the binding mode of an £-64 analog to the active site of cathepsin b

Protein Engineering vol.9 no. 11 pp.977-986, 1996

The binding mode of an £-64 analog to the active site ofcathepsin B

Ming-Hsiang Feng1>2, Shek Ling Chan3, Yuefang Xiang4,Carol P.Huber5 and Carmay Lim1>2'6

'institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan 11529,2Department of Chemistry, National Tsing-Hua University, Hsin-chu,Taiwan 30043, Republic of China, 'Biotechnology Research Institute,National Research Council of Canada, Montreal, 4Department ofBiochemistry, University of Toronto and department of Biochemistry,University of Ottawa, Canada

'To whom correspondence should be addressed

Two binding modes of the isobutyl-NH-Eps-Leu-Pro inhib-itor to cathepsin B have been proposed. Molecular dockingusing an empirical force field was carried out to distinguishbetween the two modes. The search began with manualdocking, followed by random perturbations of the dockingconformation and cycles of Monte Carlo minimization.Finally, molecular dynamics was carried out for the mostfavorable docking conformations. The present calculationspredict that the isobutyl-NH-Eps-Leu-Pro inhibitor prefer-entially binds to the S' rather than the S subsites ofcathepsin B. The S' binding mode prediction is supportedby the X-ray crystal structure of cathepsin B bound to aclosely related ethyl-O-Eps-Ile-Pro inhibitor, which wasfound to bind in the S' subsite with the C-terminal epoxyring carbon making a covalent bond to the sulfur atom ofCys29. This agreement, in turn, validates our dockingstrategy. Furthermore, the calculations provide evidencethat the dominant contribution to the total stabilizationenergy of the enzyme-inhibitor complex stems from thestrong electrostatic interaction between the negativelycharged C-terminal carboxylate group of the ligand andthe positively charged imidazolium rings of HisllO andHislll. The latter are stabilized and held in an optimalorientation for interactions with the C-terminal end of theligand through a salt bridge between the side chains ofHisllO and Asp22. By comparison with the crystal struc-ture, some insight into the specificity of the epoxyldipeptidefamily towards cathepsin B inhibition has been extracted.Both the characteristics of the enzyme (e.g. subsite sizeand hydrophobicity) as well as the nature of the inhibitorinfluence the selectivity of an inhibitor towards an enzyme.Keywords: cathepsin B/conformational search/cysteine pro-tease/inhibitors/molecular dynamics

Introduction

Cysteine proteases are very important since they have beenimplicated in various disease states (Prous, 1986; Katunumaand Kominami, 1987; Van Noorden et ai, 1988; North et ai,1990; Sloane et ai, 1990). They are essential in the life cyclesof a number of protozoan parasites (North et al., 1990),e.g. Trypanosoma brucei (sleeping sickness), Plasmodiumfalciparum (malaria) and Entamoeba histolytica (dysentry).Mammalian cysteine proteases, such as cathepsins B, H andL, have been implicated in diseases that involve aberrant

protein turnover, e.g. muscular dystrophy (Katunuma andKominami, 1987), bone resorption (Delaisse et al., 1991),tumor invasiveness (Sloane et al., 1990; Liu et al., 1992),pulmonary emphysema (Harris et al., 1975), arthritis andinflammatory diseases (Mort et al., 1984; Trabant et al., 1984;Van Noorden et al., 1988). The calcium-dependent cysteineproteases calpain I and II are involved in degenerative diseases,e.g. Alzheimer's disease (Nilsson et al., 1990) and myocardialtissue damage (Prous, 1986). Thus, for the treatment of thesediseases, it is very desirable to develop highly selective andpotent inhibitors targeted specifically for the cysteine proteaseinvolved in the disease, without concurrent inactivation ofother cysteine or serine proteases.

It has been shown that l-[L-A^-(fra«5--epoxysuccinyl)leucyl]-amino-4-guanidinobutane, commonly known as E-64(Figure la), isolated from the culture of Aspergillus japonicusTRP-64 (Hanada et al., 1978a,b), is a potent irreversibleinhibitor towards several cysteine proteases. Although E-64has no preference for any particular cysteine protease, thepotency of this inhibitor coupled with its low toxicity indicatesa potential use for the epoxide and its derivatives as a clinicaldrug for the treatment of diseases involving cysteine proteases.Studies by Prous (1986), Noorden et al. (1988), Yamamotoet al. (1989) and North et al. (1990) have shown that E-64analogs such as loxistatin and NCO-700 may be beneficialfor muscular dystrophy (Yamamoto et al., 1989) and acutemyocardial infarction (Prous, 1986) respectively. However, theselectivity of these inhibitors must be enhanced for thera-peutic uses.

Recently, a novel type of epoxysuccinyl peptide (E-64analogs), R-Eps-Ile/Leu-Pro (Figure lb), with the terminalcarboxylic acid of the epoxide (R) either esterified or convertedto an amide functionality, has been reported to show strongselectivity towards cathepsin B over other cysteine proteases(Murata et al., 1991; Towatari et al., 1991; Sumiya et al.,1992). For example, the analog with R = n-propyl-NH has aninhibitory activity value of 2 nM for cathepsin B but is of theorder of 105 nM for cathepsin L and H and calpain II (Sumiyaet al., 1992). By analogy to the binding mode of E-64to papain obtained from the papain/E-64 crystal structure(Varughese et al, 1989), Sumiya et al. (1992) assumed thatE-64 analogs bind to the S subsites of cathepsin B (seeFigure Id). In contrast, Buttle et al. (1992) and Gour-Salinet al. (1993) proposed that E-64 analogs bind to the S' subsitesof cathepsin B (see Figure lc) with the negatively charged C-terminal carboxylate group of the inhibitor interacting withthe positively charged HisllO and Hislll residues. This isbecause derivatization of the carboxylate on the epoxide ring(R = ethoxy or isobutyl-NH) confers selectivity for cathepsinB over papain only when it is linked to a dipeptidyl moietywith a free negatively charged C-terminal residue (Gour-Salinet al., 1993).

