.org/bj/429/bj4290379add.htm). Not
surprisingly, the activity of cathepsin K alone was relatively
unstable in aqueous solution, having a half-life of approx. 7 min.
Increasing the ionic strength of the incubation mixture to 300 mM
with NaCl decreased the half-life by 7-fold. The presence of CS
and DS slightly reduced the stability of cathepsin K (half lives of
5.5 and 6.0 min respectively), whereas HP had a strong stabilizing
effect and increased the enzyme’s half-life by more than 5-fold
(38 min). The same effect was achieved with DxS, indicating that
binding at the secondary HP-binding site is directly involved in
regulating cathepsin K stability.
The experimental results shown in Supplementary Table S1
used enzyme incubated in the absence of substrate to determine
the effect of GAGs in the absence of other substances binding
to the enzyme. In vivo, however, the concentration of proteins is
high and binding of substrate to the active centre can also stabilize
the enzyme. Indeed, a macromolecular substrate (5 mg/ml soluble
ETNA-elastin) substantially increased the half-life of cathepsin
K (33 compared with 7 min). Addition of HP to this reaction
mixture resulted in further stabilization of the enzyme (a halflife of 190 min), indicating synergy between substrate and HP in
protecting its activity. These pooled observations suggest that
stabilization of cathepsin K activity by HP and by substrate
may occur by protection from thermal denaturation and/or by
protection from autoproteolysis. As shown in Supplementary
Figure S4 (http://www.BiochemJ.org/bj/429/bj4290379add.htm),
the enzyme undergoes autodegradation by proteolysis at pH 7.40,
whereas addition of HP protects the enzyme from self-digestion.
Thus the major factor mediating the enhanced stability of
cathepsin K at neutral pH and 37 ◦C in presence of elastin, HP or
DxS is likely to be protection from autoproteolysis.
Collagenolytic and elastinolytic activity at neutral pH
All the experiments presented above relied on low-molecularmass synthetic substrates to report the activity of cathepsin K.
However, its activity on physiologically relevant macromolecular
substrates may differ from that observed with low-molecular-mass
substrates. Therefore we also examined the activity of cathepsin
K on collagen and elastin, two abundant extracellular structural
proteins, which have been long known as (patho)physiological
substrates of cathepsin K.
Collagenolytic assays (Figure 7A) showed that cathepsin K is
capable of digesting type I collagen on its own. Heparin increased
the collagenolytic activity of cathepsin K, whereas, interestingly,
CS/DS decreased the extent of collagen digestion. Collagenolysis
was also decreased in the presence of 300 mM NaCl, whereas
DxS had no effect.
Total elastinolytic activity was also measured with the
fluorescamine method (Figure 7B). The activity of cathepsin K
at pH 7.40 was 60 % of that observed at pH 6.20, indicating that
cathepsin K retains most of its potent elastinolytic activity at this
pH. Increasing the salt concentration again reduced the activity of
cathepsin K and all GAGs acted as potent activators in this assay.
CS/DS increased the elastinolytic activity by 4-fold, whereas HP
stimulated the activity by as much as 12-fold.
Stabilization of cathepsin K activity
DISCUSSION
Apart from directly affecting the catalytic properties of an
enzyme, effector molecules can also regulate its stability
by other means. We measured the activity of cathepsin
The results of the present study show novel aspects of the
mechanism of cathepsin K activity and regulation. Despite
focusing on only one member of cysteine cathepsins, the results
c The Authors Journal compilation c 2010 Biochemical Society
Allosteric regulation of cathepsin K
Figure 7 Collagenolytic and elastinolytic activities of cathepsin K in the
presence of GAGs
(A) Calf-skin collagen (0.5 mg/ml in 50 mM Hepes, pH 7.40, containing 1 mM EDTA and 2.5 mM
DTT) was incubated with 0.5 μM cathepsin K (CatK) in the presence of different modifiers for
16 h at 25 ◦C. Samples were then separated on an 8 % polyacrylamide gel and stained with
Coomassie Brilliant Blue. Positions of calibrating proteins are given in kDa on the left-hand
side. Ctrl, control (undigested collagen). (B) Suspensions of insoluble bovine neck ligament
elastin (5 mg/ml in 50 mM Hepes, pH 7.40, containing 1 mM EDTA and 2.5 mM DTT) were
incubated with 0.1 μM cathepsin K in the presence of different modifiers/conditions for 2 h at
37 ◦C. Reactions were stopped by addition of 5 % (w/v) trichloroacetic acid, soluble peptides
reacted with fluorescamine and fluorescence of the samples measured at λex 390 nm and λem
480 nm. Peptide concentrations were determined from a standard curve of known concentrations
of L-alanine.
may be exploited for investigating similar mechanisms shared
with other enzymes of this group. Our results imply that
the structure of cathepsin K is flexible and converts between
multiple conformational states with distinctive characteristics at
physiological plasma pH. The conformational transition is a slow
process that can be manipulated in vitro. Our experimental set-up
was aimed at ‘trapping’ the enzyme in a single conformation and
a mathematical model of two discrete conformational states was
sufficient to describe the experiments. In vivo the enzyme probably
fluctuates between multiple conformational states and the T and
R states that we observed in vitro possibly represent two extreme
situations. A plausible interpretation is that in biological systems
the conformational flexibility serves the purpose of adapting the
shape of the active site to bring bulky macromolecular substrates
into position for cleavage, meaning that cathepsin K operates via
a mechanism reminiscent of the ‘induced fit’ model [34]. Indeed,
the active site can accommodate a wide variety of structurally
diverse substrates [35] and fluctuations of this region were also
observed in molecular dynamics simulations, which are presented
and discussed in Supplementary material.
