Crystal structure of cathepsin X: a flip–flop of the ring of His23
allows carboxy-monopeptidase and carboxy-dipeptidase activity
of the protease
Gregor Guncar
1, Ivica Klemencic
1, Boris Turk
1, Vito Turk
1,
Adriana Karaoglanovic-Carmona
2, Luiz Juliano
2and Dušan Turk
1*
Background: Cathepsin X is a widespread, abundantly expressed papain-like mammalian lysosomal cysteine protease. It exhibits carboxy-monopeptidase as well as carboxy-dipeptidase activity and shares a similar activity profile with cathepsin B. The latter has been implicated in normal physiological events as well as in various pathological states such as rheumatoid arthritis, Alzheimer’s disease and cancer progression. Thus the question is raised as to which of the two enzyme activities has actually been monitored.
Results:The crystal structure of human cathepsin X has been determined at 2.67 Å resolution. The structure shares the common features of a papain-like enzyme fold, but with a unique active site. The most pronounced feature of the cathepsin X structure is the mini-loop that includes a short three-residue insertion protruding into the active site of the protease. The residue Tyr27 on one side of the loop forms the surface of the S1 substrate-binding site, and His23 on the other side modulates both carboxy-monopeptidase as well as carboxy-dipeptidase activity of the enzyme by binding the C-terminal carboxyl group of a substrate in two different sidechain conformations.
Conclusions:The structure of cathepsin X exhibits a binding surface that will assist in the design of specific inhibitors of cathepsin X as well as of cathepsin B and thereby help to clarify the physiological roles of both proteases.
Introduction
Cathepsin X belongs to the group of lysosomal cysteine proteases, which constitute a numerous and important part of the family of papain-like enzymes (clan CA; reviewed in [1]). The research interest in this field is increasing rapidly, together with the number of identified enzymes, as it becomes evident that lysosomal cysteine proteases are, besides being involved in intracellular protein catabo-lism, also involved in a number of important cellular processes [2,3]. Currently 11 human cathepsins are known at the sequence level (B, C, F, H, K, L, O, S, V, W and X). Some of them are expressed in a wide variety of cells (B, C, F, H, L, O and X), whereas others (S, K, V and W) are tissue-specific and have been shown to be or proposed to be involved in more specialized processes (reviewed in [3]). They are involved in invariant chain processing and antigen presentation [4–7], in bone remodeling [8–10] and in proteolytic processing of various proteins [11], among these the most important being thyroglobulin [12–14] and granzyme B [15]. Whereas the former is an important source of thyroglobulin hormones the latter is involved in the activation of the cell death proteases, the caspases (reviewed in [3]). Besides their normal physiological roles, lysosomal cysteine proteases are involved in a number of
pathological processes such as tumor malignancy and pro-gression (reviewed in [16–18]), muscular dystrophy [19], rheumatoid arthritis [20], osteoporosis and other bone dis-orders [2,10,21] and Alzheimer’s disease [22].
Cathepsin X was discovered only recently [23–25] and its biochemical characterization is just emerging [25–27]. It is the only known lysosomal cysteine protease exhibiting monopeptidase activity. Besides monopeptidase activity cathepsin X also exhibits carboxy-dipeptidase activity and is readily inhibited by CA074, previously known as the specific inhibitor of cathepsin B, which is the only known carboxy-dipeptidase among lyso-somal cysteine proteases (I.K. et al., unpublished observa-tions). The inhibitor has been used as a standard tool to assess functions of cathepsin B in numerousin vivo andin vitro studies, for example in relation with exocytosis [28] and cancer (reviewed in [16,18,29]). Similarly, the implica-tion of cathepsin X in the development of Alzheimer’s disease is based on the use of inhibitors [27] and therefore requires confirmation.
A structural investigation of the active site of cathepsin X is, therefore, a mandatory step for understanding the specific
Addresses: 1Department of Biochemistry and
Molecular Biology, Jozvef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia and 2Departamento de
Biofisica, Escola Paulista de Medicina, Rua Tres de Maio 100, 04044-020 Sao Paulo, Brazil.
*Corresponding author. E-mail: dusan.turk@ijs.si
Key words:Alzheimer’s disease, carboxypeptidase, cathepsin B, cathepsin X, papain-like cysteine protease
Received: 1 November 1999
Revisions requested: 8 December 1999
Revisions received: 6 January 2000
Accepted: 7 January 2000
Published: 29 February 2000
Structure2000, 8:305–313 0969-2126/00/$ – see front matter
basis of the specificity of similar cathepsins, in particular cathepsin B. This will enable us to design substrates and inhibitors that will address specifically only the desired enzyme. It has already been suggested, from sequence data and a substrate study [26], that a short insertion of residues 24–26, which constitutes the tip of the mini-loop placed in the immediate vicinity of the reactive site, must be involved in interaction with substrate. It was also suggested [26] that the mini-loop residue His23 is involved in recogni-tion and binding of the C-terminal carboxylic group of a bound substrate. The crystal structure of cathepsin X pre-sented here has enabled us to elucidate the structural basis of the mechanism by which cathepsin X exhibits both carboxy-monopeptidase and carboxy-dipeptidase activity.