The aim of this work is to distinguish between the twodifferent proposed binding modes of an E-64 analog inhibitor

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NH-CH2CH2CH2CHrNH-C<(x®

(b)

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occluding loop

(c)

Fig. 1. Chemical structures of (a) E-64 and (b) E-64 analog. Schematicrepresentations of (c) the S'B binding mode in which the inhibitor binds tothe S' subsite with Cys29 S forming a covalent bond with the epoxyl ringcarbon near the butyl end and (d) the SC binding mode where the inhibitorbinds to the S subsite with Cys29 S forming a covalent bond with theepoxyl ring carbon near the C-terminal end; dashed lines partition the E-64analog into three residues: Pro, Leu and Luu (involving the butyl group).

to cathepsin B. The inhibitor studied in this work is isobutyl-NH-Eps-Leu-Pro inhibitor, which shows a 93-fold preferencefor cathepsin B over papain (Gour-Salin et al., 1993). The 3-DStructure of human liver cathepsin B (C.P.Huber, R.L.Campbell,T.Hirama, X.Lee, J.S.Morb, R.To and S.Hasnain, in prepara-tion) has been determined to a resolution of 2.15 A at pH 4.As the active-site conformation of cathepsin B at physiologicalpM could differ from that in the crystal structure, a stochasticboundary molecular dynamics (MD) simulation was carriedOut at pH 6 starting from the X-ray structure of human livercathepsin B. The average MD structure of cathepsin B withthe catalytic cysteine and histidine in the ion-pair state wasemployed in the docking of the inhibitor. The energy-minimizedisobutyl-NH-Eps-Leu-Pro inhibitor was docked into the S andS' subsites with either epoxy ring carbon making a bond with

978

Table I. Parameters used for the covalent linkage between Cys29 and theepoxide ring carbon

Parameter1 Approximation0 9°

S-CT-CS-CT-HBC-CT-S-CTHB-CT-S-CTS-CT-C-OS-CT-C-NI1

S-CT-CTS-CT-HACT-CT-S-CTHA-CT-S-CTX-CT-CC/CD-XX-CT-CC/CD-X

46.446.1

0.310.280.050.05

109.5111.3180.0

0.0180.0180.0

-_3366

The parameters for the angles and dihedrals in the first column areapproximated by available CHARMM parameters in the second column CTand C refer to tetrahedral and polar carbons respectively, CC or CD refer tothe carbonyl carbons of Asn, Asp, Gin and Glu side chains, HA and HBrefer to non-polar and backbone hydrogens respectively and X refers to anyatom.bk is the force constant.°0 is the angle corresponding to the equilibrium geometry.dn is the multiplicity of the dihedral angle.

Table II. Measured pK, values of ionizable groups in cathepsin B*

Ionizable group P*.

Cys29HisllOHislllHisl99Glu245

3.66.97.78.65.1

"From Hasnain et al. (1992).

the sulfur atom of Cys29; the inhibitors shown in Figure l(c)and (d) are referred to as B and C respectively (correspondingto the Cys29 sulfur making a covalent bond with the epoxidering carbon near the butyl or C-terminal end of the ligand).The simulation procedure and docking strategy are outlined inthe next section, followed by a description of the results forthe various binding modes. The predicted binding mode iscompared with available experimental data and the implicationsfor a structure-based inhibitor design are discussed in thefinal section.

MethodsParametersThe CHARMM version 22 all-hydrogen parameter set(Mackerell et al., 1992) was employed in the simulations. TheE-64 analog was partitioned into three residues, Pro, Leu andLuu, as shown by the dashed lines in Figure l(c) and (d). Inbinding Cys29 to one of the epoxide ring carbon atoms,the charge on the deleted cysteine hydrogen (+0.26) wasredistributed so that the charge on the sulfur atom was thesame as that found in disulfide bridges, whereas the remainingcharge was allocated to the attacked carbon. The resultingcharges on the Cys29 sulfur and the attacked carbon are—0.09 and -0.06 respectively. The missing parameters wereapproximated by the available parameters of similar atomtypes and are listed in Table I.

Protonation states of ionizable groupsThe simulations were carried out at the pH (6) of the inhibitorstudies (Gour-Salin et al., 1993), which is near physiologicalpH. The pKa values of the ionizable groups in cathepsin Bhave not been measured with the exception of Cys29, His 110,Hisl l l , His 199 and Glu245 (see Table H). To determine the


protonation states of the ionizable groups with unknown pKa

values in cathepsin B at pH 6, their electrostatic potentials atthe dissociation site were calculated and compared to those insolution. The electrostatic potentials in the enzyme and solutionwere found to be similar except for the last residue, Asp254.This is probably due to repulsion between the negativelycharged side chain and C-terminal carboxylate groups, sug-gesting that the side chain of Asp254 should be neutralized.Thus, at pH 6, Asp254 was assumed to be protonated and theother aspartic and glutamic residues were assumed to bedeprotonated. Cys29 was ionized due to its low pAT, value(3.6) (Hasnain et al., 1992), whereas the other ionizable groups(His, Lys and Arg) were assumed to be protonated.

Simulation procedureThe stochastic boundary MD (Brooks et al., 1988) simulationswere carried out using the program CHARMM (Brooks et al.,1993). The center of the system was chosen to be Cys29 Sfor the free enzyme and the enzyme-inhibitor complex. Theprotein and solvent atoms within 14 A were propagatedaccording to Newton's equation using the Verlet algorithm anda time step of 1 fs. The non-hydrogen protein and solventatoms in the volume outside 14 A and inside 18 A werepropagated according to the Langevin equation with harmonicconstraints determined from average X-ray temperature factors(Straub et al., 1994). Atoms outside 18 A were fixed. Thelong-range electrostatic forces beyond 12 A were approximatedby a multipole expansion up to the quadrupole term. The entiresolvated system was equilibrated for 20 ps followed by 54 psof dynamics for the free enzyme and 80 ps for the enzyme-inhibitor complex. The average structures of the free enzymeand enzyme complexes were obtained by averaging structuresfrom every 100 fs of the MD trajectory. Each picosecond ofdymamics takes ~4 c.p.u. h on an IBM 340.

Docking procedureThe search for the favorable docking conformations of theinhibitor was carried out in three stages. First, the target ligandwas built in an extended conformation using the BIOSYMINSIGHT II graphics package and visually docked into the Sand the S' subsites of the average MD structure of cathepsinB. In each binding mode, the sulfur of Cys29 formed a covalentbond with one of the epoxide ring carbons near the butyl orC-terminal end of the ligand. This generated four startingstructures, which were subsequently energy minimized usingthe CHARMM program (Brooks et al, 1983) to yield SB0,SQ), S'B0 or S'CQ; the first two correspond to the inhibitorbound in the S subsites and the latter two in the S' subsites.