387
Substrate inhibition is a phenomenon that we have
observed several times when using dipeptide coumarin-based
substrates to measure the activity of cysteine cathepsins. How
exactly inhibition by substrate occurs, remains uncertain. As
shown in Supplementary Figure S5 (http://www.BiochemJ.
org/bj/429/bj4290379add.htm), a substantial part of the active
site remains unoccupied when a substrate molecule is bound
in a productive manner. A second substrate molecule could
bind in this area and hinder the turnover of productively
bound substrate. This observation is in agreement with the
mathematical model of substrate inhibition, which postulates
that the second (inhibitory) substrate molecule binds to the
enzyme–substrate complex and not to enzyme alone. The presteady state phase of the progress curves might represent a
conformational adaptation of the enzyme to the bound substrate
molecule(s). This is indicated by the reversal of substrate inhibition by modifiers that affect the conformation of the enzyme
(Figure 4). Why this effect is only seen with the the substrate
Z-FR-AMC, but not with the similar Z-VVR-AMC, can be
explained by the slow turnover of the latter. As estimated from our
experiments, the turnover of Z-FR-AMC is reduced to approx.
1 s−1 at maximal inhibition. This is, however, still several-fold
higher than the kcat values measured for Z-VVR-AMC, meaning
that if a second Z-VVR-AMC molecule were to bind the enzyme
in a manner similar to Z-FR-AMC, it would not limit the turnover
of the productively bound molecule thus no substrate inhibition
would be detected experimentally.
The concept of ‘trapping’ the enzyme in a certain conformation
appears to be utilized by GAGs, natural allosteric modifiers of
cathepsin K. The results of the present study show that GAGs
increase the activity of the enzyme and promote a conformational
change (Figures 4 and 5). The crystal structure of cathepsin K
in complex with CS shows no obvious differences from that of
cathepsin K alone [12]. A plausible explanation is that the crystal
structures show cathepsin K already in the R state and therefore
no further conformational change is caused by CS. Altogether
this indicates that GAGs act by affecting the distribution of a preexisting equilibrium of conformational states. This behaviour has
been observed in other allosterically regulated systems [36].
Cathepsin K has thus far been attributed several physiological
and pathological functions. Whereas bone resorption occurs in
an acidic environment deemed to be optimal for enzyme activity,
other functions of cathepsin K occur in an environment where the
pH is close to the physiological plasma value. The results from
the present study show that despite the fact the enzyme is relatively
susceptible to autoproteolysis at pH 7.40 it still shows substantial
activity against macromolecular substrates under these conditions.
Cathepsin K cleaves most extracellular matrix components,
including the collagen triple helix [37]. It has been reported that
the collagenolytic activity of cathepsin K at acidic pH depends on
complex formation with CS [11]. Since then there have been other
reports about diverse effects of different GAGs on this process
[32,38]. Our results show that at pH 7.40 the enzyme degrades
collagen on its own and that CS/DS reduces its collagenolytic
activity, whereas HP enhances it. Altogether this shows that the
molecular mechanism behind the unique collagenolytic activity
of cathepsin K depends on the environment. In contrast with
the diverse effects on collagen digestion, all GAGs increased
the elastinolytic activity of cathepsin K. Overall activity of
GAGs in our experiments increased in the order CS < DS < HP.
The same trend has been observed in many GAG-dependent
processes, e.g. the inhibition of lysosomal enzymes at low pH
(HP > CS > DS > hyaluronan) [15,16]. The effect of CS and DS
on cathepsin K is virtually identical, although DS binds more
rapidly and tightly than CS; the major difference between them
c The Authors Journal compilation c 2010 Biochemical Society
388
M. Novinec and others
is that DS contains iduronic acid instead of glucuronic acid and
is therefore more flexible due to fewer intramolecular hydrogen
bonds [39].
Despite being structurally similar to CS and DS, HP exerts
different effects on cathepsin K in terms of activity and stability.
A similar behaviour has been observed with two other related
peptidases, papain and cathepsin B, which interact with HP and
HS, but not with CS and DS [13,14]. At the molecular level, the
unique effect of HP on cathepsin K was attributed to the presence
of two binding sites for HP. Kinetic measurements have shown that
both sites can be bound simultaneously (Figure 4B); however, due
to the spatial proximity of the proposed binding sites (Figure 6) it
remains ambiguous whether each site is bound by a separate HP
chain or if one HP molecule simultaneously binds at both sites.
The selectivity of the second site for HP (and the synthetic DxS)
over CS or DS can be explained by its high density of positively
charged residues, which interact favourably with HP, but not with
the less densely charged CS/DS.
