©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Crystal Structures of Recombinant Rat Cathepsin B and a Cathepsin B-Inhibitor Complex
IMPLICATIONS FOR STRUCTURE-BASED INHIBITOR DESIGN (*)

(Received for publication, November 9, 1994)

Zongchao Jia (1) (2)(§) Sadiq Hasnain (1)(¶) Tomoko Hirama (1) Xavier Lee (1)(**) John S. Mort (3) Rebecca To (1) Carol P. Huber (1) (2)(§§)

From the  (1)Institute for Biological Sciences, National Research Council of Canada, Ottawa K1A 0R6, the (2)Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montreal H4P 2R2, and the (3)Joint Diseases Laboratory, Shriners Hospital for Crippled Children and Department of Surgery, McGill University, Montreal H3G 1A6, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The lysosomal cysteine proteinase cathepsin B (EC 3.4.22.1) plays an important role in protein catabolism and has also been implicated in various disease states. The crystal structures of two forms of native recombinant rat cathepsin B have been determined. The overall folding of rat cathepsin B was shown to be very similar to that of the human liver enzyme. The structure of the native enzyme containing an underivatized active site cysteine (Cys) showed the active enzyme conformation to be similar to that determined previously for the oxidized form. In a second structure Cys was derivatized with the reversible blocking reagent pyridyl disulfide. In this structure large side chain conformational changes were observed for the two key catalytic residues Cys and His, demonstrating the potential flexibility of these side chains. In addition the structure of the complex between rat cathepsin B and the inhibitor benzyloxycarbonyl-Arg-Ser(O-Bzl) chloromethylketone was determined. The complex structure showed that very little conformational change occurs in the enzyme upon inhibitor binding. It also allowed visualization of the interaction between the enzyme and inhibitor. In particular the interaction between Glu and the P(2) Arg residue was clearly demonstrated, and it was found that the benzyl group of the P(1) substrate residue occupies a large hydrophobic pocket thought to represent the S`(1) subsite. This may have important implications for structure-based design of cathepsin B inhibitors.


INTRODUCTION

Cathepsin B (EC 3.4.22.1) is a lysosomal cysteine proteinase belonging to the papain superfamily (1) but is unique in its ability to act as both an endo- and an exopeptidase. It is synthesized as an inactive zymogen (2, 3) which, in the case of the rat enzyme, is substituted with two N-linked oligosaccharide units, one in the proregion and a second at Asn. Following the modification of mannose residues to mannose 6-phosphate, the proenzyme is targeted to the lysosome through the mannose 6-phosphate receptor-mediated transport pathway(4) .

Recently, procathepsin B has been expressed in the yeast Saccharomyces cerevisiae(5) and used to study the mechanism of proenzyme processing. Autoprocessing of the recombinant proenzyme occurs in acidic environments yielding a single chain form of cathepsin B with a 6-residue N-terminal extension relative to the fully processed lysosomal form. Since the N-linked oligosaccharides synthesized by S. cerevisiae are extremely heterogeneous, the consensus sequence for oligosaccharide substitution in cathepsin B was mutated yielding a homogeneous, non-glycosylated protein which was shown to be functionally equivalent to cathepsin B purified from rat liver(6) .

Activation of procathepsin B in the mammalian cell, with concomitant propeptide removal, occurs on acidification of the transport vesicles and inside the lysosome. Additional processing steps include N-terminal trimming and the removal of 6 residues from the C terminus to yield the mature, single chain, 254 residue protein. In a subsequent but much slower processing step, the single chain form of cathepsin B is cleaved internally with excision of residues 48 and 49 to generate a two-chain form of the enzyme(3) .

The three-dimensional structure of the two-chain form of human liver cathepsin B, determined recently by two different groups (7) , (^1)reveals the overall similarity between this enzyme and the stereotypical cysteine proteinase, papain. Several important differences are, however, apparent. In particular, a large insertion consisting of an 18-residue surface loop that occludes the active site cleft was found to be present in the primed substrate binding region in cathepsin B. The region expected to define the S(2) pocket was seen to contain a negatively charged residue, Glu, which has been shown to be involved in the binding of Arg residues(8) .