Results
Overall structureThe structure of human cathepsin X comprises the sequence of the whole mature single-chain protein of 242 amino acid residues, which are numbered consecutively from Leu1 to Val242. The asymmetric unit of orthorhom-bic crystals contains two molecules. The three-dimen-sional structures of the two molecules are almost identical. The complete chains of both molecules, with the excep-tion of a few sidechain residues, are well resolved in the electron-density maps. The superimposed structures show a root mean square deviation (rmsd) of 0.55 Å for all Cα atoms; when only residues included into noncrystallo-graphic symmetry (NCS) constraints are used for Cαatom superposition (235 residues), the resulting rms fitting pro-cedure yields a considerably lower value (0.26 Å).
The structure possesses the typical fold of a papain-like cysteine protease, a two domain structure [30,31]. The predominant secondary structural features of the N-termi-nal domain on the left, or L domain, are αhelices, whereas the C-terminal domain, or R domain, has a β-barrel fold
interface, reminiscent of a book with the spine in front. At the top the two domains separate and form a ‘V-shaped’ active-site cleft with the reactive site residues, Cys31 and His180, each coming from a different domain.
The structure of cathepsin X, as judged from a number of superimposed pairs, is rather similar to the structures of human cathepsin K, rat cathepsin B, zymogen of cathepsin L and papain (yielding respectively 145, 148, 146 and 141 equipositioned Cαpairs at a threshold distance of 1.5 Å). It is less similar to cathepsin L, actinidin, glycyl-endopep-tidase I and cruzipain (yielding 122, 116, 128 and 130 pairs) and least similar to human cathepsin B and porcine cathepsin H (with 89 and 73 pairs; Figure 2). Although by numerical criteria the structure of cathepsin X does not superimpose on the structure of cathepsin B as well as on those of other related enzymes, a visual inspection reveals that the structures of cathepsin B, and to a smaller extent cathepsin H, are the most similar in the position and archi-tecture of the surface loops to the structure of cathepsin X. Some outstanding loops of the cathepsin X structure (110–123 and 189–193) appear at similar positions to some cathepsin B loops (132–139 and 208–212). Particularly striking is the exposed position of the ring of Phe115, placed at the tip of the 110–123 loop. The short three-residue insertion (Ile24, Pro25 and Gln26) appears to be of particular importance for the specificity of cathepsin X. It is located within the mini-loop (Figure 3), an otherwise conserved loop between Gln19 and Cys25 (using papain numbering), forming the wall of the S2′substrate-binding site [32]. An exception is the loop of glycyl-endopepti-dase, in which a tyrosine residue replaces the conserved Ser21 (papain numbering).
The mature form of human cathepsin X contains 11 cys-teine residues. Ten of them are involved in disulfide bonds (28–71, 65–103, 93–109, 112–118 and 153–235). The only
Figure 1
Structure of cathepsin X.(a)Ribbon diagram of cathepsin X (blue ribbons) in the standard view with the mini-loop shown in green. The His23, Tyr27 mini-loop sidechains are shown in green and the Cys31 catalytic residue sidechain in yellow. Disulfide bridges are
sulfhydryl group of the enzyme that is accessible to solvent is the active site Cys31, which appears to be blocked by a small unidentified group as revealed by the final difference electron-density maps (2Fo–Fc; Figure 3). The disulfides 65–103 and 28–71 are well conserved among the papain-like enzyme structures. Like cathepsin B, cathepsin X does not have the disulfide 153–200 (using papain number-ing) to fix the upper loops defining the S2 and S1′ sub-strate binding sites. Instead there is a disulfide 153–235 much lower in the core of the β barrel domain, which is absent in the related structures. The disulfide 93–109 has an equivalent only in the cathepsin B structure (100–132), whereas the cathepsin X disulfide 112–118 appears to be unique within the family of papain-like enzymes.
Two cisprolines, Pro25 and Pro53, have been resolved in the structure. The Pro25 is positioned on the tip of the mini-loop.
Active-site cleft
The active-site cleft of cathepsin X is reminiscent of related endopeptidases. It is a V-shaped canyon with no features similar to the occluding loop of carboxy-dipepti-dase cathepsin B [33] or the mini-chain of amino-pepti-dase cathepsin H [34], which block access to a substrate. A closer inspection, taking into account the sidechains, shows that the positions of the otherwise conserved residues (Gly20 and Ser21 using papain numbering) are occupied by His23 and Tyr27. The Gly20 and Ser21 in all Figure 2
Structural based alignment of the sequences of cathepsin X (catx) with cathepsin B (1huc [33]), cathepsin H (8pch [34]), cathepsin L (1icf [53]) and papain (9pap [54]). Identical residues are shaded in black and similar residues are in gray.