The strong effect of HP on both the activity and stability of
cathepsin K suggests that it may also be an important factor
in cathepsin K regulation in vivo. Endogenous HP is produced
exclusively in mast cells as part of the proteoglycan serglycin
[40], and free HP is widely used as an anticoagulant drug. It
has been shown that prolonged use of HP can cause osteoporosis
[41]. Given cathepsin K is one of the major factors contributing
to osteoporosis [10] it seems a plausible target for orally
administered HP. Owing to its restricted pattern of production,
the action of endogenous HP on cathepsin K is probably limited
in vivo. However, the effect of HP can be extended to HS, which is
an abundant component of the extracellular matrix (for a review on
HS see [42]). HS molecules are highly heterogeneous. However, at
least a fraction of them can be expected to exhibit a HP-like effect
on cathepsin K, as has been the case with papain and cathepsin B
[13,14].
Conformational flexibility and allosteric regulation are novel
concepts in the regulation of cysteine cathepsins but are by
no means surprising. In fact, there is growing evidence that
most, if not all, globular proteins possess a certain degree of
conformational flexibility [43]. Several findings, such as the DNAinduced conformational changes in the closely related cathepsin
V [44] or binding of CS at a site distinct from the active site
in cathepsin K [12], have already indicated such mechanisms of
regulation, even though it was not interpreted as such. Targeting
the allosteric sites of enzymes is becoming increasingly popular
as a strategy in drug development [36,43,45]. Even though activesite-directed cathepsin K inhibitors gave some promising results
in clinical trials, the possibility of allosteric regulation opens novel
perspectives for the design of drugs aimed at regulating the activity
of this enzyme.
AUTHOR CONTRIBUTION
Antonio Baici designed the project. Marko Novinec designed, performed and interpreted
most of the experiments under supervision of Antonio Baici and Brigita Lenarčič. Lidija
Kovačič performed and interpreted the cross-linking experiments. Marko Novinec wrote
the paper with editorial supervision from Antonio Baici.
FUNDING
This work was supported by the Swiss National Science Foundation [grant number 31113345/1]; the Slovenian Research Agency; and the Olga Mayenfisch Foundation.
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Received 5 March 2010/28 April 2010; accepted 7 May 2010
Published as BJ Immediate Publication 7 May 2010, doi:10.1042/BJ20100337
c The Authors Journal compilation c 2010 Biochemical Society
Biochem. J. (2010) 429, 379–389 (Printed in Great Britain)
doi:10.1042/BJ20100337
SUPPLEMENTARY ONLINE DATA
Conformational flexibility and allosteric regulation of cathepsin K
Marko NOVINEC*†, Lidija KOVAČIȇ, Brigita LENARČIȆ§ and Antonio BAICI*1
*Department of Biochemistry, University of Zürich, 8057 Zürich, Switzerland, †Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of
Ljubljana, 1000 Ljubljana, Slovenia, ‡Department of Molecular and Biomedical Sciences, Jožef Stefan Institute, 1000 Ljubljana Slovenia, and §Department of Biochemistry and
Molecular and Structural Biology, Jožef Stefan Institute, 1000 Ljubljana, Slovenia
complexes, respectively, and are defined as:
THEORETICAL BACKGROUND
Slow substrate inhibition
The slow inhibition of enzyme (E) by substrate (S) was analysed
by the mechanism in Scheme S1.
K si =
k−3
k3
K si∗ = K si
(5)
k−4
k−4 + k4
(6)
Conformational equilibrium of cathepsin K
The equilibrium of enzyme between two conformational states
in the presence of substrate was analysed by the mechanism in
Scheme S2.
Scheme S1
This describes the ‘classical’ example of substrate inhibition,
which is analogous to uncompetitive inhibition [1], except that
binding of the second substrate molecule occurs in two steps: an
initial rapid binding step yields the inhibited ES2 complex which
then slowly rearranges to the more tightly bound ES2 ∗ complex.
The progress curves have typical exponential profiles that can be
described by eqn (1):
vz − vs
(1 − e−λt )
[P] = vs t +
λ
(1)
where vz and vs are reaction rates at zero time and at steadystate and λ is an apparent first-order rate constant. Individual
expressions for vz , vs and λ are given by eqns (2–4):
vz =
V [S]
[S]
K m + [S] 1 +
K si
vs =
V [S]
[S]
K m + [S] 1 + ∗
K si
λ = k−4 +
K si
k4 [S]
Km
+ [S]
1+
[S]
(2)
This mechanism takes into account two enzyme species ET
and ER (‘tense’ and ‘relaxed’) that slowly interconvert both in
the unbound and substrate-bound states. The progress curves are
again described by eqn (1). The apparent first-order rate constant
λ for this mechanism is defined as:
λ=
K mR
KT
k3 m + k6
+ k−6
[S]
[S]
+
K mR
KT
1+
1+ m
[S]
[S]
k−3
(7)
where
(3)
K mT =
k−1 + k2
k1
and
(4)
where V is the limiting rate and K m is the Michaelis constant. K si
and Ksi∗ are equilibrium dissociation constants of the ES2 and ES2 ∗
1
Scheme S2
K mR =
k−4 + k5
k4
are Michaelis constants for the T and R state, respectively.
