sign in sign up
<<
Home , Cathepsin S, Cruzipain, Cystatin, Cystatin A, Cystatin B, Cysteine protease

Vol. 48 No. 1/2001

1–20

QUARTERLY

Review

Structural studies of and their inhibitors*.

Zbigniew Grzonka1½, El¿bieta Jankowska1, Franciszek Kasprzykowski1, Regina Kasprzykowska1, Leszek £ankiewicz1, Wies³aw Wiczk1, Ewa Wieczerzak1, Jerzy Ciarkowski1, Piotr Drabik1, Robert Janowski2, Maciej Kozak3, Mariusz Jaskólski2,4 and Anders Grubb5

1Faculty of Chemistry, University of Gdañsk, Gdañsk, Poland; 2Faculty of Chemistry, A. Mickiewicz University, Poznañ, Poland; 3Faculty of Physics, A. Mickiewicz University, Poznañ, Poland; 4Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznañ, Poland; 5Department of Clinical Chemistry, University Hospital, Lund, Sweden Received: 12 December, 2000; accepted: 29 January, 2001

Key words: cysteine proteases, , synthetic inhibitors, structure–activity relationship

Cysteine proteases (CPs) are responsible for many biochemical processes occurring in living organisms and they have been implicated in the development and progression of several diseases that involve abnormal turnover. The activity of CPs is regulated among others by their specific inhibitors: cystatins. The main aim of this review is to dis- cuss the structure–activity relationships of cysteine proteases and cystatins, as well as of some synthetic inhibitors of cysteine proteases structurally based on the binding frag- ments of cystatins.

CYSTEINE PROTEASES (CPs) metallo-proteases [1, 2]. Cysteine proteases (CPs) are with molecular mass about 21–30 Proteases are classified according to their cata- kDa. They show the highest hydrolytic activity at lytic site into four major classes: serine proteases, pH 4–6.5. Because of the high tendency of the cysteine proteases, aspartic proteases and group to oxidation, the environment of the