In the second stage, each of the four starting structureswas subjected to random perturbations, then solvent overlayfollowed by energy minimization, as summarized in FlowchartI. Random perturbation was achieved by performing 10 stepsof MD (using a 1 fs time step) at a high temperature of1000 K; this was carried out in the absence of solvent toenable a thorough search of the conformational space aroundthe starting point. Since the ligand is small (~15 A) relativeto the enzyme (-30 A X -45 A X -60 A), its binding willlikely have a negligible effect on atoms far away from thebinding site. Hence, atoms greater than a cut-off distance of8 A from any ligand atom were fixed during the perturbationstep. The perturbed structure from the final step of MD at1000 K was overlaid with an 18.5 A sphere of equilibratedTIP3P water molecules (Jorgensen et al., 1983) centered onthe Cys29 sulfur atom. Water molecules that overlapped with

Binding mode of an E-64 analog to cathepsin B

Flowchart I.

Initial visually dockedstructures.

No waters included.

V

Random perturbation ofatomic position within cutoff R.

(executed by M.D. at 1000K)

F

i

Overlay perturbed structurewith a sphere of water molecules.Delete waters that overlap with

original structure.

Energy minimization.

Energy minimizedstructure.

the host atoms were deleted. The overlay procedure wasrepeated four times with different orientations. The entiresolvated system was energy minimized using steepest descentwith a 5 A non-bond cut-off for 300 steps followed by anadopted-basis Newton—Raphson with an 8 A cut-off for 200steps and a 10 A cut-off for another 200 steps. The entireprocedure outlined in Flowchart I was repeated 1000 times.In order to compare the energies of the resulting structures,the number of atoms in the system was unified by deletingwater molecules furthest from the Cys29 sulfur atom untileach structure contained the same number of (1200) watermolecules. The resulting system was further energy minimizedusing an adopted-basis Newton-Raphson with a non-bond cut-off of 13 A until successive energies differed by <0.1 kcal/mol. Each perturbation—minimization cycle took —2.5 h on anIBM 340.

The procedure outlined above (Flowchart I) gives a coarsesearch for the favorable docking positions; the resulting lowestenergy structures corresponding to the starting structures, SBo,SCo, S'B0 and S'Q, are denoted by SB,, SC,, S'B, and S'C,respectively. The latter were employed as the starting pointsfor structure refinement using a Monte Carlo minimization(MCM) procedure (Caflisch et al., 1992), as summarized inFlowchart II. Atoms within 6 A of any ligand atom were

.979


M.-H.Feng et al.

Flowchart II.

Random perturbation of atomicpositions (executed by MD ')

Use Metropolisalgorithm to decide

whether to keep newconformation-.

Monte Carlo minimization repeatedSO times per grand cycle.

Retain the conformations from the grand cyclewhich hold the record low of the energy for

more than 1 MC minimization cycle.

Full energy minimization usingadopted-basis Newton-Ralphson.

Choose lowest energy coafonnarionjjj

Table m . Energies before and after Monte Carlo minimization1

'At 1000 K for the first grand cycle, at 500 K for the next two grandcycles, then at 250 K for the fourth grand cycle onwards.2Temperarure at 100 K for the first three grand cycles, then at 50 K for thefourth grand cycle onwards.

perturbed by running MD at a temperature of 1000 K for threesteps. The resulting structure was then energy minimized for10 conjugate gradient steps using a non-bond cut-off of 13 A.If this gave a lower energy conformer, the new conformationwas used as the starting point for the next step, otherwise itwas accepted with a probability e^^ where 1/p is Boltzmann'sconstant multiplied by the simulated annealing temperatureand AE is the energy difference between the new conformationand the starting one. Pilot runs showed that for a simulatedannealing temperature of 100 K, MD at 500 K gave perturba-tions that would yield an acceptance rate of ~50%. TheMonte Carlo dynamics and subsequent short minimization wasrepeated 50 times. After each cycle, the energy of the structuremay increase or decrease with respect to the previous one.The structures which hold the record low energy for morethan one cycle were subjected to full energy minimizationemploying an adopted-basis Newton-Raphson method with acut-off of 13 A until successive energies differ by less than0.1 kcal/mol. The thoroughly relaxed structure that had thelowest energy was chosen as the starting point for the nextseries of Monte Carlo runs.

The above MCM procedure was repeated in series and thefully relaxed structure with the lowest energy in each serieswas chosen as the starting point for the next series of MonteCarlo runs. From the fourth series onwards, the dockingconformation was fine-tuned by subjecting the system torelatively small perturbations, performed by MD at 250 K anddropping the simulated annealing temperature to 50 K. Finally,the procedure in Flowchart II was stopped when the drop in

980

Startingstructure

Energy(kcal/mol)

Finalstructure

Energy(kcal/mol)

Number ofMCM cycles

SB,SC,S'B,S'C,

-21 228-21 177-21 318-21 303

SB2SC2

S'B2

S'C2

-21 228-21 212-21 331-21 308

1584

"Energies computed in the presence of 1200 waters using a constantdielectric of unity and a non-bond cut off of 13 A.

the energy of the thoroughly relaxed conformation was below1 kcal/mol over one series of runs. The numbers of MCMcycles required to obtain the four final structures, denoted bySB2, SC2, S'B2 and S'C2 and their final energies are tabulatedin Table HI. The S'B and S'C structures corresponding to theS' subsites are significantly lower in energy than the SB andSC structures corresponding to the S subsites (Table III). Thus,these two structures were subjected to stochastic boundaryMD simulations (as described above) to obtain the averageMD structures. In addition, an 80 ps simulation of cathepsinB complexed to "OOC-Eps-Leu-Pro in the S'C binding mode,starting from the S'C average structure with the NH-isobutylgroup removed, was carried out to study the effect of derivatiz-ation of the N-terminal carboxylate on its binding to cathep-sin B.