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2010 Biochemical Society
M. Novinec and others
The plot of v0 /vA against σ /(1 + σ ) always produces straight lines
with intersection points at v0 /vA = 1, regardless of the interaction
mechanism. The values of parameters α, β and K A are determined
by replotting the extrapolated values of straight lines at σ /(1 + σ )
= 0 (a) and σ /(1 + σ ) = 1 (b) against 1/[A] in the form:
The steady-state reaction rate is given by eqn (8):
vs =
VT
K mT
+ k6 K
K mR
[S]
+
1+
1
+
KR
[S]
[S]
k−3 m + k−6
[S]
T
m
+
k3
α KA 1
α
a
=
+
a−1
α − β [A] α − β
(12)
R
V
K mR
+ k−6 k−3
K mT
KR
[S]
1
+
1+ m +
KT
[S]
[S]
k3 m + k6
[S]
(8)
b
α KA 1
1
=
+
b−1
1 − β [A] 1 − β
where V T = k2 [E]t and V R = k5 [E]t are limiting rates of the T
and R states respectively. If we assume that ET and ER are in
equilibrium in the absence of substrate, the reaction rate at zero
time can be described by eqn (9):
[S]
[S]
VR R
T
Km
Km
vz =
+
k3
k−3
1+
1+
k−3
k3
and
VT
(13)
Activation of cathepsin K by CS (chondroitin sulfate) and DS
(dermatan sulfate)
CS and DS act as slow non-essential activators of cathepsin K.
The minimal mechanism that describes this kind of interaction is
shown in Scheme S4.
(9)
General modifier mechanism and the specific velocity plot
The general modifier mechanism [2] describes the interaction of
modifier (A) with enzyme (E) according to Scheme S3.
Scheme S4
Scheme S3
For the mechanism in Scheme S3 the reaction rate in the presence
of modifier, vA , is defined as:
[A]
v0 (1 + σ ) 1 + β
α KA
(10)
vA =
[A]
[A]
1+
+σ 1+
KA
α KA
where v0 is the reaction rate in the absence of modifier, K A is
the equilibrium dissociation constant of the EA complex, α and
β are dimensionless coefficients and σ = [S]/K m . The equation
was derived under the assumptions of quasi-equilibrium for the
binding of A to E and ES, and steady-state for the fluxes around
ES and ESA.
The specific velocity plot [3] is a handy graphical method for
plotting kinetic results and determining interaction parameters.
For this purpose eqn (10) is rewritten as:
1
1
[A]
−
[A]
1+
σ
v0
α KA
KA
KA
+
=
(11)
[A]
[A]
vA
1+σ
1+β
1+β
α KA
α KA
c The Authors Journal compilation c 2010 Biochemical Society
Scheme S4, which takes into account the binding of modifier A
to enzyme E in two steps, with both steps affecting the catalytic
properties of the enzyme and its affinity for substrate S. The
first step occurs rapidly, whereas the second involves a slow
isomerization. As discussed by Szedlacsek and Duggleby [4], an
analytical expression for such a system is difficult to obtain and
would be of little practical use due to a large number of variables.
Given we are primarily interested in studying the net effect
of GAGs on cathepsin K, we have simplified the mechanism in
Scheme S4 by separately analysing the effect of GAGs at zero
time and at steady-state. If we only consider a short interval at
the beginning of the reaction (t = 0) when no EA∗ complex is
yet formed, and we assume that all components present are in
quasi-equilibrium, the mechanism can be described by the general
modifier mechanism [2], as shown in Scheme S5.
Scheme S5
Allosteric regulation of cathepsin K
Figure S1
Progress curves of Z-FR-AMC hydrolysis after cathepsin K pre-incubation in (A) high-salt buffer or (B) low-salt buffer
All measurements were performed in low-salt buffer (50 mM Hepes, pH 7.40, containing 1 mM EDTA and 2.5 mM DTT) at 25 ◦C at a final enzyme concentration of 0.1 nM. Prior to the experiments
enzyme was kept on ice in (B) low-salt buffer or (A) high-salt buffer (50 mM Hepes, pH 7.40, containing 300 mM NaCl, 1 mM EDTA and 2.5 mM DTT) at concentration of 0.2 μM AU, arbitrary units.
Figure S2
Effect of GAGs on the intrinsic fluorescence of cathepsin K
Change in intrinsic tryptophan fluorescence at 340 nm upon binding of (A) CS, (B) DS or (C) HP. The time point of GAG addition is shown by arrows and the final concentration of the three GAGs
was 0.2 mM. All experiments were performed in 50 mM Hepes, pH 7.40, containing 1 mM EDTA and 2.5 mM DTT at 25 ◦C and at a protein concentration of 0.1 μM. To minimize enzyme activity loss
by autoproteolysis, all samples were supplemented with 1 μM E-64 prior to the experiments. AU, arbitrary units.