Figure 3
short loop with the serine sidechain pointing away from the active-site cleft, whereas the three residue insertion (Ile24, Pro25 and Gln26) forces the His23 and Tyr27 into an extended conformation with their sidechains intruding into the active site cleft of the enzyme. The imidazole ring of His23 attaches to the surface of the mini-loop (Gln22–Cys31; Gln19–Cys25, using papain numbering) and actually occupies the place, which in equivalent struc-tures is the S2′substrate-binding site. The arrangement is similar to that in cathepsin H, in which Thr83P fills the S2 substrate-binding site. The difference is that in cathepsin H the C-terminal carboxylic group binds the N terminus of a bound substrate, whereas in cathepsin X the car-boxylic group of a bound substrate is attracted by the posi-tively charged sidechain of His23.
The active-site cleft in cathepsin X thus comprises sub-strate-binding sites S2, S1 and S1′[31,32]. The conserved catalytic residues, Cys31 and His180, and the functional groups of residues involved in interaction with a substrate mainchain are observed at the expected positions. The amide proton on Nε1 of Trp202 is in a position to bind the carbonyl of the P1′residue. The oxyanion hole between the Gln22 sidechain and the amide of Cys31 positioned at the N terminus of the central α helix from the L domain can bind the carbonyl of the P1 residue. Gly74 (Gly66 in papain) provides its amide proton as well as carbonyl oxygen to form a short antiparallel β-leader hydrogen bonded arrangement with the mainchain carbonyl and amide group of a bound P2 residue. The interaction between the car-bonyl and the electrostatic positive potential at the edge of the highly conserved Trp32 is evident [32].
With the extended mainchain of Ala157 and Thr158 forming a substantial part of the surface of the S1′ site, cathepsin X joins the group of cathepsins B and H, in con-trast to other related enzymes (cathepsins K, L and S, papain, actinidin and others), in which an additional residue twists the chain-trace into a short curve. In cathep-sin B the S1′binding site is enclosed from the top by the unique trace of the loop between Phe174 and His199, whereas cathepsin H has a valine residue at the position of Ala157. The S1′ binding site of cathepsin X is thus the largest among the related cathepsins of which the struc-tures are known.
In the related papain-like proteases the sidechain of a bound substrate P1 residue is in contact with the shallow groove of the S1 binding site formed between Ser21, Cys22 and Gly23 (papain numbering) and Cys65, Asn64, Gly66 on the other side bridged by the disulfide Cys22–Cys65 (papain numbering). (An exception is glycyl-endopeptidase, in which the Glu23 of the enzyme fills the space that is normally occupied by the moiety of the sidechain.) In the cathepsin X structure, however, the
which points downwards towards the catalytic cysteine residue, demarcating the region between the S1 and S2′ binding site. The Glu72 sidechain, which points upwards on the other side of the S1 pocket, provides a negative charge, which is not unique for a papain-like enzyme. A similar negative charge is provided by Asp72 in actinidin, whereas the occluding loop residue Glu122 in cathepsin B enters the S1 binding site from the opposite side (the side of Tyr27 in cathepsin X). The interactions between the P1 residue and the S1 binding site of cathepsin X thus appear to be tighter and also more selective than in the other related proteases.
The place where the P2 residue sidechain binds to the cathepsin X is, as in other related proteases, the only sub-strate site that can be called a pocket [32]. In most papain-like cysteine proteases this site is hydrophobic. The surface of the pocket in cathepsin X also has predomi-nantly hydrophobic character with Val181 at the bottom and Gly154 and Ile178 on the right. Ser205 (papain num-bering) is in general quite conserved, exceptions being cathepsin B and cruzipain with a glutamic acid. Out of those related enzymes for which structures are known, cathepsin X is the only one with a histidine residue (His234) at this position. In addition, the specific character of the S2 pocket is modulated in cathepsin X by Asp76, which is a hydrophobic residue in other related structures, usually methionine or proline. The Asp76–His234 arrange-ment presumably enables cathepsin X to process substrates with long sidechains with a hydrophilic tail at position P2.
