Biochem. J. (2010) 429, 379–389 (Printed in Great Britain) doi:10.1042/BJ20100337 379

Conformational flexibility and allosteric regulation of cathepsin KMarko NOVINEC*†, Lidija KOVACIC‡, Brigita LENARCIC†§ and Antonio BAICI*1

*Department of Biochemistry, University of Zurich, 8057 Zurich, Switzerland, †Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University ofLjubljana, 1000 Ljubljana, Slovenia, ‡Department of Molecular and Biomedical Sciences, Jozef Stefan Institute, 1000 Ljubljana, Slovenia, and §Department of Biochemistry andMolecular and Structural Biology, Jozef Stefan Institute, 1000 Ljubljana, Slovenia

The human cysteine peptidase cathepsin K is a key enzymein bone homoeostasis and other physiological functions. Inthe present study we investigate the mechanism of cathepsinK action at physiological plasma pH and its regulation bymodifiers that bind outside of the active site. We show that atphysiological plasma pH the enzyme fluctuates between multipleconformations that are differently susceptible to macromolecularinhibitors and can be manipulated by varying the ionic strengthof the medium. The behaviour of the enzyme in vitro canbe described by the presence of two discrete conformationswith distinctive kinetic properties and different susceptibilityto inhibition by the substrate benzyloxycarbonyl-Phe-Arg-7-amino-4-methylcoumarin. We identify and characterize sulfatedglycosaminoglycans as natural allosteric modifiers of cathepsinK that exploit the conformational flexibility of the enzyme to

regulate its activity and stability against autoproteolysis. Allsulfated glycosaminoglycans act as non-essential activators inassays using low-molecular-mass substrates. Chondroitin sulfateand dermatan sulfate bind at one site on the enzyme, whereasheparin binds at an additional site and has a strongly stabilizingeffect that is unique among human glycosaminoglycans.All glycosaminoglycans stimulate the elastinolytic activity ofcathepsin K at physiological plasma pH, but only heparin alsoincreases the collagenolytic activity of the enzyme under theseconditions. Altogether these results provide novel insight into themechanism of cathepsin K function at the molecular level and itsregulation in the extracellular space.

Key words: chondroitin sulfate, conformational change, dermatansulfate, heparin, non-essential activation, substrate inhibition.

INTRODUCTION

Allostery is the coupling of conformational changes between twoseparated sites. Initially described in oligomeric systems [1–3],allostery is known today as a widely used mechanism inthe regulation of monomeric proteins. The ability to undergoconformational changes in response to ligand binding is anintrinsic property of many, if not all, non-fibrous proteins.Conformational changes triggered by an allosteric modifier canrange from major structural movements to subtle, even virtuallyunrecognizable, rearrangements within the protein [4].

Although long believed to be relatively unstable in the extra-cellular milieu, it has become clear in recent years that cysteinecathepsins are major players in extracellular proteolysis. Muchwork has been dedicated in investigating the regulation of theseenzymes by proteinaceous competitive inhibitors [5], but little isknown about other mechanisms involved in the regulation of theiractivity.

Cathepsin K is one of the most potent mammalian peptidases. Itis the major proteolytic enzyme involved in bone metabolism andits deficiency causes pycnodysostosis, a rare disease characterizedby bone abnormalities [6]. It is also expressed in several other celltypes of the fibroblast and haemopoietic lineages, as well as someepithelioid cells and aortic smooth muscle cells. Besides boneresorption, it plays important roles in embryonic development,spermatogenesis and thyroid hormone release [7]. Previousfindings also suggest roles for cathepsin K in schizophrenia [8] andobesity [9]. Excessive cathepsin K activity has also been reportedin association with cardiovascular and pulmonary diseases, aswell as arthritis and cancer [7]. Because of its role in bone matrixdegradation it has received considerable attention in recent years

as a target for osteoporosis therapy and at least two cathepsin Kinhibitors are currently in clinical trials [10].

Cathepsin K can cleave most extracellular substrates, includingthe collagen triple helix and it has been reported that itscollagenolytic activity depends on complex formation with CS(chondroitin sulfate), which increases the activity and stability ofthe enzyme [11]. The crystal structure of cathepsin K in complexwith a chondroitin-4-sulfate hexasaccharide, however, shows thatthe CS-binding site is distant from the active site and no effect onenzyme activity was observed [12].

There have been several other reports about the influenceof GAGs (glycosaminoglycans) on the activity of cysteinecathepsins. HP (heparin) and HS (heparan sulfate) were foundto increase the activity and stability of papain and cathepsinB [13,14], whereas, and in contrast, intralysosomal membrane-bound GAGs act as inhibitors of lysosomal enzymes [15–17].Furthermore, GAGs facilitate the autocatalytic conversion of pro-cathepsins B and S into their mature forms [18,19].

Bone resorption by osteoclasts occurs at acidic pH, which hasalso been found to be optimal for the stability of cathepsin K [20].Other extracellular actions of cathepsin K, such as degradationof elastic fibres in the walls of blood vessels, occur howeverat pH values equal or close to the physiological plasma pH.This indicates that cathepsin K retains significant activity undersuch suboptimal conditions. In the present study we investigatethe activity of cathepsin K and its regulation at physiologicalplasma pH, as is found in the extracellular matrix. We report thatunder these conditions the enzyme exists in multiple functionallydistinct conformational states in vitro. We identify sulfated GAGsas allosteric modifiers of cathepsin K and investigated their effecton the activity and stability of cathepsin K under these conditions.

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 abaici@bioc.uzh.ch).

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EXPERIMENTAL

Materials

Recombinant human cathepsin K was produced according tothe procedure described by D’Alessio et al. [21]. Enzymeconcentration was determined by active-site titration withthe irreversible inhibitor E-64 (Bachem). The fluorogenicsubstrates Z-FR-AMC (benzyloxycarbonyl-Phe-Arg-7-amino-4-methylcoumarin) and Z-VVR-AMC (benzyloxycarbonyl-Val-Val-Arg-7-amino-4-methylcoumarin) were from Bachem. DS(dermatan sulfate) from porcine intestinal mucosa was fromCalbiochem. Heparin sodium salt, DxS (dextran sulfate) sodiumsalt, leupeptin hydrochloride and chondroitin-4-sulfatesodium salt from bovine trachea were from Sigma–Aldrich.Although labelled ‘chondroitin-4-sulfate’ this sample was aco-polymer of the 4- and 6-isomers along the same chain andcontained 69 % 4-sulfate and 25% 6-sulfate; DS contained 98%4-sulfate and the balance to 100% was non-sulfated materialfor both CS and DS. The uronic acid moieties were consistentlyglucuronic acid and iduronic acid in CS and DS, respectively, asmeasured by HPLC analysis of the unsaturated disaccharides asdescribed 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 concentrationsof disaccharide units. Bovine neck ligament elastin and solubleETNA-elastin were from Elastin Products Company. Solublecalf-skin collagen was from the Worthington BiochemicalCorporation. The cross-linking reagent sulfo-SBED {sulfo-N-hydroxysuccinimidyl-2-[6-(biotinamido)-2-(p-azido benza-mido)-hexanoamido] ethyl-1,3′-dithioproprionate} was fromThermo Scientific. Recombinant human stefin A and recom-binant human N2TY (thyroglobulin type 1 domain 1 of humannidogen-2) were produced according to published procedures[24,25].

Kinetic measurements

Prior to the reactions the enzyme was kept on ice either inlow-salt buffer [50 mM Hepes, pH 7.40, containing 1 mM EDTAand 2.5 mM DTT (dithiothreitol)] or in high-salt buffer (50 mMHepes, pH 7.40, containing 300 mM NaCl, 1 mM EDTA and2.5 mM DTT), as described in the text. Buffers were preparedand used at 25 ◦C. All measurements were performed in low-salt buffer in single-use acrylic cuvettes (1 cm × 1 cm) that werekept at a constant temperature of 25 +− 1 ◦C and subject tomagnetic stirring. Z-FR-AMC was used as the substrate, exceptwhere indicated otherwise. Reactions were started by addingenzyme to the reaction-mixture-containing buffer, substrate andmodifier, where appropriate. Reaction progress was monitoredfluorimetrically at an excitation wavelength (λex) of 383 nm andan emission wavelength (λem) of 455 nm. The final enzyme activesite concentration in the assays was 0.1 nM. Blanks containingstandard concentrations of AMC (7-amino-4-methylcoumarin)were recorded under reaction conditions to assure that totalsubstrate consumption in the experiments was less than 10%.

