st8308.qxd 03/22/2000 11:36 Page 305 Research Article 305 Crystal structure of cathepsin X: a flip–flop of the ring of His23 allows carboxy-monopeptidase and carboxy-dipeptidase activity of the protease Gregor Guncar1, Ivica Klemencic1, Boris Turk1, Vito Turk1, Adriana Karaoglanovic-Carmona2, Luiz Juliano2 and Dušan Turk1* Background: Cathepsin X is a widespread, abundantly expressed papain-like Addresses: 1Department of Biochemistry and v mammalian lysosomal cysteine protease. It exhibits carboxy-monopeptidase as Molecular Biology, Jozef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia and 2Departamento de well as carboxy-dipeptidase activity and shares a similar activity profile with Biofisica, Escola Paulista de Medicina, Rua Tres de cathepsin B. The latter has been implicated in normal physiological events as Maio 100, 04044-020 Sao Paulo, Brazil. 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 *Corresponding author. E-mail: [email protected] two enzyme activities has actually been monitored. Key words: Alzheimer’s disease, carboxypeptidase, Results: The crystal structure of human cathepsin X has been determined at cathepsin B, cathepsin X, papain-like cysteine 2.67 Å resolution. The structure shares the common features of a papain-like protease enzyme fold, but with a unique active site. The most pronounced feature of the Received: 1 November 1999 cathepsin X structure is the mini-loop that includes a short three-residue Revisions requested: 8 December 1999 insertion protruding into the active site of the protease. The residue Tyr27 on Revisions received: 6 January 2000 one side of the loop forms the surface of the S1 substrate-binding site, and Accepted: 7 January 2000 His23 on the other side modulates both carboxy-monopeptidase as well as Published: 29 February 2000 carboxy-dipeptidase activity of the enzyme by binding the C-terminal carboxyl group of a substrate in two different sidechain conformations. Structure 2000, 8:305–313 Conclusions: The structure of cathepsin X exhibits a binding surface that will 0969-2126/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. View metadata, citationassist and insimilar the design papers of at specificcore.ac.uk inhibitors of cathepsin X as well as of cathepsin brought to you by CORE B and thereby help to clarify the physiological roles of both proteases. provided by Repositório Institucional UNIFESP Introduction pathological processes such as tumor malignancy and pro- Cathepsin X belongs to the group of lysosomal cysteine gression (reviewed in [16–18]), muscular dystrophy [19], proteases, which constitute a numerous and important part rheumatoid arthritis [20], osteoporosis and other bone dis- of the family of papain-like enzymes (clan CA; reviewed orders [2,10,21] and Alzheimer’s disease [22]. in [1]). The research interest in this field is increasing rapidly, together with the number of identified enzymes, Cathepsin X was discovered only recently [23–25] and its as it becomes evident that lysosomal cysteine proteases biochemical characterization is just emerging [25–27]. It is are, besides being involved in intracellular protein catabo- the only known lysosomal cysteine protease exhibiting lism, also involved in a number of important cellular carboxy-monopeptidase activity. Besides carboxy- processes [2,3]. Currently 11 human cathepsins are known monopeptidase activity cathepsin X also exhibits carboxy- at the sequence level (B, C, F, H, K, L, O, S, V, W and X). dipeptidase activity and is readily inhibited by CA074, Some of them are expressed in a wide variety of cells (B, previously known as the specific inhibitor of cathepsin B, C, F, H, L, O and X), whereas others (S, K, V and W) are which is the only known carboxy-dipeptidase among lyso- tissue-specific and have been shown to be or proposed to somal cysteine proteases (I.K. et al., unpublished observa- be involved in more specialized processes (reviewed in tions). The inhibitor has been used as a standard tool to [3]). They are involved in invariant chain processing and assess functions of cathepsin B in numerous in vivo and in antigen presentation [4–7], in bone remodeling [8–10] and vitro studies, for example in relation with exocytosis [28] in proteolytic processing of various proteins [11], among and cancer (reviewed in [16,18,29]). Similarly, the implica- these the most important being thyroglobulin [12–14] and tion of cathepsin X in the development of Alzheimer’s granzyme B [15]. Whereas the former is an important disease is based on the use of inhibitors [27] and therefore source of thyroglobulin hormones the latter is involved in requires confirmation. the activation of the cell death proteases, the caspases (reviewed in [3]). Besides their normal physiological roles, A