Figure 4
Discussion
Carboxy-monopeptidase and carboxy-dipeptidase activities of cathepsin X
A kinetic study has shown that cathepsin X can act not only as a carboxy-monopeptidase as shown by Nägler et al. [26] but also as carboxy-dipeptidase (I.K. et al., unpublished observations) Of the substrates DnpGFFFW-OH, DnpGRFFW-OH, DnpGFRFW-OH and DnpGFFRW-OH (sequences in single-letter amino acid code), it has been found that cathepsin X cleaves the last two C-terminal residues from DnpGFRFW-OH. In addition, cathepsin X can be readily inhibited by CA074 (I.K. et al., unpublished observations), which was previously believed to be specific for cathepsin B. CA074 is an epoxysuccinyl-based inhibitor (Figure 4) with its isoleucine–proline dipeptide mimicking substrate binding in the S1′and S2′ binding sites [35–37]. We therefore used the crystal structure of CA030 in
complex with cathepsin B and the proposed substrate model [37] as a template for the modeling of the geometry of substrates bound into the active cleft of cathepsin X (Figure 5). Models of AFFW and AFRFW peptide sequences were used to investigate the mechanisms of carboxy-peptidase and carboxy-dipeptidase activities. The results of the modeling study suggest that the proton on Nε2 of His23, together with the amide proton on Nε2 of Trp202, can form the anchor for a carbonyl as well as for a carboxyl group of the C-terminal P1′ substrate residue (Figures 6 and 7). This confirms the proposal based on a homology model of cathepsin X [26]. In order to create an open S2′substrate-binding site, the His23 ring has to move out of the place occupied in the crystal structure. Simple rotation about the χ1and χ2angles of the histidine can bring the imidazole ring into a position equivalent to that occu-pied by His110 in cathepsin B, where the two Figure 5
The structure of cathepsin B (thin lines) in complex with the CA030 inhibitor (thick lines) superimposed on the structure of cathepsin X (medium lines). The His23 ring that can be rotated to the cathepsin-B-like position is shown in bold dashed lines.
Figure 6
Carboxy-peptidase activity of cathepsin X.
(a)The surface of the active-site cleft region of cathepsin X is colored according to electrostatic potential, whereas the surface of the catalytic Cys31 residue is shown in yellow. The modeled substrate AFFW is shown in ball-and-stick representation. Atoms are represented as small spheres, color-coded as in Figure 3. This figure was prepared with WebLab ViewerLite (Molecular Simulations Incorporated, San Diego, CA).
histidines His110 and His111 [33,37] bind the carboxyl group of a substrate (Figure 5). Cathepsin X has no equiva-lent residue to His111 of cathepsin B and therefore inter-acts with only one carboxyl group oxygen atom of a substrate. This explains why cathepsin X does not differen-tiate between the substrates with blocked and free C-termi-nal carboxylic group (I.K. et al., unpublished observations).
The flip-flop of the His23 sidechain between the confor-mation observed in the crystal structure and the cathepsin B-like conformation is most probably a substrate-induced mechanism. The carboxy-dipeptidase activity appears to be defined by the affinity of cathepsin X for the residue binding into the S1 binding site. Arginine is preferred over phenylalanine to such an extent that, instead of accepting an arginine residue in the S2 binding site, the enzyme modifies the binding pattern by shifting the arginine into the P1 position (I.K. et al., unpublished observations). Thereby two binding sites on the prime site are made available and carboxy-dipeptidase activity is exhibited. From our modeling study it is not evident whether a bound P1 residue sidechain results in a shift of the Tyr27 ring, which then assists in displacement of the His23 sidechain. This is an issue that requires further investigation.
Binding of protein inhibitors
In contrast to the initial report [26], cathepsin X can be inhibited with various cystatin-type protease inhibitors such as cystatin C and stefin A with K
i values in the nanomolar range (I.K. et al., unpublished observations), similar to those obtained for the inhibition of cathepsin B [30]. A modeling study based on the papain–stefin B complex [38] has shown that the sidechains of His23 and Tyr27 clash with residues Val55 and Ala56 in the QVVAG region (data not shown). A similar situation occurs in
cathepsin B, in which a cystatin-type inhibitor competes with occluding loop residues for binding into the active-site cleft [39,40]. The obvious solution appears to be that the sidechains of His23 and Tyr27 are displaced when a complex with a cystatin inhibitor is formed. Our modeling study suggests that the ring of His23 flips away into its cathepsin B-like position and the ring of Tyr27 rotates into the S1 substrate-binding site.
Specific inhibitor design
The primary goal of the design of specific inhibitors of lysosomal cysteine proteases is to establish the biological role of a particular enzyme. Cross reactivity is likely to lead to wrong conclusions. The relatively wide tissue dis-tribution and abundance of cathepsin X [23,24] are similar to those of cathepsin B. Because CA074 has been shown to interact similarly with cathepsins X and B, it is evident that physiological studies based on the use of this inhibitor have to be repeated in order to clarify whether cathepsin B or cathepsin X, or perhaps both enzymes, are implicated.
The structure of cathepsin X in a putative complex with CA030, which was generated by superposition of the structure of cathepsin B complexed with CA030 [37] on the structure of cathepsin X, provides the first hints for the design of inhibitors able to differentiate between the two enzymes (Figure 5). CA030 is a very close analog of CA074 (Figure 4): the inhibitors do not differ in the parts that interact with the primed binding sites of the enzymes. The only difference is in the nonprimed side, where an ester-tail instead of an amide-tail points towards the S2 binding site. In order to convert CA074 into a selective inhibitor of cathepsin X, the following modifica-tions appear evident. Firstly, a compound with a single amino acid residue instead of two could be attached to an
Carboxy-dipeptidase activity of cathepsin X. His23 in the cathepsin X active site has been rotated to the cathepsin-B-like position.