*Presented at the International Conference on “Conformation of Peptides, Proteins and Nucleic Acids”, Debrzyno, Po- land, 2000. .This work was supported by the State Committee for Scientific Research (KBN, Poland) grant No. 279/P04/97/13. ½Corresponding author: Prof. Zbigniew Grzonka, Department of Organic Chemistry, Faculty of Chemistry, University of Gdañsk, J. Sobieskiego 18, 80-952 Gdañsk, Poland; tel.: (48 58) 345 0369; fax.: (48 58) 344 9680; e-mail: [email protected] Abbreviations: Boc, t-butoxycarbonyl; CP, cysteine ; DAM, diazomethylketone; dArg, desaminearginine; E64, (2S,3S)-trans-epoxysuccinyl-L-leucyl-agmatine; hCA, hCB, hCC, hCD, hCE, hCF, hCS, hCSA, hCSN, A, B, C, D, E, F, S, SA, SN, respectively; hHK, hLK, human high or low molecular mass , respectively; ValY[CH2NH], (S)-1-isopropyl-1,2-etanediamine; Z, benzyloxycarbonyl. 2 Z. Grzonka and others 2001 should contain a reducing component. in all CPs. Following a proposal by Schechter & Glutathione serves as an activating agent in cells, Berger [6], the substrate pocket of binds at whereas addition of mercaptoethanol or dithio- least seven amino-acid residues in appropriate threitol is required for in vitro experiments. subsites (Fig. 2). Recently, Turk et al. [7] have pro- Cysteine proteases are present in all living or- posed, on the basis of kinetic and structural stud- ganisms. Till now, 21 families of CPs have been ies, that only 5 subsites are important for sub- discovered [3, 4], almost half of them in viruses. strate binding. The S2,S1, and S1¢ pockets are im- Many of these are found in bacteria (e.g. portant for both backbone and side-chain binding, in Clostridium histolyticum, whereas S3 and S2¢ are crucial only for amino in Porhyromonas gingivalis), fungi ( B in acid side-chain binding. Aspergillus flavus, proteinease ysc F in yeast), pro- tozoa ( in , amoebo- pain in Entamoeba histolytica) and plants (papain and in Carica , ficin in Ficus glabrata, in Ananas comosus, actinidin in Actinidia chinesis, calotropin DI in Calotropis gigantea). In mammals, two main groups of cysteine proteases are present: cytosolic ( type I, calpain type II) and lysosomal Figure 2. Substrate subsites of papain [6]. (cathepsins: B, C, H, K, L, M, N, S, T, V, and W) [3–5]. The best characterized family of cysteine pro- Enzymatic activity of cysteine proteases is re- teases is that of papain. The papain family con- lated to the presence of a catalytic diad formed by tains peptidases which are structurally related to the cysteine and residues which in the papain, like for example lysosomal cathepsins. pH interval 3.5–8.0, exists as an ion-pair Papain is characterized by a two-domain structure –S–...H+Im– [8, 9]. Formation of an intermedi- (Fig. 1). The (catalytic pocket), where ate, S-acyl-enzyme moiety, is a fundamental step in . This intermediate is formed via nucleophilic attack of the thiolate group of the cysteine residue on the of the hy- drolyzed peptide bond with a release of the C-terminal fragment of the cleaved product. In the next step, a water molecule reacts with the inter- mediate, the N-terminal fragment is released, and the regenerated free papain molecule can begin a new catalytic cycle [10]. CPs are responsible for many biochemical pro- cesses occurring in living organisms. The main physiological role of CPs is metabolic degradation of peptides and proteins. Mammalian cysteine proteases have been implicated in the develop- ment and progression of many diseases that in- Figure 1. Structure of papain [100]. volve abnormal protein turnover [11–15]. The ac- tivity of cysteine proteases is regulated by proper the substrate is bound, is located between the do- transcription and the rate of protease syn- mains. The catalytic residues of papain are Cys25 thesis and degradation, as well as by their specific and His159, and they are evolutionarily preserved inhibitors. Vol. 48 Structural studies of cysteine proteases and their inhibitors 3 CYSTATINS been assigned to 3 [29], and that for human stefin B to [30]. Many natural protein inhibitors of cysteine pro- teases, called cystatins, have been isolated and Cystatin family characterized. They act both intra- and extra- cellularly forming complexes with their target en- The cystatin family comprises the following hu- zymes. Maintenance of appropriate equilibrium man cystatins: C (hCC), D (hCD), E (hCE), F between free cysteine proteases and their com- (hCF), S (hCS), SA (hCSA) and SN (hCSN). Their plexes with inhibitors is critical for proper func- homologues have also been found in other mam- tioning of all living systems. In this role, cystatins malian organisms and birds. Chicken cystatin has are general regulators of harmful cysteine prote- been used in defining the superfamily of cystatins ase activities. The roles of cystatins in health and [31, 32]. Human cystatins are coded on chromo- disease have been reviewed by Henskens et al. some 20 [33]. They consist of 120–122 amino-acid [13] and Grubb [14]. residues and are synthesized as proproteins con- The human superfamily of cystatins is divided taining a signal peptide (20 residues), which sug- into three families. Family I, called stefins, com- gests that cystatins display an extracellular activ- prises intracellular cystatins A and B. Family II ity [34]. The cystatins contain two includes extracellular and/or transcellular cysta- bridges and most of them are not glycosylated. tins (cystatins: C, D, E, F, S, SA, and SN). Kinino- Cystatins S, SA, and SN (S-type cystatins) consist- gens, the intravascular cystatins, form family III ing of 121 amino-acid residues with molecular of cystatins. mass of 14.2–14.4 kDa display high (90%). Post-translational phosphoryla- tion of cystatins S and SA leads to formation of Stefin family several isoforms. Expression of these cystatins is The stefin family comprises the following inhibi- very restricted: cystatin SN has been found only tors: human stefin A (hCA), human stefin B (hCB) in saliva and tears, whereas variants S and SA are [16–18], as well as their analogues from rat [19], also