The validity of these assumptions has been discussed by
Topham and Brocklehurst [5]. The reaction rate for Scheme S5 is
described by eqn (14):
[A]
v0 (1 + σ ) 1 + βz
αz K A,z
vz =
[A]
[A]
1+
+σ 1+
K A,z
αz K A,z
(14)
where vz and v0 are the reaction rates in the presence and absence
of modifier A, K A,z is the equilibrium dissociation constant of the
EA complex, α z and β z are dimensionless coefficients and σ =
[S]/K m ; the subscript z consistently indicates that the variables
refer to zero time.
When the system has reached steady-state the whole mechanism shown in Scheme S4 is necessary to accurately describe all
species present. To simplify the mathematical treatment Scheme
S4 can be rewritten by combining the two steps involving the
c The Authors Journal compilation c 2010 Biochemical Society
M. Novinec and others
Figure S3
Progress curves of Z-FR-AMC hydrolysis in the presence of increasing concentrations of (A) chondroitin sulfate, (B) dermatan sulfate or (C) HP
All measurements were performed in 50 mM Hepes buffer, pH 7.40, containing 1 mM EDTA and 2.5 mM DTT at 25 ◦C at a final enzyme concentration of 0.1 nM. AU, arbitrary units.
formation and isomerization of the EA complex into one step and
assuming that all species are at quasi-equilibrium as shown in
Scheme S6.
Figure S4
of HP
Scheme S6
In Scheme S6 species EA and EA S are defined as:
EA = [EA] + EA∗
(15)
and
Autodegradation of cathepsin K in the absence and presence
Samples of cathepsin K (CatK; 3 μM final concentration) were incubated at 37 ◦C in 50 mM
Hepes, pH 7.40, containing 1 mM EDTA and 2.5 mM DTT for 15 min, 30 min or 60 min in the
absence or presence of HP (0.2 mM final concentration). Reactions were stopped by addition of
5 μM E-64 and analysed by SDS/PAGE (17 % gel). Protein bands were stained with Coomassie
Brilliant Blue. Positions of calibrating proteins (in kDa) are given on the left-hand side.
α s and β s . The reaction rate for Scheme S6 is given by:
EA S = [EAS] + EA∗ S
(16)
K A,s in this case is not a true equilibrium constant, but a composite
parameter that includes information about both steps in the
mechanism (EA and EA∗ ). The same is true for the coefficients
c The Authors Journal compilation c 2010 Biochemical Society
[A]
v0 (1 + σ ) 1 + βs
αs K A,s
.
vs =
[A]
[A]
1+
+σ 1+
K A,s
αs K A,s
(17)
Allosteric regulation of cathepsin K
Interaction of cathepsin K with HP (heparin)
Heparin binds cathepsin K at two sites. One binding mode is
identical to that observed with CS/DS and the effect of slow
binding is still observed in progress curves recorded at very low
HP concentrations (Figure S3C). However, as the steady-state
treatment would result in complex expressions beyond practical
use, we analyse only the steady-state parts of the curves where
we assume quasi-equilibrium conditions, as discussed above for
CS/DS. The secondary site is bound in a rapid manner and is not
exclusive with respect to the primary binding site. The overall
binding of HP was analysed with a modified version of the model
describing simultaneous binding of two modifiers to one enzyme,
adapted from the equation described by Schenker and Baici [6],
as shown in Scheme S7:
Figure S5 Computer model of Z-FR-AMC bound into the active site of
cathepsin K
The substrate is shown as sticks. The enzyme is shown in surface representation and the catalytic
residues Cys25 and His162 are coloured green and blue respectively.
Scheme S7
A and A represent two molecules of HP that bind to a single
molecule of enzyme E. The reaction rate for the system in Scheme
S7 is given by eqn (18):
vA =
[A]
[A]
[A]2
v0 (1 + σ ) 1 + β1
+ β2
+ β12
α1 K A,1
α2 K A,2
eK A,1 K A,2
[A]
[A]
[A]2
[A]
[A]
[A]2
1+
+
+
+σ 1+
+
+
K A,1
K A,2
cK A,1 K A,2
α1 K A,1
α2 K A,2
eK A,1 K A,2
(18)
where α 1 , β 1 and K A,1 describe the binding of the first molecule
of A, α 2 , β 2 and K A,2 describe binding of the second molecule,
β 12 describes the catalytic properties of the enzyme when both
molecules of A are bound, the coefficient c defines the interaction
between both molecules of A and e is a combined interaction
constant for the formation of the quaternary complex [6].