Kinetic models and data analysis

The detailed descriptions of kinetic models used in this workare available in the Supplementary Theoretical backgroundsection (at http://www.BiochemJ.org/bj/429/bj4290379add.htm).All mathematical analyses and graphical manipulations wereperformed with GraphPad Prism 5.0 software.

Intrinsic fluorescence spectroscopy

Intrinsic fluorescence spectra of cathepsin K were recordedin 50 mM Hepes, pH 7.40, containing from 0 to 300 mMNaCl in single-use acrylic cuvettes (1 cm × 1 cm) at 25 ◦Cwith magnetic stirring. Samples were excited at 295 nm andemitted fluorescence spectra were either recorded from 310to 400 nm or the fluorescence was monitored continuously at340 nm (5 nm bandwidth). The final enzyme concentration inall experiments was 0.2 μM. When recording time-dependentchanges in fluorescence, recording was started immediately upondiluting the enzyme from a stock solution (1 mg/ml cathepsin Kin 50 mM Hepes, pH 5.0, containing 500 mM NaCl, 1 mM DTT,25 μg/ml DxS) into the reaction mixture.

Stability of the cathepsin K activity

Cathepsin K was incubated at 37 ◦C at a final enzymeconcentration of 0.2 μM in 50 mM Hepes, pH 7.40, containing1 mM EDTA and 2.5 mM DTT, in the presence or absence of0.2 mM GAGs and 300 mM NaCl, or 5 mg/ml soluble ETNA-elastin. Residual enzyme activity was determined by removingaliquots from the reaction mixture at regular time intervals andmeasuring their activity using the substrate Z-FR-AMC (10 μMfinal concentration).

Elastinolytic assays

Suspensions of bovine neck ligament elastin (5 mg/ml) wereprepared in 50 mM Hepes, pH 7.40, containing 1 mM EDTAand 2.5 mM DTT. GAGs (0.2 mM final concentration) wereadded to the suspensions just prior to addition of the enzyme(final concentration 0.1 μM). The same experiments wherealso performed in two other buffers, 50 mM Hepes, pH 7.40,containing 300 mM NaCl, 1 mM EDTA and 2.5 mM DTT and50 mM Mes, pH 6.20, containing 1 mM EDTA and 2.5 mMDTT. All mixtures were incubated in an Eppendorf ThermomixerCompact at 37 ◦C with shaking (1200 rev./min) and reactionswere stopped after various incubation times by addition oftrichloroacetic acid to a final concentration of 5% (w/v). Aftercentrifugation (14000 g for 10 min), clear supernatants (200 μl)were diluted to 3.0 ml with 0.2 M borate buffer, pH 8.50, andreacted with 1.0 ml of a fluorescamine solution (0.15 mg/ml inacetone). The fluorescence of the samples was measured at λex

390 nm and λem 480 nm. Peptide concentrations were determinedfrom a standard curve produced in the same manner using astandardized 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 mMGAGs and digestion started by addition of cathepsin K (finalconcentration 0.25 μM). All reactions were incubated for 16 h at25 ◦C and then stopped by addition of SDS/PAGE sample buffer.Polypeptides were separated by SDS/PAGE (8% gels) and stainedwith Coomassie Brillant Blue R-250.

Docking of GAG octasaccharides to cathepsin K

Octasaccharide models of DS (consisting of four 4-sulfo-N-acetylgalactosamine-iduronic acid disaccharides) wereconstructed with the CambridgeSoft ChemOffice 11 Suite.Torsion angles between monosaccharides were adjusted manuallyaccording to published data [26] and the structures then optimizedby energy minimization using the MMFF94 force-field. Heparin

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Allosteric regulation of cathepsin K 381

Figure 1 Dependence of the inhibitory efficiency of macromolecularinhibitors of cathepsin K on ionic strength

Reactions were performed by adding 0.1 nM cathepsin K to a reaction mixture containing thesubstrate10 μM Z-FR-AMC and inhibitor at 25◦C. (A) Examples of progress curves of substratehydrolysis in the presence of stefin A (20 nM) recorded in 50 mM Hepes, pH 7.40, containingthe indicated concentrations of NaCl. (B) Steady-state reaction rates vs (left-hand panel) andvalues of the first-order rate constant λ (right-hand panel) for the interaction between enzymeand inhibitor. The inhibitors used were 20 nM stefin A (stfA) or 50 nM N2TY. The values ofvs and λ were determined by non-linear regression using eqn (1) (Supplementary material athttp://www.BiochemJ.org/bj/429/bj4290379add.htm).

models were retrieved from the PDB (PDB code 1HPN). Co-ordinates of cathepsin K were extracted from the crystal structureof the cathepsin K–CS complex (PDB code 3C9E). All dockingcalculations were performed with AutoDock 4 [27] using theLamarckian genetic docking algorithm. A detailed description ofthe procedure is available in the Supplementary section. Surfacepotentials were calculated with the Adaptive Poisson–BoltzmanSolver [28]. All images were created with PyMOL (DeLanoScientific; http://www.pymol.org).

RESULTS

Interaction of cathepsin K with macromolecular inhibitors

The initial observation that led us to investigate the phenomenapresented in this paper was that macromolecular inhibitors failto inhibit cathepsin K under certain experimental conditions atpH 7.40. In the present study we show results obtained withthe slow-binding inhibitors of cathepsin K, cystatin stefin A andN2TY. Figure 1(A) shows several examples of progress curvesof the hydrolysis of a fluorogenic substrate by cathepsin K inthe presence of a large excess of stefin A (20 nM inhibitorand 0.2 nM enzyme) measured in buffers with increasing ionicstrength. The ionic strength had a large impact on the efficiencyof the inhibitor and similar progress curves were obtained withN2TY. Figure 1(B) shows that both the residual enzyme activityand the value of the apparent first-order rate constant for thebinding of inhibitor (λ) were affected. Only 50% inhibition wasachieved at the lowest ionic strength (50 mM Hepes, pH 7.40,I 19 mM), and over 90% inhibition, comparable with thatobserved at acidic pH values, was achieved when the ionic strengthwas more than 0.15 M and reached the maximum at I � 0.3 M.

Intrinsic tryptophan fluorescence

To investigate whether the different susceptibility towardsmacromolecular inhibitors results from a conformational change,intrinsic fluorescence spectra of cathepsin K were recorded.Cathepsin K contains four tryptophan residues, three of whichare located in or near the active centre (Figure 2A). It is thereforereasonable to assume that conformational changes of this part ofthe molecule will be reflected in a change of intrinsic fluorescence.Initially, near-UV emission spectra were recorded in two differentenvironments: a low-salt buffer (50 mM Hepes, pH 7.40), wherethe effect of macromolecular inhibitors was minimal, and a high-salt 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 intensityand a red-shift of 3 nm upon increasing the ionic strength.

Time-dependent measurements were performed to investigatefurther the effect of ionic strength on the intrinsic fluorescence ofcathepsin K. These showed a slow decrease of fluorescenceemission intensity at 340 nm upon dilution of the enzymewith a stock solution (1 mg/ml enzyme concentration, pH 5.00,500 mM NaCl and 25 μg/ml DxS) into the reaction mixturescontaining increasing concentrations of NaCl (Figure 2C).Concentrations of up to 300 mM showed increasing effectson the decrease of fluorescence intensity, whereas increasingfurther the concentration of NaCl had no additional effect. In theseexperiments the reaction mixtures were supplemented with thereversible inhibitor leupeptin. Even though its presence may havecontributed to the overall effect it was necessary to minimize theprobability of enzyme autodegradation during the measurements.