In addition to its role in normal intracellular protein breakdown, the action of cathepsin B has also been implicated in several pathological conditions, in particular arthritis(9, 10) , muscular dystrophy(11) , and tumor invasion and metastasis(12) . This has stimulated the search for specific cathepsin B inhibitors for therapeutic use. While rational, structure-based design of specific inhibitors requires a detailed description of the native enzyme, it also depends on the information available from the structures of inhibitor complexes which demonstrate the different binding sites in the enzyme(13) . Here, in addition to the structure of the single chain form of rat cathepsin B, both as the free enzyme and as the 2-pyridyl disulfide adduct, we present the structure of a complex between the enzyme and the peptidyl inhibitor, benzyloxycarbonyl-Arg-Ser(O-Bzl)-chloromethylketone. This inhibitor was chosen to allow investigation of the structural basis for the unique ability of cathepsin B, relative to other cysteine proteinases, to accept an Arg at P(2)(1) and for the previously reported high affinity of cathepsin B for Ser(O-Bzl) in the P(1) position(14, 15) .


EXPERIMENTAL PROCEDURES

Materials

Rat cathepsin B was expressed in yeast as an alpha-factor fusion construct as described previously(5) . To optimize the homogeneity of the recombinant protein the following mutations were introduced: SerAla to eliminate N-linked oligosaccharide substitution in the mature region and GlnTer (termination codon) to eliminate the 6-residue C-terminal extension coded for by the cDNA but normally absent in the mature lysosomal enzyme(16) . The recombinant proteinase was purified by affinity chromatography using a Sepharose 4B 6-aminohexanoyl-Gly-Phe-Gly-semicarbazone column and elution with 2,2`-pyridyl disulfide, which reacts covalently with the active site thiol (17) yielding the pure enzyme as a 2-pyridyl disulfide complex. The enzyme was stored at 4 °C in this form.

The propeptide of cathepsin B (residues -57 to -7) was prepared using an Applied Biosystems 431A synthesizer and purified as described previously(18) . The inhibitor CBZ-Arg-Ser(O-Bzl)-chloromethylketone was custom synthesized by Enzyme Systems Products Ltd. (Dublin, CA).

Crystal Preparation

Crystals of recombinant rat cathepsin B blocked with 2-pyridyl disulfide were prepared as described previously (19) . This reversibly inactivated form was crystallized at pH 4.4 and is designated as Native Enzyme 1.

A second native enzyme crystal was obtained at pH 4.6. Our intention was to obtain a complex of rat cathepsin B with the propeptide segment of procathepsin B. Before crystallization, dithiothreitol was added to the inactivated cathepsin B to eliminate the pyridyl sulfide group, regenerating active free enzyme. Droplets containing 5 µl of a mixture of 5.25 mg/ml cathepsin B, 3.27 mg/ml synthetic propeptide, and 5 µl of reservoir solution were equilibrated with reservoir solution of 20.5% ammonium sulfate, 0.1 M sodium acetate (pH 4.6), 1% PEG 4000. A washed seed was then introduced into the drop 1 day later. Crystals usually grew in a few days to a maximum size of 0.4 times 0.1 times 0.1 mm and proved to contain the unoxidized native enzyme uncomplexed with the propeptide, which most probably was hydrolyzed because of the low pH(18) . This form is designated as Native Enzyme 2.

The cathepsin B-inhibitor complex was formed through the reaction between cathepsin B and the peptidyl inhibitor, as follows. Dithiothreitol was added to freshly purified enzyme in 50 mM sodium phosphate, 1 mM EDTA, 0.001% Brij-35 (pH 6.0), to a final concentration of 0.2 mM to activate the enzyme, and the solution was allowed to stand for 30 min at 4 °C. A 3-fold excess of benzyloxycarbonyl-Arg-Ser(O-Bzl)-chloromethylketone dissolved in water was added to the activated enzyme solution and the mixture allowed to incubate at 4 °C for 30 min. Residual enzyme activity was monitored spectrophotometrically and determined to be no more than 0.2% of the initial active enzyme concentration. The enzyme-inhibitor covalent complex was crystallized using the hanging drop method, in 50 mM phosphate buffer (pH 6.0), 0.001% Brij-35, and 0.05% NaN(3) at a protein concentration of 7.7 mg/ml. A mixture of 5 µl of the complex solution and 5 µl of the reservoir solution was equilibrated against the reservoir solution of 10% PEG 8000, 0.2 M ammonium sulfate, and 0.1 M sodium citrate buffer (pH 4.0). Crystals usually appeared in 24 h and are designated as Complex.

Structure Determination

X-ray diffraction data from single crystals were collected on a San Diego Multiwire twin-detector system mounted on a Rigaku RU-200 rotating anode x-ray generator operated at 40 kV, 120 mA. No explicit correction for absorption or decay was applied to the data.