present in seminal fluid [35]. Cystatin D has bovine [20, 21] and porcine [22] tissues, and from also been found mostly in saliva and tears. Fully some plants [23]. Stefins are proteins of about active cystatin D, formed after removal of the 100 amino-acid residues (molecular mass about 20-residue signal peptide, consists of 122 amino- 11 kDa) which do not contain any sugar moiety or acid residues with molecular mass about 13.8 disulfide bridge. There is one cysteine residue in kDa. The protein exists in 2 polymorphic forms: the stefin B sequence, and it can be converted into [Cys26]hCD and [Arg26]hCD which have identical 3 3¢ an intermolecular disulfide bridge (–Cys –Cys –) activity, stability, and distribution. The quite low resulting in formation of inactive dimers, easily homology with other cystatins (51–55%) suggests transformed back into active monomers at reduc- that, on a phylogenetic tree, cystatin D is located ing conditions [24, 25]. Immunohistochemical between cystatins S and C[36, 37]. Human studies have shown the presence of stefin A in cystatin E (hCE, [38]), also described in the litera- skin and , suggesting that the major ture as cystatin M [39], and human cystatin F function of stefin A is related to protection of (hCF, [40]), also called leukocystatin [41], are re- these organs against overreactivity of cysteine leased from appropriate proproteins containing proteases [26]. On the other hand, stefin B is dis- signal peptides (28-mer for hCE and 19-mer for tributed in many tissues, which suggests that this hCF). Cystatins E and F are glycoproteins built of inhibitor interacts with cathepsins liberated from 122 and 126 amino-acid residues, respectively. [27]. Both inhibitors have been found Their structures display low homology to the sec- in all human fluids, but at a small concentration ond family of cystatins: 26–34% in a case of hCE [28]. The gene coding for human has and 30–34% in a case of hCF. Unlike other mem- 4 Z. Grzonka and others 2001 bers of the second family, cystatin F contains an tory function, have a cystatin-like three-dimen- additional, third, disulfide bridge stabilizing the sional structure [48, 49]. N-terminal fragment of the protein. Tis- sue-distribution profile studies have shown that –cystatin interaction the highest concentration of hCE is in the uterus and liver [38] and that of hCF in spleen and leuko- Numerous spectroscopic, kinetic, and crystallo- cytes [40]. graphic studies have been carried out to explain Human (hCC), formed after removal the mechanism of cysteine protease inhibition by of a 26-residue signal peptide, is a protein of 120 cystatins. The results have shown that the inhibi- amino-acid residues with molecular mass of 13.4 tor binds in a one-step process that is simple, re- kDa [42, 43]. In contrast to other members of the versible, and second-order type. In addition, those family, hCC is a basic protein (pI = 9.3) [44]. It is studies have revealed that enzymes with a blocked widely distributed in all physiological fluids. The active centre could still bind cystatins, albeit with highest amounts were found in seminal plasma lower affinity [50–52]. This indicates that and cerebro-spinal fluid, and much lower concen- cysteine protease–cystatin interactions are not trations were observed in tears, amniotic fluid, sa- based on a simple reaction with the catalytic liva, milk, and blood plasma [45]. The wide distri- cysteine residue of the enzyme, as is typical of bution and high inhibitory potency of cystatin C substrates, but that they consist of hydrophobic suggest that this protein is a major cysteine prote- contacts between the binding regions of cystatins ase inhibitor. and the corresponding residues forming the bind- ing pockets of the enzyme. Despite their struc- tural homology and similar mode of inhibition, cystatins display quite different enzyme affinities The third family consists of 3 members: human (Table 1). high molecular mass kininogen (hHK), about 120 From functional studies of cystatin Cit was con- kDa; human low molecular mass kininogen (hLK), cluded that the N-terminal fragment containing about 68 kDa; and kininogen T, discovered so far 11 amino-acid residues is important for the inhibi- only in rats [46]. Kininogens hHK and hLK are tory activity of hCC [44]. Our early studies with glycoproteins released as proproteins containing synthetic peptides corresponding to the N-ter- a signal peptide (18 amino-acid residues) [3]. The minal sequence of hCC showed that they were highest concentration of kininogens is found in very good substrates of papain, and that the cleav- blood plasma and synovial fluid [45]. age took place at the Gly11–Gly12 peptide bond. We have concluded that Arg8, Leu9 and Val10 Other cystatins and cystatin-like proteins from the N-terminal segment of cystatin Cinter - act with papain substrate-pocket subsites S4,S3 A number of proteins have been described and S2, respectively [56]. This was further con- which, in spite of high sequence homology, show firmed when the three-dimensional structure of distinct differences in structure and biological ac- cystatins was solved. tivity in comparison with cystatins. Histidine-rich So far, only three 3D-structures of cystatins glycoproteins (HRG) and fetuins are examples of have been published: the crystallographic struc- such cystatin-like proteins. Both HRG and fetuins tures of N-truncated chicken cystatin C[57] and did not inhibit cysteine proteases. The subject of of a complex of human (stefin B) with cystatins and cystatin-like proteins has been re- papain [58], as well as an NMR-structure of hu- viewed by Brown & Dziegielewska [47]. Convers- man cystatin A (stefin A) [59, 60]. From the struc- ely, there are also proteins, like the intensely ture of the complex between papain and stefin B, sweet plant protein monellin which, in spite of it is evident that the interactions between the en- very low sequence homology and lack of inhibi- zyme and cystatins are formed by the amino-acid Vol. 48 Structural studies of cysteine proteases and their inhibitors 5