EXPERIMENTAL
Docking and molecular dynamics simulations
Molecular dynamics simulations were performed using cathepsin
K alone or in complex with the substrate Ala-Gly-Leu-GluGly-Gly-Asp-Ala (the cleavage site is after the first glutamate
residue). The octapeptide was constructed with PyMOL (DeLano
Scientific; http://www.pymol.org) and then docked into the active
centre of the enzyme (PDB code 1ATK) using AutoDock 4 [7]
with the Lamarckian Genetic docking algorithm. In the docking
calculation only the ligand (substrate) was defined as flexible,
whereas the receptor (enzyme) was treated as rigid, i.e. no flexible
residues were defined. The complex was solvated with the Solvate
plugin in the VMD program [8]. A molecular dynamics simulation
was then performed with the NAMD program [9] at a constant
temperature of 310 K, with periodic boundary conditions and
CHARMM 27 force-field parameters [10]. An initial 1000-step
energy minimization was included in the calculation to remove
bad contacts in the initial model. To repeat the simulation with
cathepsin K alone, its co-ordinates were extracted from the
minimum energy conformation observed in this simulation,
the molecule re-solvated and molecular dynamics re-run using
the same parameters. The same procedure was also performed
starting from the crystal structure of cathepsin K alone (PDB code
1ATK). All simulations were performed for 2 ns. Energies of the
whole system, as well as of the protein alone, were calculated
throughout the simulations to ensure that a stable conformation
of the protein had been reached.
The Z-FR-AMC molecule was constructed with the
CambridgeSoft ChemOffice 11 Suite. It was then docked into
the active site of cathepsin K using AutoDock 4 [7] with the
Lamarckian Genetic docking algorithm. All images were created
with PyMOL.
Interaction site mapping by chemical cross-linking
Pro-cathepsin K was first reacted with the cross-linking reagent
sulfo-SBED at room temperature (25 ◦C) for 1 h in the dark at a
molar ratio of 1:3. The reaction mixture was then extensively
dialysed to remove the unreacted cross-linker and sulfoSBED {sulfo-N-hydroxysuccinimidyl-2-[6-(biotinamido)-2-(pazido benzamido)-hexanoamido] ethyl-1,3 -dithioproprionate}–
pro-cathepsin K was then aliquoted and stored at −80 ◦C.
Pro-cathepsin K–HP cross-linking was performed by
incubating the protein (final concentration 2 μM) with HP
(0.8 mg/ml) in 50 mM Hepes, pH 7.4, containing 150 mM NaCl
in the dark for 30 min and then irradiating the sample with
a UV lamp. The sample was digested with LysC over night
at 37 ◦C and then applied to a CIM-QA monolithic column
(BIA Separations). HP chains were eluted from the disk in a
linear 0.5–3 M NaCl gradient and unreacted HP was removed by
c The Authors Journal compilation c 2010 Biochemical Society
M. Novinec and others
Table S1
Half-life of cathepsin K at 37 ◦C in 50 mM Hepes (pH 7.40)
The enzyme (E; 0.2 μM) was incubated in the presence or absence of different effectors (300 mM
NaCl, saturating concentrations of GAGs or DxS and/or 5 mg/ml soluble ETNA-elastin) in
50 mM Hepes, pH 7.40, containing 1 mM EDTA and 2.5 mM DTT with shaking on an Eppendorf
Thermomixer Compact. Half-lives were determined by measuring residual enzyme activity at
regular time intervals using the substrate Z-FR-AMC. Results are best fit values (+
− S. E. M.) from
non-linear regression analysis using a first-order decay function to calculate a decay constant
k , from which half-life = In2/k .
Conditions
Half-life (min)
E only
E plus NaCl (300 mM)
E plus CS
E plus DS
E plus HP
E plus DxS
E plus elastin
E plus elastin and HP
7.0 +
− 1.0
1.0 +
− 0.3
5.5 +
− 0.5
6.0 +
− 0.5
37.9 +
− 1.1
37.2 +
− 1.4
33.0 +
− 2.0
190 +
− 20
avidin-affinity chromatography. HP-linked peptides were then
released by treatment with DTT and separated by HPLC on a C18 column in a linear 0–100 % acetonitrile gradient. All peptides
were identified by N-terminal sequencing.
Docking of GAG octasaccharides to cathepsin K
A hexasaccharide model of CS (chondroitin-4-sulfate; consisting of three 4-sulfo-N-acetylgalactosamine-glucuronic acid
disaccharides) and octasaccharide models of DS (consisting of
Figure S6
four 4-sulfo-N-acetylgalactosamine-iduronic acid disaccharides)
were constructed with the CambridgeSoft ChemOffice 11 Suite.
Torsion angles between monosaccharides were adjusted manually
according to published data [11] and the structures then optimized
by energy minimization using the MMFF94 force-field. HP
models were retrieved from the PDB code 1HPN. Co-ordinates
for cathepsin K were extracted from the structure of the cathepsin
K–CS complex (PDB code 3C9E).
All docking calculations were performed with AutoDock 4
[7] using the Lamarckian Genetic docking algorithm. In all
calculations 50 runs were performed with populations of 300
individuals run for 3000 generations. Docking of DS and HP
octasaccharides to the CS-binding site identified in the crystal
structure of the cathepsin K–CS complex [12] was performed with
flexible side chains of receptor residues Lys9 , Lys10 , Lys147 , Lys173
and Lys191 . Two separate calculations were performed for each
ligand, one using only rigid glycosidic bonds in the ligand and
the other using flexible glycosidic bonds 4 and 7 in each ligand.