The structure of the inhibitor complex was solved first by molecular replacement. Program packages used included the CCP4 suite(20) , BRUTE (21) , and X-PLOR(22, 23) . These programs were used to either obtain a molecular replacement solution or confirm the results from other programs. The tetragonal form of human liver cathepsin B^1 was used as the search model. This structure represents the two chain form of human liver cathepsin B and has about 83.5% sequence identity with rat cathepsin B. Calculations with CCP4 produced two sharp peaks in the rotation search, which correspond to the two molecules in the asymmetric unit. Independent calculations using an X-PLOR conventional rotation search and then Patterson coefficient (PC) refinement gave rise to equivalent orientations of the molecule. In addition, similar results were also obtained using a ``direct search'' or a brute-force Patterson coefficient refinement as implemented in X-PLOR. X-PLOR and BRUTE were used independently for translation searching. An individual translation search was first carried out for each of the two molecules in the asymmetric unit, and the same clear translation peaks were obtained using both programs. Then a combined translation search was performed to determine the relative translation between the two molecules. The translation along the y axis of one molecule is arbitrary in the polar space group P2(1). The positioned molecules were subject to refinement using X-PLOR. After one round of X-PLOR refinement, the resulting (2F(o)-F(c)) and (F(o)-F(c)) maps showed clear density for the inhibitor. The native enzyme structures were isomorphous with the complex, and a refined set of complex coordinates was used as starting point for the native enzyme structures. The simulated annealing procedure was used in the initial refinement, but in the later stages only conventional positional refinement was carried out. The refinement was gradually extended to higher resolution and many cycles of alternating manual fitting using the graphics program FRODO (24) and refinement calculations were carried out. Unless otherwise noted figures were prepared using the program SETOR(25) .


RESULTS

Overall Structure

The structures of recombinant rat cathepsin B and of its isomorphous complex with the inhibitor benzyloxycarbonyl-Arg-Ser(O-Bzl)-chloromethylketone were solved by the molecular replacement method. In each case the crystallographic asymmetric unit contains two molecules that are almost identical and are related by a non-crystallographic 2-fold rotation axis (Fig. 1). Some crystal structure statistics are listed in Table 1.


Figure 1: Overall folding of the native structure, showing the two cathepsin B molecules in the asymmetric unit related by a non-crystallographic 2-fold axis. alpha-Helices are shown as cylinders in blue, beta-strands as arrowed ribbons, non-secondary structures as purple rope, disulfide bridges are shown in yellow, and the side chains of the active site residues are shown in green.





The overall structure of the recombinant rat cathepsin B is very similar to that of human liver cathepsin B in its tetragonal form^1 with a backbone root-mean-square deviation of only approximately 0.5 Å. It also appears to closely resemble the human liver cathepsin B structure previously reported by Musil et al.(7) . Although the latter also belongs to space group P2(1), it has very different unit cell dimensions, and thus is not isomorphous with the present structures.

In each of the three crystal structures reported here, the two molecules in the asymmetric unit are very similar with backbone root-mean-square deviations of no more than 0.35 Å. The backbone root-mean-square deviations between the three crystal structures are also very low (about 0.4 Å). As shown previously(7) , cathepsin B has a very similar overall structure to that of papain and can be divided into two domains, arbitrarily designated left and right. The long active site cleft is situated at the interface between these two domains. Well-defined major secondary structural elements are illustrated in Fig. 1. At the protein sequence level, rat cathepsin B is 83.5% identical to the human enzyme. Comparison of the three-dimensional structures shows that the non-identical residues are situated at the surface of the molecule and do not introduce significant changes in main chain folding.

The recombinant cathepsin B used in the present work differs from the previously studied human liver cathepsin B which is a completely processed lysosomal product. Autoprocessing of recombinant procathepsin B results in the single chain form containing a 6-residue N-terminal extension(5) . Insufficient electron density was observed to determine the positions of the N-terminal residues indicating high flexibility of this region. In contrast residues 48 and 49, which are lacking in the lysosomal two chain form, could be inserted into the recombinant cathepsin B structure based on difference Fourier density and are seen to form part of a turn linking the left domain major alpha-helix to a segment containing no regular secondary structure (Fig. 2). The positions of these 2 residues are consistent with their accessibility to cleavage and dipeptide excision, during processing, to yield the two chain form. The recombinant enzyme also differs from the natural enzyme in the elimination of the glycosylation consensus sequence at Asn-Ser, and it is noteworthy that the deletion of N-linked carbohydrate at Asn shows no effect on the protein folding relative to the natural enzyme.