(a)The same color coding as in Figure 6 is used. The model of substrate AFRFW is shown in ball-and-stick representation.
epoxysuccinyl reactive group and bind to His23 of cathep-sin X and thus be too short to reach the His110 and His111 residues on the surface of cathepsin B. Secondly, a bulkier residue than isoleucine of CA074 could fill the S1′ site of cathepsin X and simultaneously not be able to bind the smaller S1′site of cathepsin B. Thirdly, the carboxyl group of CA074 could be modified to interfere with the binding to cathepsin B residue His111 and binding to cathepsin X would remain unaffected.
The reverse case, the design of an inhibitor that will inhibit only cathepsin B and not cathepsin X (and others) seems a more difficult problem. One solution might be to enlarge the cathepsin B inhibitor by expanding the inter-action region to the nonprimed binding sites. It is known that the specificity of papain-like cysteine proteases is par-ticularly encoded in the S2 and S1′substrate binding sites [31,32]. A large number of small organic compounds react chemically with the reactive site sulfhydryl of a cysteine protease [31]. Most of these inhibitors interact only with one half of the active-site cleft on the ‘nonprimed’ side. A selective inhibitor of cathepsin B should probably span the active site from the S2 to the S2′binding site, as has already been suggested [37]. Such a setup has been shown to be quite successful in the cases of lysosomal cysteine endopeptidases, cathepsins L, S and K [41,42], whereas the potent double-headed inhibitors of cathepsin B might now require additional improvements [43,44]. The selec-tive cathepsin B inhibitor should, therefore, exploit the ‘primed’ substrate binding sites S1′ and S2′ as well as reach into the S2 binding pocket, in which it should opti-mally utilize the fit against the cathepsin B residues Pro76 and Glu245 and at the same time repel the equivalent cathepsin X residues Asp76 and His234.
Biological implications
Lysosomal cysteine proteases are involved in a number of physiological and pathological events. Although similar in sequence and fold, they exhibit diversity in localization, substrate and inhibitor specificity and sta-bility. With the discovery of new members of the group, the perception that the role of lysosomal cysteine teases is limited to the nonselective degradation of pro-teins in lysosomes is changing. There is a growing body of evidence that they work in concert. For example, they appear to be involved in the strictly regulated matura-tion of major histocompatibility complex (MHC) class II molecules and antigen presentation [2].
It is not surprising that the discovery of each new enzyme might require reconsideration of current knowl-edge. In the case of cathepsin X it is evident that new inhibitors should be developed in order to re-evaluate the role(s) of cathepsin B. The crystal structure of cathepsin X presented here reveals the differences and similarities between the structures of the active sites of
the two enzymes. The structure of cathepsin X with a rotated His23 ring, superimposed on the structure of the complex of CA030 with cathepsin B and models of sub-strates AFFW and AFRFW, explains the dual capabil-ity of cathepsin X to exhibit carboxy-monopeptidase and carboxy-dipeptidase activities and thus provide the basis for design of selective inhibitors of cathepsin B.
Although cathepsin X was unknown until recently, it has already been involved in an industrial application [25,27]. It is held responsible for the cleavage of β -amyloid peptide, which can, when secreted from the cells and accumulated in amyloid plaques, result in Alzheimer’s disease. Inhibition of its activity was pro-posed to slow-down the progress of the disease and, if diagnosed in time, even prevent its manifestation. It should be noted that this role of cathepsin X is based on an inhibition study and might thus require additional clarification.
Materials and methods
Crystallization and data collection
Cathepsin X was isolated from human liver (I.K. et al., unpublished
observations). The purified cathepsin X was concentrated to a concen-tration of 8.8 mg/ml in a spin concentrator (Centricon; Amicon). Crys-tals were grown using the sitting-drop vapor diffusion method. The reservoir contained 1 ml 14.4% PEG 4000, 7.2% 2-propanol and 0.072 M Na-HEPES adjusted to pH 8.0. The drop was composed of 2.2µl reservoir solution and 2.8µl protein (8.8 mg/ml) in 20 mM Na-acetate and 1 mM EDTA pH 5.1.
Diffraction data were collected from a single crystal using Cu Kα radia-tion from a Rigaku Ru200 rotating-anode X-ray generator and recorded on a 345 mm MAR Research image-plate detector. Although crystals diffracted to a higher resolution at synchrotron, we did not use synchro-tron data sets because of increased mosaicity of the frozen crystals. Auto indexing and scaling was done using the programs MOSFLM and SCALA [45,46]. The crystal diffracted to 2.67 Å resolution and belonged to the orthorhombic space group C2221 with cell dimen-sions a = 62.3 Å, b = 92.5 Å, c = 209.9 Å. The asymmetric unit con-tained two molecules.