A possible interpretation of the results presented thus faris the existence of multiple conformations of cathepsin K;the slow decay of intrinsic fluorescence can be interpreted asa slow transition between these conformational states. Thisbehaviour was originally described by Frieden [29] as a regulatorymechanism in metabolic processes. A minimal mechanismconsisting of a population of enzyme molecules that can exist intwo conformational states is sufficient to describe the experimentsin the present study, even though the treatment of cathepsin K asa fluctuating enzyme would probably be more appropriate at thesingle-molecule level [30]. In analogy with other allostericallyregulated proteins we termed these states the T (tense) and R(relaxed) states, which predominate at low and high ionic strengthrespectively. At the same time this nomenclature reflects thedifferent susceptibility of the two states towards macromolecularinhibitors. This hypothesis is supported by the plot of the valuesof the apparent rate constant (k) for the exponential decay offluorescence as a function of ionic strength shown in Figure 2(D).The hyperbolic profile describes the dependency of k upon ionicstrength and shows that k approaches zero at low ionic strength andtends asymptotically to the value 0.015 s−1 for increasing ionicstrength. This means that a certain ionic strength is necessaryto trigger the change in intrinsic fluorescence associated with aconformational change of the enzyme, whereas at lower ionicstrength the enzyme will remain in the initial conformation.

Activity measurements

To verify the hypothesis presented above, we sought evidence forfunctional differences between the two putative conformations.Experimentally this was achieved by measuring the activity ofcathepsin K in low-salt buffer following pre-incubation in eitherlow-salt buffer (enzyme assumed to be in the T state) or high-salt buffer (enzyme in R state). The use of this artificial systemwith non-physiological ionic strengths was necessary to keep the

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382 M. Novinec and others

Figure 2 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 shownas 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. Firstderivatives 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 indicatedconcentrations 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 rectangularhyperbola fit gave k = 0.015 +− 0.010 s−1 as the asymptote of the function for increasing ionic strength. AU, arbitrary units.

enzyme predominantly in a single conformation; at physiologicalionic strengths equilibration of the enzyme between both stateswould have made the kinetic analyses unfeasible.

Two different substrates (Z-FR-AMC and Z-VVR-AMC) wereused 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 Km.The T state had 2-fold higher catalytic efficiency, but also 2-fold higher Km than the R state. The behaviour with Z-FR-AMC,the most frequently used endoproteolytic substrate for cysteinecathepsins, was more complex. It did, however, provide additionalinformation about the conformational flexibility of cathepsin Kin that it showed that the conformational equilibrium is regulatednot only by ionic strength, but also by the substrate. The completereaction scheme to be considered in these experiments is shownin Figure 3. The enzyme is in equilibrium between the T andR states in both the free and substrate-bound forms. As anadditional off-path, substrate inhibition occurs at high substrateconcentrations (above Km) that locks the enzyme in a non-functional conformation. This phenomenon has been observedpreviously [31], but remained without interpretation.

To simplify the analysis, we investigated the mechanism inparts, as indicated by boxes in the outline scheme in Figure 3 (up-per panel); the progress curves are shown in Supplementary FigureS1 (at http://www.BiochemJ.org/bj/429/bj4290379add.htm) andthe concentration dependencies are shown in Figures 3(A)–3(C).The results shown in Figures 3(A) and 3(B) demonstrate that sub-strate inhibition occurs in a slow manner and affects the reactionrates at zero time (vz) and at steady-state (vs). The model usedto describe this mechanism is shown in Supplementary SchemeS1 (see http://www.BiochemJ.org/bj/429/bj4290379add.htm).

Table 1 Kinetic parameters for the hydrolysis of Z-VVR-AMC and Z-FR-AMCby cathepsin K

Experiments were performed at 25◦C in 50 mM Hepes, pH 7.40, containing 1 mM EDTA and2.5 mM DTT after pre-incubation of the enzyme in the same buffer without salt [enzyme (E) inthe T state] or with 300 mM NaCl (enzyme in the R state). Results are best fits (+− S.E.M.) fromnon-linear regression analysis using the Michaelis–Menten eqn (Z-VVR-AMC) or supplementaryeqn (3) (Z-FR-AMC).

Parameter K m (μM) k cat (s−1) k cat/K m (M−1·s−1)

Z-VVR-AMCE in T state 7.7 +− 1.3 0.37 +− 0.02 (4.8 +− 0.8) × 104

E in R state 3.2 +− 0.5 0.19 +− 0.01 (5.9 +− 0.9) × 104

Z-FR-AMCE in T state 21 +− 4 91 +− 12 (4.3 +− 1.0) × 106

E in R state 5.1 +− 1.1 39 +− 5 (7.6 +− 1.9) × 106

Comparison between experiments started from the T and Rstates shows that the former becomes affected at much highersubstrate concentrations, reflecting the higher Km value ofthe T state in comparison with the R state. The plot inFigure 3(C) shows that at low substrate concentrations a slowre-equilibration of the enzyme occurs. This is characterizedby ‘concave-up’-shaped progress curves (Supplementary FigureS1B) and is observed only when experiments are startedfrom enzyme in the T state. The model used to describethis mechanism is shown in Supplementary Scheme S2 (seehttp://www.BiochemJ.org/bj/429/bj4290379add.htm). Calcula-tion of the kinetic parameters showed that k−3�k3 and k6�k−6,meaning that free enzyme prefers the R state and substrate-boundenzyme prefers the T state. The rate constant k6 corresponds to

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Allosteric regulation of cathepsin K 383

Figure 3 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 thetop 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 reactionrates at zero time (vz) and at steady state (vs) (left-hand panels) and plots of the values of the rate constant λ (right-hand panels). The calculated values of kinetic parameters corresponding to eachpart 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 (enzymeinitially 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 SchemeS1 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 inthe intrinsic fluorescence of the enzyme that accompanies thetransition from the T to the R state in the presence of leupeptin,hence in the bound state. The results in Figure 2(D) confirms thatunder 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 conformationalflexibility 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 polysaccharidesalso influence the conformational flexibility of cathepsin K atphysiological plasma pH. We have experimentally determinedthe effects of CS, DS, HP and hyaluronan. Hyaluronan had noeffect, whereas all of the sulfated GAGs stimulated the activity ofcathepsin K.

Binding of CS, DS and HP to cathepsin K resulted ina 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

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384 M. Novinec and others

Figure 4 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 tocathepsin K. Enzyme was first added to a reaction mixture containing inhibitor (20 nM stefinA) and10 μM Z-FR-AMC as substrate (E + I). DS was added to the reaction mixture (0.2 mMfinal 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 mMfinal concentration). Both experiments were performed in 50 mM Hepes, pH 7.40, containing1 mM EDTA and 2.5 mM DTT at 25◦C with a final enzyme concentration of 0.2 nM. AU, arbitraryunits.

change of the enzyme (Figure 2). The rate of transition increasedin the order CS < DS < HP. Further experiments that provideinsight into the effect of GAGs on the conformation of cathepsinK are shown in Figure 4. In the experiment shown in Figure 4(A),cathepsin K was added to a mixture of substrate and the inhibitorstefin A. Because the enzyme is in the T state under theseconditions, the inhibitor binds only weakly. Addition of GAGinto the reaction mixture then causes a change in the enzymeconformation, resulting in rapid binding of the inhibitor. A similareffect is shown in Figure 4(B) which shows a typical example ofslow substrate inhibition of cathepsin K. Addition of DS intothis mixture again appears to cause a conformational change,which in this case relieves the enzyme from the effect of substrateinhibition.

Activation of cathepsin K by GAGs

The conformational change upon binding of GAGs to cathepsinK was reflected in its activity. CS and DS had a similar effecton cathepsin K. Progress curves had ‘concave-up’ exponentialprofiles (Supplementary Figure S3 at http://www.BiochemJ.org/bj/429/bj4290379add.htm) consistent with a mechanism ofslow non-essential activation. To describe the mechanism ofCS/DS binding to cathepsin K it was sufficient to analyseseparately the situations at zero time and at steady-state. Specificvelocity plots constructed from steady-state reaction rates hadsimilar profiles of straight lines with a positive slope and a

trend of intercepting the ordinate axis with value 1 at abscissavalues near 1 (Figures 5A and 5B). By combining results fromthe specific velocity plots with conventional plots of reactionrates in the presence of increasing concentrations of CS/DS(Figure 5C and 5D), we were able to determine the parametersα, β and KA at steady-state and zero time. Their values showthat CS/DS act by increasing the affinity of the enzyme for thesubstrate (α < 1) without major influence on the catalytic activity(β ≈ 1) thus promoting an effect similar, but not identical, tothe conformational changes undergone by the enzyme alone inresponse to low substrate concentration.