Figure 2: Stereo diagram showing the CA structure of rat cathepsin B. The side chains are shown for the active site residues and for other residues discussed in the text.



The Active Site

The interface region between the two domains houses the active site cleft. The key catalytic residues are located on the upper portion of the V-shaped cleft (Fig. 2). When compared with papain, a long insertion loop (residues 105-125) is seen to partially block the upper back aspect of the active site cleft. This has been designated as the ``occluding loop''(7) . The partially obstructed active site cleft is not a very rigid ``compartment'' structure. Toward the end of the long insertion, residues 120-124 form a solvent-exposed segment of which residues 120-122 create a high wall on the upper left side of the active site cleft. High temperature factors and relatively poor density were observed in this region indicating a high mobility. All cathepsin B structures reported here (a total of six crystallographically independent molecules) show this flexibility. Since the bottom floor and the right wall of the active site cleft are relatively rigid, the flexibility of the high covering loop may be essential for accommodating substrates of various sizes.

Comparison of the Active Site in the Two Forms of the Native Enzyme

We have determined the structure of the native enzyme under two different conditions, one at pH 4.4 with 2-pyridyl sulfide covalently bound to the active site thiol group, forming a disulfide bridge, and the second at pH 4.6 without any blocking group. In the blocked structure, the difference density for the pyridyl group is very clear. In the other structure where the active enzyme was crystallized in the presence of the synthetic propeptide, the Cys shows very little evidence of oxidation indicating that this is predominantly in the reduced state, essentially describing the true native enzyme (Fig. 3). In this structure the active site residues are oriented in a similar conformation to that seen in other cysteine proteinase structures(26, 27, 28, 29) . In the case of the pyridyl disulfide complex, however, a large local conformational change is observed. There is an approximately 120° rotation of the His side chain (1) torsion angle between the two structures and about 65° rotation of the Cys side chain (1) torsion angle. Both His and Cys in the active site of the pyridyl disulfide substituted enzyme have therefore rotated away from their ``normal'' positions, as shown in Fig. 4. The pyridyl ring now occupies the position where the His imidazole ring was previously located. Very little change in the peptide backbone of the active site accompanies this change although, understandably, there is a small backbone shift for His. In the free enzyme there is a hydrogen bond between the N2 atom of His and the Asn side chain, which helps to orient the His imidazole ring. This hydrogen bond is broken in the pyridyl disulfide complex, due to the replacement of the His imidazole by the pyridyl group, and a new hydrogen bonding interaction between the pyridyl N atom and the Asn side chain is formed. There may be a major difference between these two hydrogen bonds. The Asn side chain serves as a proton acceptor in the case of His but probably is a proton donor in the case of the pyridyl ring. Since the pK(a) of the pyridyl group is presumably very low (2.0-3.0) (30, 31) it is unlikely to be able to act as proton donor at the pH of crystallization (4.6).


Figure 3: Part of the structure around the active site of the underivatized native rat cathepsin B. Electron density from a (2F-F) map is shown in blue, and from a (F-F) map is shown in red. The contour levels are 1.5 and 3.0 . The residual positive density nearest Cys is in the wrong location to represent oxygen atoms bonded to the sulfur atom.




Figure 4: Comparison of the active site in native cathepsin B and the 2-pyridyl disulfide complex. The crystal structure without blocking reagent is shown in blue and the structure with blocking reagent bound shown in orange. The blocking reagent itself is shown in light purple.



However, the possibility that the pK(a) of the pyridyl group is significantly perturbed, as are those of Cys and His, cannot be excluded. In any case, the number of hydrogen bonding interactions is equal for the two conformations of His, and this factor does not apparently favor either conformation. Furthermore, both rotamers of His are favored conformations. After (1) angle change, the His side chain is more solvent exposed in the derivatized cathepsin B. Modeling shows that if His were to maintain its original position then it would be difficult for the pyridyl ring to find a location that is not solvent exposed. As the imidazole ring is more hydrophilic than the pyridyl ring, it is energetically less costly for the imidazole ring to occupy the more solvent exposed location.