In the first set, the lowest energy solution was selected from those
that contained the DS chain running in the same direction as in
the crystal structure. In the second set, the primary criterion for
model selection was a conformation equivalent to that seen in the
crystal structure of the cathepsin K–CS complex. Within these, the
solution with the lowest binding energy was selected. Docking of
HP octasaccharides to the second binding site as was performed
with a rigid receptor molecule and rigid glycosidic bonds in the
ligand. The selection criteria are described and discussed in the
Results and Discussion below.
Surface potentials were calculated with the Adaptive Poisson–
Boltzman Solver [13]. All images were created with PyMOL.
Conformational flexibility of cathepsin K in molecular dynamics simulations
(A) Minimum energy conformations of cathepsin K in complex with the octapeptide substrate (AGLEGGDA) (left-hand panel) and substrate-free cathepsin K (right-hand panel). The enzyme is shown
as a grey surface and the substrate is shown as sticks. (B) Superposition of the structures in (A). The substrate-bound conformation is shown in blue and the free conformation in orange. Residues
involved in catalysis and flexible residues lining the active site are shown as sticks and are labelled.
c The Authors Journal compilation c 2010 Biochemical Society
Allosteric regulation of cathepsin K
Figure S7
Conformational change of CS/DS upon binding to cathepsin K
Figure S8
Structural models of HP binding to cathepsin K
(A) Difference between the conformations of free CS in solution and CS in complex with
cathepsin K. (B) Binding of CS/DS to cathepsin K (in surface representation) illustrated
with a DS octasaccharide as an example (shown as sticks). The binding proceeds via a slow
conformational adaptation of the GAG to the structure of cathepsin K. (C) All solutions obtained
with Autodock that conform to experimental data. The models were constructed using Autodock
4. The surface potential of cathepsin K was calculated with APBS software and the structures
visualized with PyMOL.
(A) Positions of Lys10 , Lys39 and Lys77 (shown in shades of blue) that were cross-linked with
HP. (B) Model of a HP octasaccharide binding to the primary binding site on cathepsin K in
a manner analogous to CS/DS. (C) Three possible binding modes of HP at the secondary
HP-binding site on the bottom of cathepsin K. The putative binding site is composed of seven
positively charged residues (coloured in shades of blue). In all figures the enzyme is shown in
surface representation and HP is shown as sticks. In (B) the enzyme is coloured according to
the electrostatic potential calculated with APBS. All structures were visualized with PyMOL.
RESULTS AND DISCUSSION
and a second molecular dynamics simulation was performed
starting from this conformation of enzyme to mimic the conditions
after product release from the active site. Within 200 ps the
active centre of the free enzyme ‘opened’ to become wide and
shallow (Figure S6A, right-hand panel) and remained in this
conformation for the remainder of the simulation (2 ns). The
same conformation was also reached in a separate molecular
dynamics simulation of enzyme alone started directly from the
crystal structure. Altogether the simulations suggest that cathepsin
K adapts its conformation upon substrate ‘binding and release’
in a manner consistent with the ‘induced fit’ hypothesis [15]. In
the absence of substrate the enzyme adopts a conformation in
which the active centre is easily accessible. Upon binding of a
substrate molecule the enzyme adapts its conformation to bring
the substrate into the proper position for the catalytic step.
The major structural differences between the two conformations in the active centre region are illustrated in the superposition
in Figure S6B (the ‘free’ enzyme is shown in orange and substratebound enzyme in blue). The most notable change involves the
loop Gln19 –Cys22 that lines the left side of the active centre. In
the presence of substrate the loop moves over the active centre
Conformational flexibility studied by molecular dynamics
The experiments presented in Figure 3 of the main paper indicate
that cathepsin K can undergo a conformational change upon
binding of substrate. We further investigated this hypothesis in
silico by molecular dynamics simulations of free enzyme and
enzyme in complex with an octapeptide substrate (Ala-Gly-LeuGlu-Gly-Gly-Asp-Ala). This sequence was chosen based on the
specificity matrix of cathepsin K as retrieved from the MEROPS
database [14]. It represents an ‘ideal’ substrate, composed of
residues most readily accepted by the enzyme in positions P3
through P3 (Gly-Leu-Glu-Gly-Gly-Asp) flanked by additional
alanine residues at both termini to extensively cover the entire
active centre of the enzyme.
In the molecular dynamics simulation of the enzyme–substrate
complex the active centre adapted to the shape of the substrate
in the time frame within 1 ns by becoming narrower and deeper
(Figure S6A, left-hand panel) and remained in that conformation
for the rest of the simulation (2 ns). The substrate molecule was
then removed from the final conformational state of the complex
c The Authors Journal compilation c 2010 Biochemical Society
M. Novinec and others
and encloses the substrate within, whereas in absence of the latter
the loop swings in the opposite direction and widely exposes the
active centre. Binding of substrate also causes a conformational
change in the loop containing Gly65 and Gly64 that interact with
the P3 and P4 positions of the substrate and is directly connected
to Gln21 via the disulfide bond Cys22 –Cys63 . Within the active
centre the positions of the catalytic diad Cys25 –His162 do not differ
substantially between both conformations. It is, however, worth
noticing that in the unbound enzyme the side chain of Cys25
faces away from the position assumed during catalysis and only
adopts this position in the presence of substrate, which is a further
characteristic of the ‘induced fit’ model. Of the residues involved
in the catalytic mechanism, the most notable conformational
change involves rotation of Trp184 , a residue critical for the proper
positioning of the catalytic His162 [16]. In the absence of substrate
its indole ring freely rotates by approx. 30◦, but mostly remains in
the plane of the catalytic diad. Upon substrate binding the indole
ring swings out of the plane and enables residues in positions P2 and P3 to bind more tightly into the active centre. The movement
of Trp184 also causes a rearrangement of several residues lining the
right side of the active site, including Asn187 , Gln143 and Phe144 .