ResultsThe residue numbering used here corresponds to the 254-residue single chain form of cathepsin B. The backbone atomsof the unliganded cathepsin B crystal and dynamics-averagedstructures are superimposed and the conformations of active-site residues are illustrated in Figure 2. The active-site structuresof SB2, SC2, S'B2 and S'C2 resulting from the MCM runs areshown in Figures 3—6 and the hydrogen-bonding distancesbetween the Iigand and protein/water atoms are tabulated inTables IV and V.A hydrogen bond is defined by a heavyatom-heavy atom distance of *£3.5 A, a heavy atom-hydrogendistance of «2.5 A and a deviation of <60° from linearity.The dynamics-averaged structures of S'B and S'C are depictedin Figure 7.

Active-site structure of cathepsin BThere are two key differences between the crystal and theaverage MD active-site structures of cathepsin B (Figure 2):the initial Hisl99(ImH+) • • • Asn219(C=O) hydrogen bondin the X-ray structure of cathepsin B is broken and replacedby a Hisl99(ImH+) • • • Ser220(CF) hydrogen bond. This isin analogy to the papain ion-pair state simulation (Wang et al.,1994), where a sharp conformational transition is also observedat neutral pH. The second key difference is in the orientationof the thiolate ion relative to the imidazolium ring of His 199and Gln23. In the X-ray structure, Cys29(S") is 4.7, 3.5and 5.2 A from 0^23(1^) , Hisl99(N81) and Hisl99(N£2)respectively, whereas in the average MD structure, Cys29(S~)is nearly within hydrogen bonding range (3.6 A) of the amideside chain of Gln23 and is approximately equidistant (3.5 A)to the two side chain nitrogen atoms of Hisl99. This discrep-ancy may be due to the different protonation state of Cys29in particular, as well as that of the aspartic or glutamic acidresidues in general, in the crystal structure compared to theMD structure. These residues have pKa values close to the pH



ASN 219

HIS 199

SER 220

CYS29

Fig. 2. Active-site conformations of crystal (thick lines) and average MD(thin lines) structures of free cathepsin B after superposition of thebackbone atoms of the two structures.

GLY 172

Fig. 3. Hydrogen bonding network in the active-site region of SB.

^ P N * \ GLN 23

Fig. 4. Hydrogen bonding network in the active-site region of SC.

of crystallization (pH 4), so may exist in an anion—neutralequilibrium in the crystal structure, whereas they are alldeprotonated at pH 6 in the simulation. In the case of Cys29,the consequence of this anion-neutral equilibrium is thatneutral Cys29 is unable to hydrogen bond to Gln23(NE2) (asseen in the crystal structure), but anionic Cys29(S~) can interactwith Gln23(NE2) which forms the oxyanion hole (Figure 2).Apart from these differences, the active-site conformations inthe MD average and crystal structures are similar: the positivelycharged imidazolium nitrogens of HisllO form a salt bridgeto the negatively charged carboxylate side chain of Asp22,while Hislll interacts mainly with water molecules ratherthan forming hydrogen bonds with other residues.

Active-site structures of cathepsin B/isobutyl-NH-Eps-Leu-Pro inhibitor complexesS subsites. Figure 4 and Table IV show that in the SC2 MCMstructure, the ligand interacts with four water molecules: thePro carboxylate oxygens form three hydrogen bonds withwater molecules and the Luu carbonyl oxygen makes thefourth solvent hydrogen bond. Relative to the SC2 structure,the SB2 MCM structure (Figure 3) has one less hydrogen bondwith water molecules: a Pro carboxylate oxygen, Luu hydroxyloxygen and amide nitrogen each have one solvent hydrogenbond, resulting in three hydrogen bonds with water molecules(Table IV). The Luu residues of both structures are recognizedin common by Ala200; in addition, Luu interacts with Gln23and Asn72 in SC2. There are no positively charged residuesin the S subsites to stabilize the negatively charged carboxylateoxygen atoms of the inhibitor. Instead, the C-terminal of theinhibitor is stabilized by charge-dipole interactions with thebackbone amide groups of Glyl72 and Alal73 in SB2 andGly74 in SC2, in addition to its interactions with solventmolecules.

S' subsites. The hydrogen bonds found in the S' bindingpockets in the S'B2 and S'C2 MCM structures are similar(Figures 5 and 6; Table V). Both structures show hydrogenbonds between the negatively charged carboxylate oxygenatoms of the inhibitor and a water molecule as well as thepositively charged imidazolium nitrogen atoms of HisllO andHislll : H i s l l l ^ 8 2 ) makes bifurcated hydrogen bonds withboth carboxylate oxygens while HisllO(N81) hydrogen bondsto only one, Pro(OT1). Furthermore, both structures show thatthe key recognition histidine residues are stabilized by twohydrogen bonding interactions: HisllO(N81) is stabilized incommon by a salt bridge to a carboxylate oxygen of Asp22,whereas in S'B2 HisllO(N) hydrogen bonds to the carboxylateoxygen of Asp224 and in S'C2 Hislll(N) interacts with thecarbonyl oxygen of GlulO9. In both the S'B2 and S'C2

structures, the Leu carbonyl oxygen of the ligand hydrogenbonds to the side chain amide of Trp221, whereas the O4 andNT atoms of Luu are recognized by Glyl98(N) and Asn72(O)respectively. The Luu(O) and Luu(6") atoms in the S'C2

structure also form hydrogen bonding interactions with theside chain nitrogens of Gln23 and Trp30 respectively.

A comparison of the hydrogen bonds in S'B2 and S'C2 withthe corresponding dynamics-averaged MD structures (TableV) shows that the strong charge-charge interactions betweenthe ligand's Pro carboxylate oxygens and the HisllO andHis 111 imidazolium nitrogens as well as interactions stabilizingthe latter two residues are maintained. However, some of theweaker protein-ligand interactions are not preserved, e.g. theLeu(O)-Trp221(NEl) hydrogen bond in both structures and

981


M.-H.Feng et al.

Table IV. Hydrogen bonding distances (A) in SB2 and SC2 structures"

• CYS29

Fig. 5. Hydrogen bonding network in the active-site region of S'B.

HIS 111

LUU

Fig. 6. Hydrogen bonding network in the active-site region of S'C.

the LuuCOKjlnaSCN^), Luu(OH)-Trp30(N£l) and Luu(NT>-Asn72(O) hydrogen bonds in the S'C MD structure. The lossof these weak protein-ligand interactions is compensatedfor by additional solvent-ligand interactions found in thedynamics-averaged MD structures of S'B and S'C yielding atotal of six ligand-solvent hydrogen bonds for both structures.Furthermore, Luu(O) interacts with Gln23(Ne2) in the S'B MDstructure.