structural investigation of the active site of cathepsin X is, lysosomal cysteine proteases are involved in a number of therefore, a mandatory step for understanding the specific st8308.qxd 03/22/2000 11:36 Page 306 306 Structure 2000, Vol 8 No 3 features of cathepsin X in order to establish the structural (Figure 1). The two domains interact along an extended basis of the specificity of similar cathepsins, in particular interface, reminiscent of a book with the spine in front. At cathepsin B. This will enable us to design substrates and the top the two domains separate and form a ‘V-shaped’ inhibitors that will address specifically only the desired active-site cleft with the reactive site residues, Cys31 and enzyme. It has already been suggested, from sequence data His180, each coming from a different domain. and a substrate study [26], that a short insertion of residues 24–26, which constitutes the tip of the mini-loop placed in The structure of cathepsin X, as judged from a number of the immediate vicinity of the reactive site, must be superimposed pairs, is rather similar to the structures of involved in interaction with substrate. It was also suggested human cathepsin K, rat cathepsin B, zymogen of cathepsin [26] that the mini-loop residue His23 is involved in recogni- L and papain (yielding respectively 145, 148, 146 and 141 tion and binding of the C-terminal carboxylic group of a equipositioned Cα pairs at a threshold distance of 1.5 Å). bound substrate. The crystal structure of cathepsin X pre- It is less similar to cathepsin L, actinidin, glycyl-endopep- sented here has enabled us to elucidate the structural basis tidase I and cruzipain (yielding 122, 116, 128 and 130 of the mechanism by which cathepsin X exhibits both pairs) and least similar to human cathepsin B and porcine carboxy-monopeptidase and carboxy-dipeptidase activity. cathepsin H (with 89 and 73 pairs; Figure 2). Although by numerical criteria the structure of cathepsin X does not Results superimpose on the structure of cathepsin B as well as on Overall structure those of other related enzymes, a visual inspection reveals The structure of human cathepsin X comprises the that the structures of cathepsin B, and to a smaller extent sequence of the whole mature single-chain protein of 242 cathepsin H, are the most similar in the position and archi- amino acid residues, which are numbered consecutively tecture of the surface loops to the structure of cathepsin X. from Leu1 to Val242. The asymmetric unit of orthorhom- Some outstanding loops of the cathepsin X structure bic crystals contains two molecules. The three-dimen- (110–123 and 189–193) appear at similar positions to some sional structures of the two molecules are almost identical. cathepsin B loops (132–139 and 208–212). Particularly The complete chains of both molecules, with the excep- striking is the exposed position of the ring of Phe115, tion of a few sidechain residues, are well resolved in the placed at the tip of the 110–123 loop. The short three- electron-density maps. The superimposed structures show residue insertion (Ile24, Pro25 and Gln26) appears to be of a root mean square deviation (rmsd) of 0.55 Å for all Cα particular importance for the specificity of cathepsin X. It atoms; when only residues included into noncrystallo- is located within the mini-loop (Figure 3), an otherwise graphic symmetry (NCS) constraints are used for Cα atom conserved loop between Gln19 and Cys25 (using papain superposition (235 residues), the resulting rms fitting pro- numbering), forming the wall of the S2′ substrate-binding cedure yields a considerably lower value (0.26 Å). site [32]. An exception is the loop of glycyl-endopepti- dase, in which a tyrosine residue replaces the conserved The structure possesses the typical fold of a papain-like Ser21 (papain numbering). cysteine protease, a two domain structure [30,31]. The predominant secondary structural features of the N-termi- The mature form of human cathepsin X contains 11 cys- nal domain on the left, or L domain, are α helices, whereas teine residues. Ten of them are involved in disulfide bonds the C-terminal domain, or R domain, has a β-barrel fold (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 shown as orange sticks. The figure was prepared using the program ribbons) in the standard view with the mini-loop shown in green. The RIBBONS [52]. (b) A stereo plot of the Cα trace of cathepsin X. The His23, Tyr27 mini-loop sidechains are shown in green and the dotted lines represent disulphide bridges. Cys31 catalytic residue sidechain in yellow. Disulfide bridges are st8308.qxd 03/22/2000 11:36 Page 307 Research Article Crystal structure of human cathepsin X Guncv ar et al. 307 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]).