The Inhibitor Complex and the S(2) and S(1)` Subsites

The benzyloxycarbonyl-Arg-Ser(O-Bzl)-methylene moiety covalently bound to the Cys SG atom is well determined in the electron density map (Fig. 5). Compared with the active site of underivatized native rat cathepsin B, little difference is found in the conformation of the enzyme in the complex. This is also true for the His and Cys side chains. It appears, therefore, that the enzyme maintains its basic active site conformation after the complex formation. As shown in Fig. 6, the surfaces of the enzyme and the inhibitor complement each other well. Interactions between the enzyme and the inhibitor are illustrated schematically in Fig. 7. There are six main chain hydrogen bonding interactions, most of which are similar to those found previously in papain complexes(28, 32, 33) . A weak hydrogen bond (3.13 Å) forms between the P(1) carbonyl O atom and the Gln side chain. There is good evidence in papain that the equivalent residue (Gln) interacts with the oxyanion of the tetrahedral intermediate forming part of the proposed ``oxyanion hole'' which is thought to stablize the reaction intermediate(34) . The direct interaction observed here in the ground state complex provides further support for the existence of this oxyanion hole.


Figure 5: An omit map (F-F) of the inhibitor. The final structure of the active site and the superimposed inhibitor are also shown. The inhibitor was left out in both refinement and the calculation of the map which is contoured at a 3 level.




Figure 6: Surface representation of the structure of the CBZ-Arg-Ser(O-Bzl)-cathepsin B complex. The inhibitor is represented in green and the enyzme in red. The surface was calculated and illustrated using the program QUANTA(40) .




Figure 7: Scheme illustrating the interactions of the CBZ-Arg-Ser(O-Bzl) group with cathepsin B. Thick lines represent the enzyme, and thin lines represent the inhibitor. Hydrogen bonds and electrostatic interactions are illustrated as dashed lines.



A guanidinium N of the Arg P(2) side chain is salt bridged to the Glu carboxylate at an average distance of 2.90 Å. This salt bridge has been widely expected(7, 8) . There are no other interactions between the P(2) Arg residue and the S(2) subsite. However, as observed in the crystal structure of the complex, a bulky P(2) residue such as Phe might interact with some or all of residues Pro, Ala, Ala, and the side chain of Glu.

It is also interesting to note that the carbonyl portion of the benzyloxycarbonyl (CBZ) group, which could be considered as a pseudo-P(3) carbonyl, makes no direct interaction with the enzyme. However, the benzyl ring of the CBZ moiety makes a vertical or edge-on aromatic-aromatic interaction with Tyr, with the shortest distance from aromatic atom to aromatic atom of 3.71 Å. This type of interaction, with positively polarized hydrogen atoms of one ring interacting with the `` -electron cloud of a second ring, has been described by Burley and Petsko(35, 36) .

As outlined above the main chain of the P(1)O-Bzl-Ser residue interacts as in previous inhibitor complexes of papain, however, the side chain O-benzyl group is located in a hydrophobic pocket bounded by Trp, His, Leu, Phe, Met, Val, His, Phe, and Gly. Among these residues, Met partially covers the hydrophobic pocket and shields it from external solvent (Fig. 6).


DISCUSSION

Structure of the Active Site

To date the crystal structures of a series of cysteine proteinases of the papain superfamily have been determined(7, 26, 27, 29, 37) . However, in all cases inactive enzyme was observed in that the active site cysteine thiol was at least partially oxidized(7, 26, 27, 29) . (^2)For detailed molecular modeling studies, the structure of the fully active enzyme is preferred(38) . The present study describes a structure in which the active site sulfur of Cys appears predominantly in the reduced form and, therefore, the conformations of the active site side chains, Cys and His, may more reliably reflect the conformations in the active molecule. As a number of investigations have shown the probability of the existence of a Cys-His ion pair in the catalytically competent enzyme, it can be assumed that, if the ion pair is interrupted by the oxidation of the thiol, the possibility exists for an altered geometry, particularly of Cys. It has been shown previously that the pK(a) of Cys is about 3.6(6) , very similar to the pK(a) of the active site cysteine of papain. Therefore, it can be assumed that even at the low pH of crystallization, Cys of cathepsin B would largely exist as the thiolate anion, and thus could form a salt bridge with the imidazolium cation. From this work, it appears that oxidation of the active site thiol does not significantly change the conformation of the catalytic residues while dramatic reorientation of His is seen when the active site thiol is substituted as a pyridyl disulfide. Despite the dramatic conformational change, the SG29. . .ND1(199) distance increases very little, from an average of 3.34 Å in the underivatized structure to an average of 3.61 Å in the pyridyl disulfide case. This change is less than the differences between SG29. . .ND1(199) distances in the two crystallographically independent molecules in either structure.