In addition to naturally occurring GAGs, cathepsin K wasalso bound by DxS, a semi-synthetic highly sulfatedanhydroglucose polymer. Progress curves in the presence ofDxS were linear and plots of reaction rates against DxSconcentration at different substrate concentrations (Figure 5E)show that DxS acts as an activator at low substrateconcentrations (below a half of the Km) and as an inhibitorat substrate concentrations above Km. This behaviour can bedescribed as hyperbolic mixed-type inhibition or non-essentialactivation with a combination of parameters 0 < α < β < 1in the general modifier mechanism (Supplementary SchemeS3 at http://www.BiochemJ.org/bj/429/bj4290379add.htm). Theoverall effect of DxS on the activity was small ( +− 25% at most).Altogether this indicates that DxS binds at a different site thanCS/DS. Despite the fact that the interaction of cathepsin K withDxS is not physiologically relevant, it is instrumental for analysingand interpreting the effect of HP on the enzyme.

The interaction of cathepsin K with HP is more complexthan that of CS/DS or DxS. At low HP concentrations theshapes of progress curves and the effect on enzyme activity wereanalogous to that of CS/DS, whereas higher concentrations causeda decrease in cathepsin K activity (Figure 5F). This behaviourcan be interpreted by a simultaneous interaction of HP with twosites on cathepsin K, yielding a composite effect on enzymeactivity. This mechanism is shown in Supplementary SchemeS7 (at http://www.BiochemJ.org/bj/429/bj4290379add.htm). It isreasonable to assume that one binding site is the site bound byCS/DS, as indicated by the slow activation of the enzyme at lowHP concentrations. The second binding site seems to be identicalto the DxS-binding site, as indicated by the similar effects of HPand DxS on the stability of cathepsin K (see the Stabilization ofcathepsin K activity section).

Considering the effect of HP as a composite effect of thoseobserved with CS/DS and DxS, we used the values of coefficientsα and β determined for DxS and for CS/DS as constantsin Supplementary eqn (18) (at http://www.BiochemJ.org/bj/429/bj4290379add.htm). Thereby eqn (18) could be fittedto the experimental data at all substrate concentrations used(Figure 5F). The fitted curves showed no significant systematicdeviation from the experimental points, indicating that themathematical model was indeed appropriate. The concentrationdependencies were biphasic and consisted of a hyperbolicactivation phase followed by a phase of declining activity. Boththe maximal vA/v0 ratio and the vA/v0 ratio at saturating HPconcentrations depended on substrate concentration. Moreover,the latter fell under 1 at substrate concentrations above Km,i.e. the net effect of HP became inhibitory above a certainsubstrate concentration. The calculated KA values were oneorder of magnitude lower than those calculated for CS, DSand DxS, showing that the effect of HP is much strongerthan that of CS, DS or DxS. Owing to the complexity of thesystem and the heterogeneity of HP, experimental data wererelatively disperse and therefore we were unable to calculateshared values for all parameters in Supplementary eqn (18). The

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Allosteric regulation of cathepsin K 385

Figure 5 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 atzero time (vz) and steady-state (vs) 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 mMHepes, 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 greaterthan 1, meaning that binding at one site hinders bindingat the other [33]. Furthermore, the general trend observed

in the experiments was unequivocal, confirming that theproposed model is overall adequate for the description of thesystem.

c© The Authors Journal compilation c© 2010 Biochemical Society

386 M. Novinec and others

Figure 6 Structural models of GAGs binding to cathepsin K

(A) Binding of CS/DS to cathepsin K (in surface representation, coloured according to theelectrostatic potential) illustrated on the example of a DS octasaccharide (consisting of four4-sulfo-N-acetylgalactosamine-iduronic acid disaccharides; shown as sticks). The binding isproposed to proceed via a slow conformational adaptation of the GAG to the structure ofcathepsin K. (B) A possible binding mode of HP at the second HP-binding site on the bottomof 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 surfacepotential of cathepsin K was calculated with APBS software and the structures visualized withPyMOL (DeLano Scientific; http://www.pymol.org).

Structural interpretation of GAG binding

Taken together, activity and intrinsic fluorescence measurementsdemonstrate that GAGs act as allosteric regulators that alter theconformation and activity of cathepsin K by binding at a site otherthan the active centre. The location of this site has been revealedby the crystal structure of a cathepsin K/CS complex [12].Comparing the structure of CS in this complex with the calculatedstructure of CS in solution [26] shows that a rearrangement of theCS chain occurs upon binding to the enzyme. This indicates thatthe slow activation of cathepsin K observed in our experimentsresults from a slow conformational change of CS/DS, as illustratedin Figure 6(A) for DS, which allows for the formation of a moretightly bound complex between enzyme and GAG.

Binding kinetics show that HP binds cathepsin K at twodifferent sites. At very low concentrations, the effect of HP wasanalogous to CS/DS. It is therefore logical to assume that underthese conditions HP binds to cathepsin K in a manner analogous toCS/DS. The location of the second HP-binding site was predictedto be on the bottom of the molecule and the energetically mostfavourable docking pose is shown in Figure 6(B). The central partof the second binding site is composed of six basic residues (Lys40,Lys41, Arg108, Arg111, Arg127 and Lys214) organized in a ring-shapedstructure on the bottom of cathepsin K. In addition to these, theoctasaccharide forms contacts with Lys10, which is also involvedin binding of GAGs at the primary activation site.

A more detailed description and discussion of docking resultsis available in the Supplementary material.

Stabilization of cathepsin K activity

Apart from directly affecting the catalytic properties of anenzyme, effector molecules can also regulate its stabilityby other means. We measured the activity of cathepsin

K as a function of incubation time at pH 7.40 and at37 ◦C and the half lives of the enzyme in the presenceand absence of GAGs are shown in Supplementary TableS1 (at http://www.BiochemJ.org/bj/429/bj4290379add.htm). Notsurprisingly, the activity of cathepsin K alone was relativelyunstable in aqueous solution, having a half-life of approx. 7 min.Increasing the ionic strength of the incubation mixture to 300 mMwith NaCl decreased the half-life by 7-fold. The presence of CSand DS slightly reduced the stability of cathepsin K (half lives of5.5 and 6.0 min respectively), whereas HP had a strong stabilizingeffect and increased the enzyme’s half-life by more than 5-fold(38 min). The same effect was achieved with DxS, indicating thatbinding at the secondary HP-binding site is directly involved inregulating cathepsin K stability.

The experimental results shown in Supplementary Table S1used enzyme incubated in the absence of substrate to determinethe effect of GAGs in the absence of other substances bindingto the enzyme. In vivo, however, the concentration of proteins ishigh and binding of substrate to the active centre can also stabilizethe enzyme. Indeed, a macromolecular substrate (5 mg/ml solubleETNA-elastin) substantially increased the half-life of cathepsinK (33 compared with 7 min). Addition of HP to this reactionmixture resulted in further stabilization of the enzyme (a half-life of 190 min), indicating synergy between substrate and HP inprotecting its activity. These pooled observations suggest thatstabilization of cathepsin K activity by HP and by substratemay occur by protection from thermal denaturation and/or byprotection from autoproteolysis. As shown in SupplementaryFigure 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 ofcathepsin K at neutral pH and 37 ◦C in presence of elastin, HP orDxS is likely to be protection from autoproteolysis.

Collagenolytic and elastinolytic activity at neutral pH

All the experiments presented above relied on low-molecular-mass synthetic substrates to report the activity of cathepsin K.However, its activity on physiologically relevant macromolecularsubstrates may differ from that observed with low-molecular-masssubstrates. Therefore we also examined the activity of cathepsinK on collagen and elastin, two abundant extracellular structuralproteins, which have been long known as (patho)physiologicalsubstrates of cathepsin K.

Collagenolytic assays (Figure 7A) showed that cathepsin K iscapable of digesting type I collagen on its own. Heparin increasedthe collagenolytic activity of cathepsin K, whereas, interestingly,CS/DS decreased the extent of collagen digestion. Collagenolysiswas also decreased in the presence of 300 mM NaCl, whereasDxS had no effect.