Discussion

Binding mode: S' versus SThe binding affinity of the isobutyl-NH-Eps-Leu-Pro inhibitorfor the S versus the S' subsites of" cathepsin B depends on therelative free energies of the S versus the S' enzyme-inhibitorcomplexes. This difference can be approximated by the energydifference between the two sites, assuming that inhibitorbinding is dominated by favorable energetic interactions withthe solvated enzyme. Table VI gives the total and interactionenergies of the ligand with the protein and solvent in theS'B, S'C, SB and SC MCM structures as well as the S'B andS'C-minimized MD average structures. Note that in order tocompare the energies, the number of water molecules in allthe structures was unified (see the Methods section).

As shown in Table VI, the total energies of S'B2 (-21 331kcal/mol) and S'C2 (-21 308 kcal/mol) are significantly morenegative (by 103 and 96 kcal/mol) than those of SB2 (-21 228kcal/mol) and SC2 (-21 212 kcal/mol). Thus, the inhibitor ispredicted to bind to the S' subsites of cathepsin B rather than

982

Donor/acceptor Donor/acceptor SB2 SC,

Pro(OTI)

Prc^O72)

Leu(N)Luu(O)Luu(O")

Luu(Cr»)

Luu(NT)

Gly74<N)Alal73(N)Wat 13(0)Glyl72(N)Wat29(O)Wat33(O)Wat59(O)Ala200(O)Ala200(N)Asn72(O)Ala200(N)Watl(O)

Wat 1(0)Wat2(O)

-2.7_2.9_-2.63.2__2.82.8-_

2.7-2.6_2.62.6--3.02.7_-3.12.7

3.0

"Structures obtained after Monte Carlo minimization; a dash denotes theabsence of a hydrogen bond.

the S subsites. This is in accord with the proposed bindingmode of Buttle et al. (1992) and Gour-Salin et al. (1993).Table VI also shows that the inhibitor prefers to bind in theS' subsites due to the presence of favorable interactions withthe protein atoms in that region (-213 kcal/mol for S'B2 and-228 kcal/mol for S'C2) compared to the S subsites (-112kcal/mol for SB2 and - 8 5 kcal/mol for SCJ. In contrast toligand binding in the S' subsites, which is dominated byfavorable ligand-protein rather than ligand-water interactions,both the former (-112 kcal/mol for SB2 and - 8 5 kcal/mol forSC^ and the latter (-73 kcal/mol for SB2 and -138 kcal/molfor SC2) contribute to inhibitor binding in the S subsites.

The protein-ligand interactions in S' are predominantlyelectrostatic in origin (-184 kcal/mol for S'B2 and -197kcal/mol for S'CJ, arising mainly from stabilizing interactionsbetween the negatively charged carboxylate group of theinhibitor and the positively charged imidazolium side chainsof HisllO and Hislll (see Figure 7 and Table V). This isevidenced by the very favorable ligand-protein energies ofPro (-159 kcal/mol) compared to Leu (-10 to - 1 5 kcal/mol)and Luu (-14 to —24 kcal/mol). It is also reflected by thestability of the hydrogen bonding interactions between the Procarboxylate oxygens of the inhibitor and the His 110 and His 111imidazolium nitrogens during the 80 ps MD trajectories ofS'B and S'C: the average distances and root mean squaredeviations for Pro(OT1)-Hisll0(N81), PitKC^'H-IisllltN62)and Pro(OT2)-Hisl 11(1^) in the MD S'C simulations are 2.63(0.07), 2.78 (0.14) and 2.78 (0.13) respectively. Since themajor contribution to inhibitor binding in the S' subsitestems from the strong charge-charge interactions involvingthe inhibitor's C-terminal carboxylate oxygens, the approxi-mate angle and dihedral parameters used for Luu (Table I),which do not contribute significantly to the stabilization energyof the complex (Table V), would not be expected to alter theconclusion that the isobutyl-NH-Eps-Leu-Pro inhibitor bindsto the S' subsites.

Compared to the significant energy difference (96-103kcal/mol) in inhibitor binding between the S versus S' subsites,the energy difference between S'B and S'C (23 kcal/mol forMCM and 13 kcal/mol for MD) is an order of magnitude less.There appears to be no energetic discrimination between thetwo final product states, S'B and S'C. This is probably becausethe key groups distinguishing the two states, namely the Luu



Table V. Hydrogen

Donor/acceptor

Pro(OTI)

ProtO1"2)

Leu/Ile(O)

Luu(O)

Luu(OH)

Luu(O4)

Luu(NT)Luu(OT1)

HisllO(NE2)HisllO(N)Hislll(N)

bonding distances (A) in S

Donor/acceptor

HisllO(N51)HislU(NE2)Wat34(O)Wat 160(0)Hislll(NE2)Wat24(O)Wat34(O)Wat37(O)Wat 160(0)Trp221(NEl)Watl(O)Wat24(O)Wat34(O)Wat37(O)Gln23(NE2)Cys29(N)Trp30(NEl)Wat 1(0)Wat2(O)Wat6(O)Wat7(O)Gly74(N)Glyl98(N)Wat3(O)Asn72(O)Wat21(O)Wat31(O)Asp22(O51)Asp224(O52)Glu109(0)

'B and S'C structures3

S'B2b

2.62.7--2.9-2.6_-3.22.9-_-------_-3.2-2.8

2.62.9-

<S'B>C

2.63.3-2.62.6-2.7-2.9-2.7-2.7-2.9--2.7-__-2.8-2.9

2.63.0-

S'C2b

2.63.2--2.62.6_--3.02.7---2.9-3.2-2.7---2.8-2.9

2.6-3.0

< S ' O C

2.62.82.6_2.72.6-2.6--2.8--2.8---_2.8-_-2.9--

2.6_2.8

<S 'CH> c d

2.63.4--2.62.6-_--2.73.1-----_-2.82.7

2.72.6-2.62.62.6-3.0

X-raye

2.7(2.6)3.0(2.9)

3.2(2.8)3.0(-)

3.1(3.0)

2.3(2.8)3.1(3.1)

3.1(3.1)

aA dash denotes the absence of a hydrogen bond.^Structures obtained after Monte Carlo minimization.'Structures averaged over the production trajectory.dAverage structure of cathepsin B-"OOC-Eps-Leu-Pro complex.cX-ray structure of cathepsin B inhibited with ethyl-Eps-Ile-Pro taken from Turk et al. (1995); a blank indicates that no distance information is available;numbers in parentheses correspond to the distances in molecule 2 of the crystal structure.