When the structural data on the cathepsin B-inhibitor complex are considered in light of the results of previous kinetic studies using different synthetic substrates and inhibitors(6, 8, 39) , it is clear that two relatively well defined substrate-binding subsites can be described in this enzyme. The S(2) subsite is a wide pocket extending from the active site cleft toward Glu and is largely open to solvent. A large P(2) side chain, such as Phe, could be accommodated comfortably in the space available. Kinetic studies have shown that cathepsin B has a broad S(2) specificity, accepting both Phe and Arg at the P(2) position in substrates but the former is preferred 7-fold over Arg. In the case of a P(2) Arg residue, site-directed mutagenesis studies have indicated that the electrostatic interaction with Glu is important for substrate binding and contributes to transition state-complex stabilization(8) .

There is also another pocket on the left of the cleft, defined by the segment Asn to Tyr and residue Asp. If the main chain of a longer peptidyl inhibitor (with P(3) and P(4) residues) occupied the active site cleft, the P(3) side chain could fit into the pocket on the left side, as suggested by the orientation of the CBZ group, simulating a P(3) residue, in the present complex, This arrangement would be better for the binding of a longer peptide or protein substrate, since the lower part of the active site cleft floor would be made available to bind the main chain of the peptide or protein to achieve S(3)/P(3) main chain interaction, thus maximizing interaction between cathepsin B and its substrate. One way of achieving the maximum S(3)/P(3) backbone interaction might be to alter the P(3) residue to D-configuration, thus forcing the P(3) side chain to take an alternative position which might occupy the left-hand pocket (Asn, Tyr, and Asp) and thereby leave space for the P(3) main chain atoms in the lower active site cleft.

While there appears to be no strong preference in the S(1) subsite, kinetic evidence (39) indicates that the S(1)` subsite is relatively large and certainly very hydrophobic. The crystal structure of the inhibitor complex shows the P(1)O-benzyl group to be located at the entrance to a large hydrophobic pocket. The volume enclosed by this pocket would be capable of accommodating a bulky hydrophobic side chain, such as Phe and Trp, comfortably and could therefore be considered to represent the S(1)` subsite suggested by Ménard et al.(39) . Because of the different main chain tracing in the region 191-198 in cathepsin B relative to papain and other similar cysteine proteinases the S(1)` subsite in cathepsin B is more hydrophobic and less solvent exposed. Thus, in the absence of a P(1)` residue, if the P(1) side chain is long and hydrophobic it could reach into the hydrophobic S(1)` pocket and achieve some van der Waals contacts. In the case of a normal polypeptide substrate, however, the P(1) side chain would be expected to project out from the active site cleft and might interact with the flexible high wall on the left.

Implications for Inhibitor Design

Based on the structural information obtained from the analysis of the active site of the native enzyme and the interactions of the inhibitor, there are several considerations that should be of practical significance for inhibitor design. The following modifications to the current inhibitor (benzyloxycarbonyl-Arg-Ser(O-Bzl)-chloromethylketone) would be expected to increase potency. Increasing the size of the P(1) hydrophobic side chain, and the length of the arm attached to the hydrophobic benzyl ring, would gain more van der Waals contacts with the S(1)` subsite. For example, the benzyl group could be replaced by an indole moiety, and an additional methylene group could be inserted to create a longer arm. Second, our modeling suggests that if P(2) Arg were replaced by a Lys, good electrostatic interaction could be achieved with Glu with the side chain in a fully extended conformation, which would be more energetically favored than the somewhat curled conformation observed with Arg. Third, since the benzyloxy group does not appear to achieve good interactions with cathepsin B, a relatively long, positively charged moiety might be useful here as there are 2 residues (Asp and Asn) for potential interaction in this pocket.


FOOTNOTES

*
This is National Research Council of Canada Publication 38521. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Lab. of Molecular Biophysics, Dept. of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, United Kingdom.

Present address: Marketing Services, National Research Council of Canada, Ottawa K1A 0R6, Canada.

**
Present address: The Cleveland Clinic Foundation, Cleveland, OH 44195.

§§
To whom correspondence and reprint requests should be addressed: Biotechnology Research Institute, c/o Institute for Biological Sciences, National Research Council of Canada, Ottawa K1A 0R6, Canada. Tel.: 613-990-0856; Fax: 613-941-4475.

(^1)
C. P. Huber, R. L. Campbell, T. Hirama, X. Lee, J. S. Mort, R. To, and S. Hasnain, manuscript in preparation.

(^2)
R. Hilgenfeld, personal communication.


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