Total elastinolytic activity was also measured with thefluorescamine method (Figure 7B). The activity of cathepsin Kat pH 7.40 was 60% of that observed at pH 6.20, indicating thatcathepsin K retains most of its potent elastinolytic activity at thispH. Increasing the salt concentration again reduced the activity ofcathepsin K and all GAGs acted as potent activators in this assay.CS/DS increased the elastinolytic activity by 4-fold, whereas HPstimulated the activity by as much as 12-fold.

DISCUSSION

The results of the present study show novel aspects of themechanism of cathepsin K activity and regulation. Despitefocusing on only one member of cysteine cathepsins, the results

c© The Authors Journal compilation c© 2010 Biochemical Society

Allosteric regulation of cathepsin K 387

Figure 7 Collagenolytic and elastinolytic activities of cathepsin K in thepresence of GAGs

(A) Calf-skin collagen (0.5 mg/ml in 50 mM Hepes, pH 7.40, containing 1 mM EDTA and 2.5 mMDTT) was incubated with 0.5 μM cathepsin K (CatK) in the presence of different modifiers for16 h at 25◦C. Samples were then separated on an 8 % polyacrylamide gel and stained withCoomassie Brilliant Blue. Positions of calibrating proteins are given in kDa on the left-handside. Ctrl, control (undigested collagen). (B) Suspensions of insoluble bovine neck ligamentelastin (5 mg/ml in 50 mM Hepes, pH 7.40, containing 1 mM EDTA and 2.5 mM DTT) wereincubated with 0.1 μM cathepsin K in the presence of different modifiers/conditions for 2 h at37◦C. Reactions were stopped by addition of 5 % (w/v) trichloroacetic acid, soluble peptidesreacted 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 concentrationsof L-alanine.

may be exploited for investigating similar mechanisms sharedwith other enzymes of this group. Our results imply thatthe structure of cathepsin K is flexible and converts betweenmultiple conformational states with distinctive characteristics atphysiological plasma pH. The conformational transition is a slowprocess that can be manipulated in vitro. Our experimental set-upwas aimed at ‘trapping’ the enzyme in a single conformation anda mathematical model of two discrete conformational states wassufficient to describe the experiments. In vivo the enzyme probablyfluctuates between multiple conformational states and the T andR states that we observed in vitro possibly represent two extremesituations. A plausible interpretation is that in biological systemsthe conformational flexibility serves the purpose of adapting theshape of the active site to bring bulky macromolecular substratesinto position for cleavage, meaning that cathepsin K operates viaa mechanism reminiscent of the ‘induced fit’ model [34]. Indeed,the active site can accommodate a wide variety of structurallydiverse substrates [35] and fluctuations of this region were alsoobserved in molecular dynamics simulations, which are presentedand discussed in Supplementary material.

Substrate inhibition is a phenomenon that we haveobserved several times when using dipeptide coumarin-basedsubstrates to measure the activity of cysteine cathepsins. Howexactly inhibition by substrate occurs, remains uncertain. Asshown in Supplementary Figure S5 (http://www.BiochemJ.org/bj/429/bj4290379add.htm), a substantial part of the activesite remains unoccupied when a substrate molecule is boundin a productive manner. A second substrate molecule couldbind in this area and hinder the turnover of productivelybound substrate. This observation is in agreement with themathematical model of substrate inhibition, which postulatesthat the second (inhibitory) substrate molecule binds to theenzyme–substrate complex and not to enzyme alone. The pre-steady state phase of the progress curves might represent aconformational adaptation of the enzyme to the bound substratemolecule(s). This is indicated by the reversal of substrate in-hibition by modifiers that affect the conformation of the enzyme(Figure 4). Why this effect is only seen with the the substrateZ-FR-AMC, but not with the similar Z-VVR-AMC, can beexplained by the slow turnover of the latter. As estimated from ourexperiments, the turnover of Z-FR-AMC is reduced to approx.1 s−1 at maximal inhibition. This is, however, still several-foldhigher than the kcat values measured for Z-VVR-AMC, meaningthat if a second Z-VVR-AMC molecule were to bind the enzymein a manner similar to Z-FR-AMC, it would not limit the turnoverof the productively bound molecule thus no substrate inhibitionwould be detected experimentally.

The concept of ‘trapping’ the enzyme in a certain conformationappears to be utilized by GAGs, natural allosteric modifiers ofcathepsin K. The results of the present study show that GAGsincrease the activity of the enzyme and promote a conformationalchange (Figures 4 and 5). The crystal structure of cathepsin Kin complex with CS shows no obvious differences from that ofcathepsin K alone [12]. A plausible explanation is that the crystalstructures show cathepsin K already in the R state and thereforeno further conformational change is caused by CS. Altogetherthis indicates that GAGs act by affecting the distribution of a pre-existing equilibrium of conformational states. This behaviour hasbeen observed in other allosterically regulated systems [36].

Cathepsin K has thus far been attributed several physiologicaland pathological functions. Whereas bone resorption occurs inan acidic environment deemed to be optimal for enzyme activity,other functions of cathepsin K occur in an environment where thepH is close to the physiological plasma value. The results fromthe present study show that despite the fact the enzyme is relativelysusceptible to autoproteolysis at pH 7.40 it still shows substantialactivity against macromolecular substrates under these conditions.Cathepsin K cleaves most extracellular matrix components,including the collagen triple helix [37]. It has been reported thatthe collagenolytic activity of cathepsin K at acidic pH depends oncomplex formation with CS [11]. Since then there have been otherreports about diverse effects of different GAGs on this process[32,38]. Our results show that at pH 7.40 the enzyme degradescollagen on its own and that CS/DS reduces its collagenolyticactivity, whereas HP enhances it. Altogether this shows that themolecular mechanism behind the unique collagenolytic activityof cathepsin K depends on the environment. In contrast withthe diverse effects on collagen digestion, all GAGs increasedthe elastinolytic activity of cathepsin K. Overall activity ofGAGs in our experiments increased in the order CS < DS < HP.The same trend has been observed in many GAG-dependentprocesses, e.g. the inhibition of lysosomal enzymes at low pH(HP > CS > DS > hyaluronan) [15,16]. The effect of CS and DSon cathepsin K is virtually identical, although DS binds morerapidly 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 andis therefore more flexible due to fewer intramolecular hydrogenbonds [39].

Despite being structurally similar to CS and DS, HP exertsdifferent effects on cathepsin K in terms of activity and stability.A similar behaviour has been observed with two other relatedpeptidases, papain and cathepsin B, which interact with HP andHS, but not with CS and DS [13,14]. At the molecular level, theunique effect of HP on cathepsin K was attributed to the presenceof two binding sites for HP. Kinetic measurements have shown thatboth sites can be bound simultaneously (Figure 4B); however, dueto the spatial proximity of the proposed binding sites (Figure 6) itremains ambiguous whether each site is bound by a separate HPchain 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 positivelycharged residues, which interact favourably with HP, but not withthe less densely charged CS/DS.

The strong effect of HP on both the activity and stability ofcathepsin K suggests that it may also be an important factorin cathepsin K regulation in vivo. Endogenous HP is producedexclusively in mast cells as part of the proteoglycan serglycin[40], and free HP is widely used as an anticoagulant drug. Ithas been shown that prolonged use of HP can cause osteoporosis[41]. Given cathepsin K is one of the major factors contributingto osteoporosis [10] it seems a plausible target for orallyadministered HP. Owing to its restricted pattern of production,the action of endogenous HP on cathepsin K is probably limitedin vivo. However, the effect of HP can be extended to HS, which isan abundant component of the extracellular matrix (for a review onHS see [42]). HS molecules are highly heterogeneous. However, atleast a fraction of them can be expected to exhibit a HP-like effecton cathepsin K, as has been the case with papain and cathepsin B[13,14].

Conformational flexibility and allosteric regulation are novelconcepts in the regulation of cysteine cathepsins but are byno means surprising. In fact, there is growing evidence thatmost, if not all, globular proteins possess a certain degree ofconformational flexibility [43]. Several findings, such as the DNA-induced conformational changes in the closely related cathepsinV [44] or binding of CS at a site distinct from the active sitein cathepsin K [12], have already indicated such mechanisms ofregulation, even though it was not interpreted as such. Targetingthe allosteric sites of enzymes is becoming increasingly popularas a strategy in drug development [36,43,45]. Even though active-site-directed cathepsin K inhibitors gave some promising resultsin clinical trials, the possibility of allosteric regulation opens novelperspectives for the design of drugs aimed at regulating the activityof this enzyme.