Table VI. Total and interaction energies8 (kcal/mol) of the enzyme-inhibitor complexes

S'B2b <S'B>m i n

c S'C2b <S'C>m i n

c SC2b

Ligand + protein + waterdLigand-proteineLigand-protein(elec)f

Luu-proteinLeu—proteinPro-proteinLigand-watergLuu-waterLeu-waterPro-water

-21 3318

-212.66

-183.6-14.2-10.1

-159.2-24.9

2-7.0-5.2

-12.7

-21 35810

-221.14

-190.3-39.9-6.9

-143.5-74.7

6-9.0

-13.5-52.2

-21 30811

-227.98

-197.2-23.7-14.6

-158.9^*2.7

3-17.3

-5.3-20.0

-21 34510

-190.74

-159.2-10.2-9.8

-139.2-84.0

6-11.1

-9.2-63.7

-21 2287

-111.94

-59.0-18.7-9.2

-31.1-72.5

3-9.6-3.4

-59.5

-21 2128

-85.34

-51.2-4.1

-12.7-34.4

-137.64

-1.3-2.8

-133.6

aEnergies computed in the presence of 1200 waters using a constant dielectric of unity and a non-bond cut-off of 13 A.bStructures obtained after Monte Carlo minimization.cStructures averaged over the production trajectory followed by energy minimization.••Total number of ligand-protein and ligand-solvent hydrogen bonds.Hbtal number of ligand-protein hydrogen bonds.'Electrostatic component of the interaction energy.gTotal number of ligand-solvent hydrogen bonds.

hydroxyl group of the inhibitor and Cys29(S), are involved inweak interactions with protein atoms and/or water molecules.Based on the present results alone, it is not possible to predictwhether cathepsin B would react with the isobutyl-NH-Eps-Leu-Pro inhibitor to yield S'B or S'C since the outcome is

kinetically controlled by the activation free energies for thetwo reactions. In the epoxide ring-opening reaction, the S"nucleophile is expected to attack preferentially the moreelectropositive and less sterically hindered epoxide ring carbon.In the case of the isobutyl-NH-Eps-Leu-Pro ligand, both

983


M.-H.Feng et al.

Fig. 7. The average MD structures of (a) inhibitor B and (b) inhibitor Cbound to the S' subsites of cathepsin B. The ligand is colored in yellow, theprotein in blue and HisllO and Hislll are highlighted in green. The S andS' binding clefts are shown from the top view in (b); the orientation ofHisllO and Hislll with respect to the ligand's Pro carboxylate is seen in(a) from the side view.

epoxide ring carbon atoms have similar electronic and stericeffects (R-NH-CO-epoxide-CO-NH-R'), so their reactivitiesare expected to be similar.

Both minimized MD average structures of S'B and S'Cexhibit lower energies relative to the S'B2 and S'C2 MCMstructures (by 27 and 37 kcal/mol respectively). This stabiliza-tion energy stems mainly from the ligand-water interactions(Table VI), i.e. from - 2 5 (MCM) to - 7 5 (MD) kcal/mol forS'B and from - 4 3 (MCM) to - 8 4 (MD) kcal/mol forS'C, consistent with the increase in the number of solventinteractions, particularly with the Pro carboxylate oxygens andthe Leu carbonyl oxygen of the ligand (Table V). The penetra-tion of solvent into the ligand's binding site is probably dueto the loss of the hydrogen bond between the Leu(O) of theligand and the bulky hybrophobic side chain N*1 of Trp221,which was present in both MCM structures. It appears thatthe MD simulation has relaxed water molecules that weretrapped in local minima in the MCM procedure.

Comparison with the crystal structure of the cathepsin B—CA030 inhibitor complexValidation of binding mode and methodology. Recently, Turket al. (1995) solved the crystal structure of the ethyl-O-Eps-De-Pro inhibitor (referred to as CA030) complexed withcathepsin B at pH 5 to a resolution of 2.0 A. The ethyl-O-Eps-Ile-Pro inhibitor is very similar to the isobutyl-NH-Eps-Leu-Pro used in this work, thus the binding mode of the two

984

ligands to cathepsin B is expected to be the same. Indeed, thecrystal structure shows that the ethyl-O-Eps-Ile-Pro inhibitoris bound in the S' subsites of cathepsin B. This supports ourS' binding mode prediction for the isobutyl-NH-Eps-Leu-Proinhibitor complexed with cathepsin B. It also suggests that thefree energy difference between inhibitor binding in the Sversus S' subsites of cathepsin B correlates with the correspond-ing energy difference and the associated entropy differencedoes not seem to alter the energetic preference for the S'subsites of cathepsin B. Furthermore, the agreement serves tocalibrate the effectiveness of our docking strategy in searchingfor the lowest energy conformation of the enzyme-inhibitorcomplexes.

Crystal versus MCM versus MD. In the cathepsin B-CA030crystal structure, the Cys29 sulfur atom forms a covalent bondto the C2 epoxy ring carbon near the C-terminal end of theinhibitor. A comparison of the ligand-protein and ligand-solvent interactions between the cathepsin B-CA030 crystalstructure and the MCM S'C2 and MD S'C structures (TableV) shows that all four hydrogen bonds involving the Procarboxylate oxygens of the.ligand seen in the crystal structureare also found in the MCM and MD structures. The relativelyweaker interactions involving the rest of the ligand (i.e.excluding the C-terminal carboxylate oxygens) are more vari-able. The two hydrogen-bonding interactions involving thecarbonyl oxygens of the middle and N-terminal residues ofthe ligand with the side chain nitrogens of Trp221 and Gln23respectively found in the crystal structure are preserved in theMCM S'C2 structure, but not in the MD S'C structure.The remaining two hydrogen bonds involving the N-terminalresidue of the ligand in the crystal structure are not maintainedin the MCM or MD S'C structure; this is probably due to thedifferent derivative groups on the epoxide ring in the cathepsinB-CA030 X-ray and S'C model structures. In particular, theO4 atom of the inhibitor hydrogen bonds to the backbonenitrogen of Gly74 in the crystal structure, but to the backbonenitrogen of Glyl98 in the MCM and MD S'C structures (seeTable V and Figure 6).