Comparing these simulations with experimental results
presented in the main paper, it is feasible to say that the substratebound and free enzyme states show structural differences that
would be expected to exist between the T and R states
respectively. The narrower active-site groove of the substratebound conformation parallels the lower substrate affinity of the T
state, whereas proper positioning of the catalytic Cys25 explains
the higher catalytic constant. Moreover, the shape of the active
site in the substrate-bound conformation is too narrow to allow
efficient binding of a relatively bulky macromolecular inhibitor.
Structural interpretation of GAG binding
Activity assays (Figures 4 and 5 of the main paper) and intrinsic
fluorescence measurements (Figure S2) demonstrate that GAGs
act as allosteric regulators that alter the conformation and activity
of cathepsin K by binding at a site other than the active centre.
The location of this site has been revealed recently by the crystal
structure of a cathepsin K–CS complex [12]. Comparing the
structure of CS in this complex with the calculated structure of
CS in solution [11] (Figure S7A) shows that rotations of multiple
glycosidic linkages are needed to commit the GAG chain to the
conformation seen in the crystal structure. A local change of GAG
conformation upon binding to a receptor is not unusual and can
be considered as a specific recognition motif for target protein
binding [17]. On the basis of this result, the slow activation of
cathepsin K observed in our experiments can be attributed to a
slow conformational adaptation of CS/DS as illustrated in Figure
S7(B) using DS as an example. The enzyme initially interacts
with an extended GAG molecule and this interaction is sufficient
to increase the enzyme’s activity. The initial complex then slowly
rearranges to allow for a more tight interaction between enzyme
and GAG. It should be noted that the docking calculations
were performed with GAG octasaccharides, whereas the crystal
structure shows a CS hexasaccharide bound to cathepsin K [12].
In the latter, the N-acetyl-galactosamine at the non-reducing end
is positioned in an orientation that would prevent the GAG chain
from extending beyond this residue. To account for the polymer
nature of GAGs, a chain with two additional monosaccharides at
the non-reducing end was therefore chosen for the dockings. Of
course, the binding mode shown in Figure S7(B) was not the only
docking solution obtained. Our primary criterion for filtering the
results was that the orientation of the GAG chain in the model
corresponded to that in the crystal structure and further ranking
c The Authors Journal compilation c 2010 Biochemical Society
of the obtained solutions was performed based on the calculated
binding energies. To illustrate the degree of binding flexibility at
this site, Figure S7(C) shows all the docking solutions obtained
that satisfy the primary criterion obtained with a rigid GAG chain
(Figure S7C, left-hand panel) and a flexible GAG chain (Figure
S7C, right-hand panel).
Binding kinetics show that HP binds cathepsin K at two
different sites. Their locations were determined by chemically
cross-linking the enzyme with the ligand using the photoreactive cross-linker sulfo-SBED. After proteolytic digestion of
the complex, three HP-linked peptides were identified on the
bottom and back sides of cathepsin K, containing the crosslinker bound to Lys10 , Lys39 and Lys77 (Figure S8A). At very low
concentrations, the effect of HP was analogous to CS/DS. It is
therefore logical to assume that under these conditions HP binds
to cathepsin K in a manner analogous to CS/DS (Figure S8B).
For clarity, only one docking solution is shown and it should be
stressed that this model is purely illustrative, as it shows a HP
chain sulfated at every possible position, whereas in nature the
degree and patterns of sulfation are highly heterogeneous.
The location of the second HP-binding site was predicted on
the bottom of the molecule. As there is no experimental evidence
about the length of the HP fragment that interacts with the
protein, docking of HP octasaccharides to this site was performed
with non-rotatable glycosidic bonds to avoid unrealistic twisting
of the HP chain in the docking solutions. Three clusters of
docking results were identified that conformed with cross-linking
experiments, i.e. distance of the HP chain was no more than
16 Å from the ε-amino group of Lys77 . The energetically most
favourable docking poses from each of these clusters are shown
in Figure S8(C). The central part of the second binding site is
composed of six basic residues (Lys40 , Lys41 , Arg108 , Arg111 , Arg127
and Lys214 ) organized in a ring-shaped structure on the bottom
of cathepsin K. In addition to these, the octasaccharide forms
contacts with Lys10 , which is also involved in binding of GAGs at
the primary GAG-binding site.
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Received 5 March 2010/28 April 2010; accepted 7 May 2010
Published as BJ Immediate Publication 7 May 2010, doi:10.1042/BJ20100337
c The Authors Journal compilation c 2010 Biochemical Society