AUTHOR CONTRIBUTION

Antonio Baici designed the project. Marko Novinec designed, performed and interpretedmost of the experiments under supervision of Antonio Baici and Brigita Lenarcic. LidijaKovacic performed and interpreted the cross-linking experiments. Marko Novinec wrotethe paper with editorial supervision from Antonio Baici.

FUNDING

This work was supported by the Swiss National Science Foundation [grant number 31-113345/1]; the Slovenian Research Agency; and the Olga Mayenfisch Foundation.

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Received 5 March 2010/28 April 2010; accepted 7 May 2010Published 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 DATAConformational flexibility and allosteric regulation of cathepsin KMarko NOVINEC*†, Lidija KOVACIC‡, Brigita LENARCIC†§ and Antonio BAICI*1

*Department of Biochemistry, University of Zurich, 8057 Zurich, Switzerland, †Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University ofLjubljana, 1000 Ljubljana, Slovenia, ‡Department of Molecular and Biomedical Sciences, Jozef Stefan Institute, 1000 Ljubljana Slovenia, and §Department of Biochemistry andMolecular and Structural Biology, Jozef Stefan Institute, 1000 Ljubljana, Slovenia

THEORETICAL BACKGROUND

Slow substrate inhibition

The slow inhibition of enzyme (E) by substrate (S) was analysedby the mechanism in Scheme S1.

Scheme S1

This describes the ‘classical’ example of substrate inhibition,which is analogous to uncompetitive inhibition [1], except thatbinding of the second substrate molecule occurs in two steps: aninitial rapid binding step yields the inhibited ES2 complex whichthen slowly rearranges to the more tightly bound ES2

∗ complex.The progress curves have typical exponential profiles that can bedescribed by eqn (1):

[P] = vst + vz − vs

λ(1 − e−λt ) (1)

where vz and vs are reaction rates at zero time and at steady-state and λ is an apparent first-order rate constant. Individualexpressions for vz, vs and λ are given by eqns (2–4):

vz = V [S]

Km + [S]

(1 + [S]

Ksi

) (2)

vs = V [S]

Km + [S]

(1 + [S]

K ∗si

) (3)

λ = k−4 + k4 [S]

Ksi

(1 + Km

[S]

)+ [S]

(4)

where V is the limiting rate and Km is the Michaelis constant. Ksi

and K∗si are equilibrium dissociation constants of the ES2 and ES2

∗

complexes, respectively, and are defined as:

Ksi = k−3

k3

(5)

K ∗si = Ksi

k−4

k−4 + k4

(6)

Conformational equilibrium of cathepsin K

The equilibrium of enzyme between two conformational statesin the presence of substrate was analysed by the mechanism inScheme S2.

Scheme S2

This mechanism takes into account two enzyme species ET

and ER (‘tense’ and ‘relaxed’) that slowly interconvert both inthe unbound and substrate-bound states. The progress curves areagain described by eqn (1). The apparent first-order rate constantλ for this mechanism is defined as:

λ =k−3

K Rm

[S]+ k−6

1 + K Rm

[S]

+k3

K Tm

[S]+ k6

1 + K Tm

[S]

(7)

where

K Tm = k−1 + k2

k1

and

K Rm = k−4 + k5

k4

are Michaelis constants for the T and R state, respectively.

1 To whom correspondence should be addressed (email abaici@bioc.uzh.ch).

c© The Authors Journal compilation c© 2010 Biochemical Society

M. Novinec and others

The steady-state reaction rate is given by eqn (8):

vs = V T

1 + K Tm

[S]+

k3

K Tm

[S]+ k6

k−3

K Rm

[S]+ k−6

(1 + K R

m

[S]

)

+ V R

1 + K Rm

[S]+

k−3

K Rm

[S]+ k−6

k3

K Tm

[S]+ k6

(1 + K T

m

[S]

)(8)

where V T = k2 [E]t and V R = k5 [E]t are limiting rates of the Tand R states respectively. If we assume that ET and ER are inequilibrium in the absence of substrate, the reaction rate at zerotime can be described by eqn (9):

vz =V T

[S]

K Tm

1 + k3

k−3

+V R

[S]

K Rm

1 + k−3

k3

(9)

General modifier mechanism and the specific velocity plot

The general modifier mechanism [2] describes the interaction ofmodifier (A) with enzyme (E) according to Scheme S3.

Scheme S3

For the mechanism in Scheme S3 the reaction rate in the presenceof modifier, vA, is defined as:

vA =v0 (1 + σ )

(1 + β

[A]

αKA

)

1 + [A]

KA

+ σ

(1 + [A]

αKA

) (10)

where v0 is the reaction rate in the absence of modifier, KA isthe equilibrium dissociation constant of the EA complex, α andβ are dimensionless coefficients and σ = [S]/Km. The equationwas derived under the assumptions of quasi-equilibrium for thebinding of A to E and ES, and steady-state for the fluxes aroundES and ESA.

The specific velocity plot [3] is a handy graphical method forplotting kinetic results and determining interaction parameters.For this purpose eqn (10) is rewritten as:

v0

vA

=[A]

(1

αKA

− 1

KA

)

1 + β[A]

αKA

σ

1 + σ+

1 + [A]

KA

1 + β[A]

αKA

(11)

The plot of v0/vA against σ /(1 + σ ) always produces straight lineswith intersection points at v0/vA = 1, regardless of the interactionmechanism. The values of parameters α, β and KA are determinedby 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:

a

a − 1= αKA

α − β

1

[A]+ α

α − β(12)

and

b

b − 1= αKA

1 − β

1

[A]+ 1

1 − β(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 isshown in Scheme S4.

Scheme S4

Scheme S4, which takes into account the binding of modifier Ato enzyme E in two steps, with both steps affecting the catalyticproperties of the enzyme and its affinity for substrate S. Thefirst step occurs rapidly, whereas the second involves a slowisomerization. As discussed by Szedlacsek and Duggleby [4], ananalytical expression for such a system is difficult to obtain andwould be of little practical use due to a large number of variables.

Given we are primarily interested in studying the net effectof GAGs on cathepsin K, we have simplified the mechanism inScheme S4 by separately analysing the effect of GAGs at zerotime and at steady-state. If we only consider a short interval atthe beginning of the reaction (t = 0) when no EA∗ complex isyet formed, and we assume that all components present are inquasi-equilibrium, the mechanism can be described by the generalmodifier mechanism [2], as shown in Scheme S5.

Scheme S5

c© The Authors Journal compilation c© 2010 Biochemical Society

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 experimentsenzyme 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 GAGswas 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 lossby 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 byTopham and Brocklehurst [5]. The reaction rate for Scheme S5 isdescribed by eqn (14):

vz =v0 (1 + σ )

(1 + βz

[A]

αz KA,z

)

1 + [A]

KA,z

+ σ

(1 + [A]

αz KA,z

) (14)

where vz and v0 are the reaction rates in the presence and absenceof modifier A, KA,z is the equilibrium dissociation constant of theEA complex, αz and βz are dimensionless coefficients and σ =[S]/Km; the subscript z consistently indicates that the variablesrefer to zero time.

When the system has reached steady-state the whole mech-anism shown in Scheme S4 is necessary to accurately describe allspecies present. To simplify the mathematical treatment SchemeS4 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 andassuming that all species are at quasi-equilibrium as shown inScheme S6.

Scheme S6

In Scheme S6 species EA′ and EA′S are defined as:

[EA′] = [EA] + [

EA∗] (15)

and

[EA′S

] = [EAS] + [EA∗S

](16)

KA,s in this case is not a true equilibrium constant, but a compositeparameter that includes information about both steps in themechanism (EA and EA∗). The same is true for the coefficients

Figure S4 Autodegradation of cathepsin K in the absence and presenceof HP

Samples of cathepsin K (CatK; 3 μM final concentration) were incubated at 37◦C in 50 mMHepes, pH 7.40, containing 1 mM EDTA and 2.5 mM DTT for 15 min, 30 min or 60 min in theabsence or presence of HP (0.2 mM final concentration). Reactions were stopped by addition of5 μM E-64 and analysed by SDS/PAGE (17 % gel). Protein bands were stained with CoomassieBrilliant Blue. Positions of calibrating proteins (in kDa) are given on the left-hand side.