Structure determination and refinement
The structure of cathepsin X was solved using the molecular replace-ment method implereplace-mented in EPMR program [47]. The crystal struc-ture of actinidin [48] was used as the search model. Two solutions with a correlation factor of 0.311 and an R value of 0.54 were found using data in the 15–4 Å resolution range.
coordinate. After the crystallographic R value dropped below 0.25 at 2.7 Å resolution, the temperature-factor refinement was applied. Water molecules were generated using an automatic procedure in MAIN and then corrected manually.
The final refinement included all reflections (cutoff 1σ) in the resolution range 10–2.67 Å, with the crystallographic R value being 0.183. The geometry of the final model was inspected with MAIN and PROCHECK [51]. All residues lie in the allowed regions of the Ramachandran plot, 0.825 of residues in most favored regions and 0.175 in additional allowed regions. Other structural parameters for the structure refined at this resolution show no significant deviations from the expected values (overall G factor is 0.2, which is better than expected for 2.67 Å resolution). The crystallographic data and refine-ment statistics are summarized in (Table 1).
Accession numbers
The coordinates have been deposited in the Protein Data Bank with accession code 1EF7 and will be on hold for one year.
Acknowledgements
We thank CNR for providing us beam time at synchrotron Elettra and the CNR team for help with data collection. We thank Roger H Pain for thought-ful reading of the manuscript. This work was supported by the Slovenian Ministry of Science and Technology.
References
1. Barrett, A.J., Rawlings, N.D. & Woessner, J.F. Jr. (1998), Handbook of proteolytic enzymes. (Barrett, A.J., Rawlings, N.D. & Woessner, J.F. Jr., eds) Academic Press Ltd., London.
2. Chapman, H.A., Riese, R.J. & Shi, G.P. (1997), Emerging roles for
cysteine proteases in human biology. Annu. Rev. Physiol.59, 63-88.
3. Turk, B., Turk, D. & Turk, V. Lysosomal cysteine proteases: more than
scavengers. Biochim. Biophys. Acta,in press.
4. Cresswell, P. (1996). Invariant chain structure and MHC class II
function. Cell84, 505-507.
5. Nakagawa, T., et al., & Rudensky, A.Y. (1998). Cathepsin L: critical
role in Ii degradation and CD4 T cell selection in the thymus. Science
6. Nakagawa, T.Y., et al., & Rudensky, A.Y. (1999). Impaired invariant
chain degradation and antigen presentation and diminished
collagen-induced arthritis in cathepsin S null mice. Immunity10, 207-217.
7. Shi, G.P., et al., & Chapman, H.A. (1999). Cathepsin S required for
normal MHC class II peptide loading and germinal center
development. Immunity10, 197-206.
8. Drake, F.H., et al., & Gowen, M. (1996). Cathepsin K, but not
cathepsins B, L, or S, is abundantly expressed in human osteoclasts.
J. Biol. Chem.271, 12511-10006.
9. Brömme, D., Okamoto, K., Wang, B.B. & Biroc, S. (1996). Human cathepsin O2, a matrix protein-degrading cysteine protease expressed in osteoclasts. Functional expression of human cathepsin O2 in
Spodoptera frugiperdaand characterization of the enzyme. J. Biol. Chem.271, 2126-2132.
10. Saftig, P., et al., & von Figura, K. (1998). Impaired osteoclastic bone
resorption leads to osteopetrosis in cathepsin-K-deficient mice. Proc.
Natl Acad. Sci. USA95, 13453-13458.
11. Wang, P.H., et al., & Hsueh, W.A. (1991). Identification of renal
cathepsin B as a human prorenin-processing enzyme. J. Biol. Chem.
266, 12633-12638.
12. Dunn, A.D., Crutchfield, H.E. & Dunn, J.T. (1991). Thyroglobulin processing by thyroidal proteases. Major sites of cleavage by
cathepsins B, D and L.J. Biol. Chem.266, 20198-20204.
13. Brix, K., Lemansky, P. & Herzog, V. (1996). Evidence for extracellularly acting cathepsins mediating thyroid hormone liberation in thyroid
epithelial cells. Endocrinology137, 1963-1974.
14. Nagaya, T., et al., & Seo, H. (1998). Intracellular proteolytic cleavage
of 9-cis-retinoic acid receptor alpha by cathepsin L-type protease is a
potential mechanism for modulating thyroid hormone action. J. Biol.
Chem.273, 33166-33173.
15. Pham, C.T. & Ley, T.J. (1999). Dipeptidyl peptidase I is required for
the processing and activation of granzymes A and B in vivo. Proc. Natl
Acad. Sci. USA96, 8627-8632.