S' subsite specificityThe cysteine proteases possess seven subsites, each of whichcontacts with a single amino acid residue of a polypeptide(Baker and Drenth, 1987). In the present study, the correspond-ing subsites which bind to the Leu and C-terminal Pro residuesof the E-64 analog inhibitor in the S' binding mode are theS'i and S'2 subsites respectively.

The S'2 subsite with positively charged HisllO and Hislllat physiological pH favors a negatively charged C-terminalsubstrate-inhibitor residue. These are the two key residuesdetermining binding in S' rather than S subsites. In addition,Asp22, which forms a salt bridge with HisllO, may play arole in the stabilization (rigidification) of these two residuesand may also aid in maintaining an optimal orientation of thehistidines towards the negatively charged C-terminal substrate-inhibitor residue. Mutation of each of HisllO, Hislll andAsp22 to a neutral residue of similar size is expected todiminish the binding affinity of E-64 analogs to cathepsin Bseverely. Proline as a C-terminal residue of an epoxysuccinyldipeptide E-64 analog helps to preserve the strong electrostaticattraction between the carboxylate oxygens and the imidazol-ium nitrogens of HisllO and Hislll since its ring prohibitsthe rotation of the carboxylate group about the N-C" bond. Inother words, if the C-terminal carboxylate group can freely



undergo rotation, its electrostatic interaction with the twohistidines will be attenuated. However, the proline ring itselfhas no preference for binding in the S' rather than the Ssubsite as the interaction energy of the proline ring (only) withthe protein when the inhibitor is bound to the S' subsites issimilar to that when the inhibitor is bound to the S subsites.Thus, proline would lose its selectivity for binding in the S'subsites if it is a non-C-terminal residue of an endopeptidasesubstrate.

In contrast to the S'2 subsite, the S'i subsite, lined byVail76, Leul81, Met 196 and Trp221, is relatively large andhydrophobic (Menard et al., 1993), thus it favors bulky non-polar residues. This is evidenced by the favorable contactsmade by the leucine side chain in the average MD S'Cstructure, the isoleucine side chain in the cathepsin B-CA030crystal structure and the benzyl ring in the cathepsin B-CBZ-Arg-Ser(O-Bzl) crystal structure (Jia et al., 1995) with S',residues of cathepsin B.

Selectivity for cathepsin B

There are two requirements for epoxysuccinyl dipeptide E-64analogs, R-Eps-Ile/Leu-Pro (Figure lb), that show selectivitytowards cathepsin B over cathepsin H, cathepsin L and papain(Gour-Salin et al., 1993). First, the C-terminal carboxylic acidof the proline residue must be free, i.e. it should not bederivatized. Second, the N-terminal carboxylic acid of theepoxide must be either esterified (e.g. ethyl-O-Eps-Ile-Pro) orconverted to an amide functionality (e.g. isobutyl-NH-Eps-Leu-Pro).

The first requirement is due to the presence of two histidineslocated in an 'occluding' loop, HisllO and Hislll , which haveno equivalence in papain, actinidin and other mammaliancysteine proteases such as cathepsin H and L (Kamphus et al.,1985). The C-terminal carboxylate oxygens of the prolineresidue must be negatively charged to provide anchors for thetwo protonated histidines of cathepsin B at physiological pH.HisllO, in turn, is anchored by the charge-charge interactionwith a side chain carboxylate oxygen of Asp22. These inter-actions thus account, in part, for the selective binding ofepoxysuccinyl dipeptide E-64 analogs towards cathepsin Bover other cysteine proteases, as well as the unique exo-peptidase activity of cathepsin B, consistent with previousfindings (Buttle et al., 1992; Gour-Salin et al., 1993; Turket al., 1995).

A possible reason for the second requirement is that remov-ing a large derivative of the carboxylic acid on the epoxidecauses the C-terminal end of the inhibitor to become moreflexible, thus attenuating the electrostatic interactions betweenthe negatively charged C-terminus of the ligand and the twopositively charged histidines. However, the simulation resultsof cathepsin B complexed to "OOC-Eps-Leu-Pro in the S'Cbinding mode show that the hydrogen bonds involving HisllOand Hislll with the ligand's C-terminal carboxylate oxygensas well as the enzyme's Asp22 carboxylate oxygen and GlulO9carbonyl oxygen in the MD S'C structure are preservedthroughout the simulation. The dynamics-averaged structureof the cathepsin B-"OOC-Eps-Leu-Pro complex shows thatthe hydrogen-bonding interactions of "OCC-Eps-Leu-Pro aresimilar to those of isobutyl-NH-Eps-Leu-Pro, except for the N-terminal carboxylate oxygens, which interact with the backboneamide of Glyl98 and three water molecules, but no positivelycharged residues on the enzyme in contrast to the C-terminalcarboxylate oxygens. This suggests that both inhibitors have

similar enthalpic contributions to the binding free energy butthe entropy of bound "OOC-Eps-Leu-Pro may be lower thanthat of bound isobutyl-NH-Eps-Leu-Pro probably due to therestriction of water molecules around the N-terminal carb-oxylate oxygens, accounting thus for the observed decreasedinactivation rate for cathepsin B with "OOC-Eps-Leu-Prorelative to isobutyl-NH-Eps-Leu-Pro.

On the other hand, the inactivation rate for papain with"OOC-Eps-Leu-Pro was found to be greater than that withisobutyl-NH-Eps-Leu-Pro (Gour-Salin et al, 1993). In the caseof papain, epoxysuccinyl dipeptide E-64 analogs are expectedto bind in the S (instead of S') subsites due to the absence ofany positively charged residues that can stabilize the negativelycharged C-terminus of the inhibitor. In the S binding mode ofan E-64 analog to papain, the negatively charged N-terminusof "OOC-Eps-Leu-Pro can be stabilized by the side chain ofthe oxyanion hole Gin and the backbone amide and side chainnitrogen of the catalytic cysteine and histidine respectively, inanalogy to the crystal structure of the papain-E-64 complex(Varughese et al., 1989); however, there may not be enoughroom near the oxyanion hole to accommodate a large derivativeof the carboxylic acid group on the epoxide ring such asisobutyl-NH-Eps-Leu-Pro (Gour-Salin et al., 1993), resultingin the loss of inhibition with papain. Thus, the characteristicsof the enzyme (e.g. subsite size and hydrophobicity) as wellas the nature of the inhibitor can both influence the selectivityof an inhibitor towards an enzyme.