αs and β s. The reaction rate for Scheme S6 is given by:

vs =v0 (1 + σ )

(1 + βs

[A]

αs KA,s

)

1 + [A]

KA,s

+ σ

(1 + [A]

αs KA,s

) . (17)

c© The Authors Journal compilation c© 2010 Biochemical Society

Allosteric regulation of cathepsin K

Interaction of cathepsin K with HP (heparin)

Heparin binds cathepsin K at two sites. One binding mode isidentical to that observed with CS/DS and the effect of slowbinding is still observed in progress curves recorded at very lowHP concentrations (Figure S3C). However, as the steady-statetreatment would result in complex expressions beyond practicaluse, we analyse only the steady-state parts of the curves wherewe assume quasi-equilibrium conditions, as discussed above forCS/DS. The secondary site is bound in a rapid manner and is notexclusive with respect to the primary binding site. The overallbinding of HP was analysed with a modified version of the modeldescribing simultaneous binding of two modifiers to one enzyme,adapted from the equation described by Schenker and Baici [6],as shown in Scheme S7:

Scheme S7

A and A′ represent two molecules of HP that bind to a singlemolecule of enzyme E. The reaction rate for the system in SchemeS7 is given by eqn (18):

vA =

v0 (1 + σ )

(1 + β1

[A]

α1 KA,1+ β2

[A]

α2 KA,2+ β12

[A]2

eKA,1 KA,2

)

1 + [A]

KA,1+ [A]

KA,2+ [A]2

cKA,1 KA,2+ σ

(1 + [A]

α1 KA,1+ [A]

α2 KA,2+ [A]2

eKA,1 KA,2

)

(18)

where α1, β1 and KA,1 describe the binding of the first moleculeof A, α2, β2 and KA,2 describe binding of the second molecule,β12 describes the catalytic properties of the enzyme when bothmolecules of A are bound, the coefficient c defines the interactionbetween both molecules of A and e is a combined interactionconstant for the formation of the quaternary complex [6].

EXPERIMENTAL

Docking and molecular dynamics simulations

Molecular dynamics simulations were performed using cathepsinK alone or in complex with the substrate Ala-Gly-Leu-Glu-Gly-Gly-Asp-Ala (the cleavage site is after the first glutamateresidue). The octapeptide was constructed with PyMOL (DeLanoScientific; http://www.pymol.org) and then docked into the activecentre of the enzyme (PDB code 1ATK) using AutoDock 4 [7]with the Lamarckian Genetic docking algorithm. In the dockingcalculation only the ligand (substrate) was defined as flexible,

Figure S5 Computer model of Z-FR-AMC bound into the active site ofcathepsin K

The substrate is shown as sticks. The enzyme is shown in surface representation and the catalyticresidues Cys25 and His162 are coloured green and blue respectively.

whereas the receptor (enzyme) was treated as rigid, i.e. no flexibleresidues were defined. The complex was solvated with the Solvateplugin in the VMD program [8]. A molecular dynamics simulationwas then performed with the NAMD program [9] at a constanttemperature of 310 K, with periodic boundary conditions andCHARMM 27 force-field parameters [10]. An initial 1000-stepenergy minimization was included in the calculation to removebad contacts in the initial model. To repeat the simulation withcathepsin K alone, its co-ordinates were extracted from theminimum energy conformation observed in this simulation,the molecule re-solvated and molecular dynamics re-run usingthe same parameters. The same procedure was also performedstarting from the crystal structure of cathepsin K alone (PDB code1ATK). All simulations were performed for 2 ns. Energies of thewhole system, as well as of the protein alone, were calculatedthroughout the simulations to ensure that a stable conformationof the protein had been reached.

The Z-FR-AMC molecule was constructed with theCambridgeSoft ChemOffice 11 Suite. It was then docked intothe active site of cathepsin K using AutoDock 4 [7] with theLamarckian Genetic docking algorithm. All images were createdwith PyMOL.

Interaction site mapping by chemical cross-linking

Pro-cathepsin K was first reacted with the cross-linking reagentsulfo-SBED at room temperature (25 ◦C) for 1 h in the dark at amolar ratio of 1:3. The reaction mixture was then extensivelydialysed to remove the unreacted cross-linker and sulfo-SBED {sulfo-N-hydroxysuccinimidyl-2-[6-(biotinamido)-2-(p-azido 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 byincubating the protein (final concentration 2 μM) with HP(0.8 mg/ml) in 50 mM Hepes, pH 7.4, containing 150 mM NaClin the dark for 30 min and then irradiating the sample witha UV lamp. The sample was digested with LysC over nightat 37 ◦C and then applied to a CIM-QA monolithic column(BIA Separations). HP chains were eluted from the disk in alinear 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 mMNaCl, saturating concentrations of GAGs or DxS and/or 5 mg/ml soluble ETNA-elastin) in50 mM Hepes, pH 7.40, containing 1 mM EDTA and 2.5 mM DTT with shaking on an EppendorfThermomixer Compact. Half-lives were determined by measuring residual enzyme activity atregular time intervals using the substrate Z-FR-AMC. Results are best fit values (+− S. E. M.) fromnon-linear regression analysis using a first-order decay function to calculate a decay constantk , from which half-life = In2/k .

Conditions Half-life (min)

E only 7.0 +− 1.0E plus NaCl (300 mM) 1.0 +− 0.3E plus CS 5.5 +− 0.5E plus DS 6.0 +− 0.5E plus HP 37.9 +− 1.1E plus DxS 37.2 +− 1.4E plus elastin 33.0 +− 2.0E plus elastin and HP 190 +− 20

avidin-affinity chromatography. HP-linked peptides were thenreleased by treatment with DTT and separated by HPLC on a C-18 column in a linear 0–100 % acetonitrile gradient. All peptideswere identified by N-terminal sequencing.

Docking of GAG octasaccharides to cathepsin K

A hexasaccharide model of CS (chondroitin-4-sulfate; con-sisting of three 4-sulfo-N-acetylgalactosamine-glucuronic aciddisaccharides) and octasaccharide models of DS (consisting of

four 4-sulfo-N-acetylgalactosamine-iduronic acid disaccharides)were constructed with the CambridgeSoft ChemOffice 11 Suite.Torsion angles between monosaccharides were adjusted manuallyaccording to published data [11] and the structures then optimizedby energy minimization using the MMFF94 force-field. HPmodels were retrieved from the PDB code 1HPN. Co-ordinatesfor cathepsin K were extracted from the structure of the cathepsinK–CS complex (PDB code 3C9E).

All docking calculations were performed with AutoDock 4[7] using the Lamarckian Genetic docking algorithm. In allcalculations 50 runs were performed with populations of 300individuals run for 3000 generations. Docking of DS and HPoctasaccharides to the CS-binding site identified in the crystalstructure of the cathepsin K–CS complex [12] was performed withflexible side chains of receptor residues Lys9, Lys10, Lys147, Lys173

and Lys191. Two separate calculations were performed for eachligand, one using only rigid glycosidic bonds in the ligand andthe other using flexible glycosidic bonds 4 and 7 in each ligand.In the first set, the lowest energy solution was selected from thosethat contained the DS chain running in the same direction as inthe crystal structure. In the second set, the primary criterion formodel selection was a conformation equivalent to that seen in thecrystal structure of the cathepsin K–CS complex. Within these, thesolution with the lowest binding energy was selected. Docking ofHP octasaccharides to the second binding site as was performedwith a rigid receptor molecule and rigid glycosidic bonds in theligand. The selection criteria are described and discussed in theResults and Discussion below.

Surface potentials were calculated with the Adaptive Poisson–Boltzman Solver [13]. All images were created with PyMOL.

Figure S6 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 shownas 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. Residuesinvolved 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

(A) Difference between the conformations of free CS in solution and CS in complex withcathepsin K. (B) Binding of CS/DS to cathepsin K (in surface representation) illustratedwith a DS octasaccharide as an example (shown as sticks). The binding proceeds via a slowconformational adaptation of the GAG to the structure of cathepsin K. (C) All solutions obtainedwith Autodock that conform to experimental data. The models were constructed using Autodock4. The surface potential of cathepsin K was calculated with APBS software and the structuresvisualized with PyMOL.