16. Sloane, B.F., Moin, K. & Lah, T. (1994). Biochemical and molecular
aspects of selected cancers.(Pretlow, T.G. and Pretlow, T.P., eds).
Academic Press, San Diego.
17. Kos, J. & Lah, T.T. (1998). Cysteine proteinases and their endogenous inhibitors: target proteins for prognosis, diagnosis and therapy in
cancer. Oncol. Rep. 5, 1349-1361.
18. Michaud, S. & Gour, B.J. (1998). Cathepsin B inhibitors as potential
anti-metastatic agents. Exp. Opin. Ther. Patents8, 645-672.
19. Kominami, E., Kunio, I. & Katunuma, N. (1987). Activation of the intramyofibral autophagic-lysosomal system in muscular dystrophy.
Am. J. Pathol.127, 461-466.
20. Mort, J.S., Recklies, A.D. & Poole, A.R. (1984). Extracellular presence of the lysosomal proteinase cathepsin B in rheumatoid synovium and
its activity at neutral pH. Arthritis. Rheum.27, 509-515.
21. Gelb, B. D., Shi, G. P., Chapman, H. A. & Desnick, R. J. (1996). Pycnodysostosis, a lysosomal disease caused by cathepsin K
deficiency. Science273, 1236-1008.
22. Cataldo, A.M. & Nixon, R.A. (1990). Enzymatically active lysosomal proteases are associated with amyloid deposits in Alzheimer brain.
Proc. Natl Acad. Sci. USA87, 3861-3865.
23. Santamaria, I., Velasco, G., Cazorla, M., Fueyo, A., Campo, E. & López-Otín, C. (1998). Cathepsin L2, a novel human cysteine proteinase
produced by breast and colorectal carcinomas. Cancer Res.
58, 1624-1630.
24. Nägler, D.K. & Ménard, R. (1998). Human cathepsin X: a novel cysteine protease of the papain family with a very short proregion and
unique insertions. FEBS Lett.434, 135-139.
25. Tung, J.S., Sinha, S., McConlogue, L., & Semko, C.M.F. Cathepsin and methods and compositions for inhibition thereof. Athena
Neurosciences, Inc. 469362(5849711). 12-15-1998. South San Francisco,CA. US Patent.
26. Nägler, D.K., Zhang, R., Tam, W., Sulea, T., Purisima, E.O. & Menard, R. (1999). Human cathepsin X: a cysteine protease with unique
carboxypeptidase activity. Biochemistry38, 12648-12654.
27. Tung, J.S., Sinha, S., & Semko, C.M.F. Cathepsin and methods and compositions for inhibition thereof. Athena Neurosciences, Inc. 850392(5858982). 1-12-1999. South San Francisco, CA. 5-2-1997. US Patent.
28. Linebaugh, B.E. (1999). Exocytosis of active cathepsin B enzyme
activity at pH 7. 0, inhibition and molecular mass. Eur. J. Biochem.
264, 100-109.
29. Coulibaly, S., et al., & Mach, L. (1999). Modulation of invasive
properties of murine squamous carcinoma cells by heterologous
Crystallographic data and refinement statistics.
Data collection
Space group C2221 Cell parameters a = 62.3 Å, b = 92.5 Å,
c = 209.9 Å, α=β=γ= 90° Molecules in asymmetric unit 2
Limiting resolution (Å) 2.67 Measured reflections 15,2861 Independent reflections 16,732 Rsym(99–2.67 Å) 0.062 Completeness (99–2.67 Å) 0.95 Completeness in the highest shell (2.83–2.67 Å) 0.87
I/σ(99–2.67 Å) 11.9 I/σin the highest shell (2.83–2.67 Å) 3.6
Final refinement parameters
No. scattering protein atoms 3816 No. solvent molecules 118 Resolution range in refinement (Å) 10.0–2.67 Reflections used in refinement 16,433
R factor 0.183
Rfree(%) 0.226
Geometry of the final model
Rmsd of bond distances (Å) 0.012 Rmsd of bond angles (°) 1.6
B factor value (rms by bonds)
expression of cathepsin B and cystatin C. Int. J. Cancer83, 526-531. 30. Turk, B., Turk, V. & Turk, D. (1997). Structural and functional aspects
of papain-like cysteine proteinases and their protein inhibitors. Biol.
Chem.378, 141-150.
31. McGrath, M.E. (1999). The lysosomal cysteine proteases. Annu. Rev.
Biophys. Biomol. Struct.28, 181-204.
32. Turk, D., Guncar, G., Podobnik, M. & Turk, B. (1998). Revised definition of substrate binding sites of papain-like cysteine proteases.
Biol. Chem.379, 137-147.
33. Musil, D., et al., & Bode, W. (1991). The refined 2.15 Å X-ray crystal
structure of human liver cathepsin B: the structural basis for its
specificity. EMBO J.10, 2321-2330.