AcknowledgementsWe thank R.Menard, J.Mort, E.Purisima and A.Storer for stimulating discus-sions and helpful references. We are grateful to Professor M.Karplus for theCHARMM program. M.-H.Feng is supported by the Institute of BiomedicalSciences, Academia Sinica, Taiwan and S.L.Chan was supported by afellowship from the Medical Research Council of Canada when this workwas carried out. This work was supported in part by Protein EngineeringNetwork Centres of Excellence in Canada, and Academia Sinica and theNational Center for High Performance Computing in Taiwan.

ReferencesBaker.E.N. and DrenthJ. (1987) In F.A.Jurnak and A.McPherson (eds), Active

Sites of Enzymes, John Wiley & Sons, New York, Vol. 3, pp. 314-368.Brooks.B.R., Bruccoleri.R.D., Olafson,B.O., States,D.J., Swaminathan.S. and

Karplus.M.J. (1983) J. Comput. Chem., 4, 187-217.Brooks,C.L., Karplus.M. and Pettitt.B.M. (1988) Proteins: A Theoretical

Perspective of Dynamics, Structure and Thermodynamics. John Wiley &Sons, New York.

Buttle.D.J., Murata.M., Knight,C.G. and Barrett,A.J. (1992) Arch. Biochem.Biophys., 299, 377-380.

Caflisch.A., Niederer.P. and Anliker,M. (1992) Proteins, 14, 102-109.DelaisseJ.M., Ledent.P. and Vaes,G. (1991) Biochem. J., 279, 167-174.DrenthJ., Kalk.K.H. and Swen,H.M. (1976) Biochemistry, 15, 3731-3738.Gour-Salin,B.J., Lachance.P., Plouffe.C, Storer.A.C. and Menard.R. (1993) J.

Med. Chem., 36, 720-725.Hanada.K., Tamai,M., Yamagishi,M., Ohmura,S., Sawada,J. and TanakaJ.

(1978a) Agricult. Biol. Chem., 42, 523-528.Hanada,K., Tamai.M., Ohmura,S., Sawada.J., Seki.T. and TanakaJ. (1978b)

Agricult. Biol. Chem., 42, 529-536.Harris,J.O., Olsen,G.N., Castle,J.R. and Maloney.A.S. (1975) Am. Rev.

Respirat. Dis., Ill, 579-586.Hasnain.S., Hirama.T., Tam.A. and MortJ.S. (1992) J. Biol. Chem., 267,

4713-4721.Jia,Z., Hasnain.S., Hirama,T, Lee.X., Mort,J.S., To,R. and Huber.C.P. (1995)

J. Biol. Chem., 270, 5527-5533.Jorgensen,W.L., Chandrasekhar,J., Madura,J.D., Impey.R.W. and Klein,M.L.

(1983) J. Chem. Phys., 79, 926-935.KamphuisJ.G., Drenth,J. and Baker,E.N. (1985) J. Mol. Biol., 182, 317-329.Katunuma.N. and Kominami,E. (1987) Rev. Physiol. Biochem. Pharmacol.,

108, 1-20.

985


M.-H.Feng et al.

Liu.B.C, Redwood.S.M., Weiss.R.E., Hodge.D.E. and Droller.M.J. (1992)Cancer, 69, 1212-1219.

MacKerell,A.D., J r et al. (1992) FASEB J., 6, A143.Menard.R., Carmona.E., Plouffe.C, Bromme,D., Konishi.Y, Lefebvre,]. and

Storer.A.C. (1993) FEBS Lett., 328, 107-110.Mort,J.S., Recklies.A.D. and Poole.A.R. (1984) Arthritis Rheumat., 27, 509-

515.Murata,M., Miyashita.S., Yokoo,C, Tamai,M., Hanada.K.. Hatayama.K.,

Towatari,X, Nikawa.T. and Katunuma.N. (1991) FEBS Lett., 280, 307-310.Nilsson.E., Alafuzoff,!., Belnhow.K., Blomgren.K., Hall.C.M., Janson.I.,

Karlsson,!., Wallin,A., Gottfries.C.G. and KarlssonJ.O. (1990) Neurobiol.Aging, 11,425-431.

North.M.J., Mottram.J.C. and Coombs.G.H. (1990) Parasitol. Today, 6,270-275.

ProusJ.R. (1986) Drugs Future, 11, 941-943.Sloane,B.F., Moin,K., Krepela.E. and RozhinJ. (1990) Cancer Metastasis

Rev., 9, 333-352.StraubJ., Lim,C. and Karplus.M. (1994) J. Am. Chem. Soc, 116, 2591-2599.Sumiya,S., Yoneda.T., Kitamura.K., Murata.M., Yokoo.C, Tamai.M.,

Yamamoto.A., Inoue.M. and Ishida.T. (1992) Chem. Pharmacol. Bull., 40,299-303.

Towatari.T., Nikawa.T., Murata,M., Yokoo.C, Tamai.M., Hanada,K. andKatunuma,N. (1991) FEBS Lett., 280, 311-315.

Trabant,A., Gay.R.E., Fassbender.H. and Gay,S. (1984) Arthritis Rheumat.,34, 1444-1451.

Turk.D., Podobnik,M., Popovic.T., Katunuma.N., Bode.W., Huber,R. andTurk,V. (1995) Biochemistry, 34, 4791-4797.

Van Noorden,C, Smith,R.E. and Rasnik.D. (1988) J. Rheumatol., 15, 1525-1535.

Varughese.K.I., Ahmed.F.R., Carey.P.R., Hasnain,S., Huber.C.P. and Storer.A.(1989) Biochemistry, 28, 1330-1332.

Wang,J., Xiang,Y.-F. and Lim.C. (1994) Protein Engng, 7, 75-82.Yamamoto,D., Matsumoto.K., Ishida,T., Inoue,M., Sumiya,S. and Kitamura.K.

(1989) Chem. Pharmacol Bull., 10, 2577-2581.

Received May 23, 1996; revised July 9, 1996; accepted July 15, 1996

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the binding mode of an £-64 analog to the active site of cathepsin b

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