RESULTS AND DISCUSSION

Conformational flexibility studied by molecular dynamics

The experiments presented in Figure 3 of the main paper indicatethat cathepsin K can undergo a conformational change uponbinding of substrate. We further investigated this hypothesis insilico by molecular dynamics simulations of free enzyme andenzyme in complex with an octapeptide substrate (Ala-Gly-Leu-Glu-Gly-Gly-Asp-Ala). This sequence was chosen based on thespecificity matrix of cathepsin K as retrieved from the MEROPSdatabase [14]. It represents an ‘ideal’ substrate, composed ofresidues 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 entireactive centre of the enzyme.

In the molecular dynamics simulation of the enzyme–substratecomplex the active centre adapted to the shape of the substratein the time frame within 1 ns by becoming narrower and deeper(Figure S6A, left-hand panel) and remained in that conformationfor the rest of the simulation (2 ns). The substrate molecule wasthen removed from the final conformational state of the complex

Figure S8 Structural models of HP binding to cathepsin K

(A) Positions of Lys10, Lys39 and Lys77 (shown in shades of blue) that were cross-linked withHP. (B) Model of a HP octasaccharide binding to the primary binding site on cathepsin K ina manner analogous to CS/DS. (C) Three possible binding modes of HP at the secondaryHP-binding site on the bottom of cathepsin K. The putative binding site is composed of sevenpositively charged residues (coloured in shades of blue). In all figures the enzyme is shown insurface representation and HP is shown as sticks. In (B) the enzyme is coloured according tothe electrostatic potential calculated with APBS. All structures were visualized with PyMOL.

and a second molecular dynamics simulation was performedstarting from this conformation of enzyme to mimic the conditionsafter product release from the active site. Within 200 ps theactive centre of the free enzyme ‘opened’ to become wide andshallow (Figure S6A, right-hand panel) and remained in thisconformation for the remainder of the simulation (2 ns). Thesame conformation was also reached in a separate moleculardynamics simulation of enzyme alone started directly from thecrystal structure. Altogether the simulations suggest that cathepsinK adapts its conformation upon substrate ‘binding and release’in a manner consistent with the ‘induced fit’ hypothesis [15]. Inthe absence of substrate the enzyme adopts a conformation inwhich the active centre is easily accessible. Upon binding of asubstrate molecule the enzyme adapts its conformation to bringthe substrate into the proper position for the catalytic step.

The major structural differences between the two conforma-tions in the active centre region are illustrated in the superpositionin Figure S6B (the ‘free’ enzyme is shown in orange and substrate-bound enzyme in blue). The most notable change involves theloop Gln19–Cys22 that lines the left side of the active centre. Inthe presence of substrate the loop moves over the active centre

c© The Authors Journal compilation c© 2010 Biochemical Society

M. Novinec and others

and encloses the substrate within, whereas in absence of the latterthe loop swings in the opposite direction and widely exposes theactive centre. Binding of substrate also causes a conformationalchange in the loop containing Gly65 and Gly64 that interact withthe P3 and P4 positions of the substrate and is directly connectedto Gln21 via the disulfide bond Cys22–Cys63. Within the activecentre the positions of the catalytic diad Cys25–His162 do not differsubstantially between both conformations. It is, however, worthnoticing that in the unbound enzyme the side chain of Cys25

faces away from the position assumed during catalysis and onlyadopts this position in the presence of substrate, which is a furthercharacteristic of the ‘induced fit’ model. Of the residues involvedin the catalytic mechanism, the most notable conformationalchange involves rotation of Trp184, a residue critical for the properpositioning of the catalytic His162 [16]. In the absence of substrateits indole ring freely rotates by approx. 30◦, but mostly remains inthe plane of the catalytic diad. Upon substrate binding the indolering 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 theright side of the active site, including Asn187, Gln143 and Phe144.

Comparing these simulations with experimental resultspresented in the main paper, it is feasible to say that the substrate-bound and free enzyme states show structural differences thatwould be expected to exist between the T and R statesrespectively. The narrower active-site groove of the substrate-bound conformation parallels the lower substrate affinity of the Tstate, whereas proper positioning of the catalytic Cys25 explainsthe higher catalytic constant. Moreover, the shape of the activesite in the substrate-bound conformation is too narrow to allowefficient binding of a relatively bulky macromolecular inhibitor.

Structural interpretation of GAG binding

Activity assays (Figures 4 and 5 of the main paper) and intrinsicfluorescence measurements (Figure S2) demonstrate that GAGsact as allosteric regulators that alter the conformation and activityof cathepsin K by binding at a site other than the active centre.The location of this site has been revealed recently by the crystalstructure of a cathepsin K–CS complex [12]. Comparing thestructure of CS in this complex with the calculated structure ofCS in solution [11] (Figure S7A) shows that rotations of multipleglycosidic linkages are needed to commit the GAG chain to theconformation seen in the crystal structure. A local change of GAGconformation upon binding to a receptor is not unusual and canbe considered as a specific recognition motif for target proteinbinding [17]. On the basis of this result, the slow activation ofcathepsin K observed in our experiments can be attributed to aslow conformational adaptation of CS/DS as illustrated in FigureS7(B) using DS as an example. The enzyme initially interactswith an extended GAG molecule and this interaction is sufficientto increase the enzyme’s activity. The initial complex then slowlyrearranges to allow for a more tight interaction between enzymeand GAG. It should be noted that the docking calculationswere performed with GAG octasaccharides, whereas the crystalstructure shows a CS hexasaccharide bound to cathepsin K [12].In the latter, the N-acetyl-galactosamine at the non-reducing endis positioned in an orientation that would prevent the GAG chainfrom extending beyond this residue. To account for the polymernature of GAGs, a chain with two additional monosaccharides atthe non-reducing end was therefore chosen for the dockings. Ofcourse, the binding mode shown in Figure S7(B) was not the onlydocking solution obtained. Our primary criterion for filtering theresults was that the orientation of the GAG chain in the modelcorresponded to that in the crystal structure and further ranking

of the obtained solutions was performed based on the calculatedbinding energies. To illustrate the degree of binding flexibility atthis site, Figure S7(C) shows all the docking solutions obtainedthat satisfy the primary criterion obtained with a rigid GAG chain(Figure S7C, left-hand panel) and a flexible GAG chain (FigureS7C, right-hand panel).

Binding kinetics show that HP binds cathepsin K at twodifferent sites. Their locations were determined by chemicallycross-linking the enzyme with the ligand using the photo-reactive cross-linker sulfo-SBED. After proteolytic digestion ofthe complex, three HP-linked peptides were identified on thebottom and back sides of cathepsin K, containing the cross-linker bound to Lys10, Lys39 and Lys77 (Figure S8A). At very lowconcentrations, the effect of HP was analogous to CS/DS. It istherefore logical to assume that under these conditions HP bindsto cathepsin K in a manner analogous to CS/DS (Figure S8B).For clarity, only one docking solution is shown and it should bestressed that this model is purely illustrative, as it shows a HPchain sulfated at every possible position, whereas in nature thedegree and patterns of sulfation are highly heterogeneous.

The location of the second HP-binding site was predicted onthe bottom of the molecule. As there is no experimental evidenceabout the length of the HP fragment that interacts with theprotein, docking of HP octasaccharides to this site was performedwith non-rotatable glycosidic bonds to avoid unrealistic twistingof the HP chain in the docking solutions. Three clusters ofdocking results were identified that conformed with cross-linkingexperiments, i.e. distance of the HP chain was no more than16 Å from the ε-amino group of Lys77. The energetically mostfavourable docking poses from each of these clusters are shownin Figure S8(C). The central part of the second binding site iscomposed of six basic residues (Lys40, Lys41, Arg108, Arg111, Arg127

and Lys214) organized in a ring-shaped structure on the bottomof cathepsin K. In addition to these, the octasaccharide formscontacts with Lys10, which is also involved in binding of GAGs atthe primary GAG-binding site.

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Received 5 March 2010/28 April 2010; accepted 7 May 2010Published as BJ Immediate Publication 7 May 2010, doi:10.1042/BJ20100337

c© The Authors Journal compilation c© 2010 Biochemical Society