34. Guncvar, G., Podobnik, M., Pungercvar, J., Štrukelj, B., Turk, V. & Turk,
D. (1998) Crystal structure of porcine cathepsin H determined at 2.1 Å resolution: location of the mini-chain C-terminal carboxyl group
defines cathepsin H aminopeptidase function. Structure6, 51-61.
35. Towatari, T., et al., & Katunuma, N. (1991). Novel epoxysuccinyl
peptides. A selective inhibitor of cathepsin B, in vivo. FEBS Lett.
280, 311-305.
36. Buttle, D.J., Murata, M., Knight, C.G. & Barrett, A.J. (1992). CA074
methyl ester: a proinhibitor for intracellular cathepsin B. Arch.
Biochem. Biophys. 299, 377-380.
37. Turk, D., et al., & Turk, V. (1995). Crystal structure of cathepsin B
inhibited with CA030 at 2.0- Å resolution: a basis for the design of
specific epoxysuccinyl inhibitors. Biochemistry34, 4791-4007.
38. Stubbs, M.T., et al., & Turk, V. (1990). The refined 2.4 Å X-ray crystal
structure of recombinant human stefin B in complex with the cysteine proteinase papain: a novel type of proteinase inhibitor interaction.
EMBO J.9, 1939-1947.
39. Illy, C., Quraishi, O., Wang, J., Purisima, E., Vernet, T. & Mort, J.S.
(1997). Role of the occluding loop in cathepsin B activity. J. Biol.
Chem.272, 1197-1202.
40. Podobnik, M., Kuhelj, R., Turk, V. & Turk, D. (1997). Crystal structure of the wild-type human procathepsin B at 2.5 Å resolution reveals the
native active site of a papain-like cysteine protease zymogen. J. Mol.
Biol. 271, 774-788.
41. Thompson, S.K., Halbert, S.M., Bossard, M.J., Tomaszek, T.A., Levy, M.A. & Veber, D.F. (1997). Design of potent and selective human
cathepsin K inhibitors that span the active site. Proc. Natl Acad. Sci.
USA94, 14249-14254.
42. Katunuma, N., et al., & Asao, T. (1999). Structure based development
of novel specific inhibitors for cathepsin L and cathepsin S in vitro and
in vivo. FEBS Lett.458, 6-10.
43. Schaschke, N., Assfalg-Machleidt, I., Machleidt, W., Turk, D. & Moroder, L. (1997). E-64 analogues as inhibitors of cathepsin B. On the role of the absolute configuration of the epoxysuccinyl group.
Bioorg. Med. Chem.5, 1789-1797.
44. Schaschke, N., Assfalg-Machleidt, I., Machleidt, W. & Moroder, L. (1998). Substrate/propeptide-derived endo-epoxysuccinyl peptides as
highly potent and selective cathepsin B inhibitors. FEBS Lett.
421, 80-82.
45. Leslie, A.G.W. (1992). Joint CCP4 and ESF-EACMB. Newsletter on
Protein Crystallography,26, SERC Daresbury Laboratory, Warrington,
UK.
46. CCP4 (1994). Collaborative Computational Project Number 4, The
CCP4 suite: programs for proteine crystallography. Acta Crystallogr.
50, 760-763.
47. Kissinger, C.R., Gehlhaar, D.K. & Fogel, D.B. (1999). Rapid automated
molecular replacement by evolutionary search. Acta. Crystallogr. D
Biol. Crystallogr.55( Pt 2), 484-491.
48. Baker, E.N. (1980) Structure of actinidin, after refinement at 1.7 Å
resolution.J. Mol. Biol.141, 441-484.
49. Turk, D. (1992). Further Development of a Program for Molecular
Graphics and Electron Density Manipulation and its Application in Various Protein Structure Determinations. PhD Thesis, Technische
Universität München, Germany.
50. Engh, R.A. & Huber, R. (1991) Accurate bond and angle parameters
for X-ray protein structure refinement. Acta Crystallogr. A 47, 392-400.
51. Laskowski, R.A., MacArthur, M.W., Moss, D.S. & Thornton, J.M. (1993) PROCHECK: a program to check the stereochemical quality of
protein structures. J. Appl. Crystallogr.26, 283-291.
52. Carson, M. (1991) Ribbons 2.0. J. Appl. Crystallogr.24, 958-961.
53. Guncv
ar, G., Pungercv
icv
, G., Klemencic, I., Turk, V. & Turk, D. (1999) Crystal structure of MHC class II-associated p41 Ii fragment bound to cathepsin L reveals the structural basis for differentiation between
cathepsins L and S.EMBO J.18, 793-803.
54. Kamphuis, I.G., Kalk, K.H., Swarte, M.B. & Drenth, J. (1984) Structure
of papain refined at 1.65 Å resolution. J. Mol. Biol.179, 233-286.