Table 1. Dissociation constants K [nM] of enzyme–cystatin complexes i

Cystatin Cysteine protease Papain A 0.019a 8.2a 0.31a 1.3a 0.05a B 0.12a 73a 0.58a 0.23a 0.07a C0.000011 b 0.25a 0.28a <0.005a 0.008a D 1.2a >1000a 8.5a 25a 0.24a E 0.39c 32c ––– F 1.1d >1000d – 0.31d – S 108a –––– SA 0.32a –––– SN 0.016a 19a ––– H-kininogen 0.02e 400f 1.1f 0.109f – L-kininogen 0.015a 600a 0.72a 0.017a – aRef. [52]; bcalculated from association and dissociation constant velocities [53]; cref. [38]; dref. [40]; eref. [54]; fref. [55]. residues from the N-terminal segment (occupying acts with the catalytic cleft of cysteine proteases Sn subsites of the enzyme) as well as by two addi- [58]. It has been also proposed that the hydropho- tional fragments in b-hairpin loops: one in the bic amino-acid residues from the first loop, as well middle and one in the C-terminal segment of the as the tryptophan residue from the second loop, protein. These three cystatin regions, containing occupy the Sn¢ subsites of the enzyme. Kinino- evolutionarily conserved amino-acid residues (Ta- gens, which have three cystatin-like domains, dis- ble 2), form a wedge-like structure, which inter- play also high affinity for papain and cathepsins

a Table 2. Conserved amino-acid residues in binding segments of human cystatins

Cystatin N-terminus I-loop II-loop A MIPGG QVVAG B AcMMCGA QVVAG C RLVGG QIVAG VPWQ D TLAGG QIVAG VPWE E RMVGE QLVAG VPWQ F VKPGF QIVKG VPWL S IIPGG QTFGG VPWE SA IIEGG QIVGG VPWE SN IIPGG QTVGG VPWE H-kininogen 1-domain (QESQS) (TVGSD) (RSST) 2-domain DCLGC QVVAG (DIQL) 3-domain ICVGC QVVAG VPWE L-kininogen 1-domain (QESQS) (TVGSD) (RSST) 2-domain DCLGC QVVAG (DIQL) 3-domain ICVGC QVVAG VPWE aSequences in parenthesis correspond to the appropriate binding sequences of cystatins. 6 Z. Grzonka and others 2001 (Table 1). However, the first domain, which lacks was calculated from the association and dissocia- the evolutionarily conserved N-terminal and tion rate constants [53]. b-hairpin-loop residues (Table 2, sequences in pa- renthesis), has no inhibitory activity against Structure–activity relationship studies (SAR) cysteine proteases. Cystatins contain three segments which are rec- ognized as responsible for the interaction with HUMAN CYSTATIN C (hCC) cysteine proteases. These are the N-terminal frag- ment and the so-called first and second loops, Human cystatin C (hCC; also called g-trace, which are arranged at one edge of the molecule post-g-globulin, gamma-CSF and post-gamma pro- and are believed to directly interact with the cata- tein) was the first cystatin to be sequenced [42]. It lytic cleft of CPs. It has been shown in early SAR is recognized as the most physiologically impor- studies that truncation at the Gly11–Gly12 pep- tant extracellular human cystatin. Its primary tide bond decreases the affinity of hCC for papain structure consists of a single non-glycosylated by three orders of magnitude [44, 66]. The impor- polypeptide chain of 120 amino-acid residues tance of the N-terminal segment of hCC for its in- (Fig. 3). The cysteine residues at positions 73 and teraction with CPs was further confirmed by the studies of the rate of hydrolysis of appropriate synthetic peptides. Fragments comprising resi- dues Gly4–Glu21, Arg8–Asp15, and Arg8–Gly12 were all cleaved completely by papain at the Gly11–Gly12 bond within less than 60 s, whereas the corresponding bond of the peptide comprising residues Gly11–Asp15 was uncleaved even after 15-h incubation [56]. From these data, we postu- lated that the N-terminal fragment of hCC is in- volved in the inhibitor–enzyme interaction, and that the major contribution to the total affinity is through the binding of inhibitor residues Arg8, Leu9, and Val10 in the substrate subsites S ,S Figure 3. Primary structure of human cystatin C 4 3 (hCC). and S2 of the enzyme [56]. This was further cor- roborated by SAR studies with hCC variants [66–68]. The side chain of Val10 has the most im- 83 and those at positions 97 and 117 form two in- portant contribution to the affinity of the ternal disulfide bridges. The nucleotide sequence N-terminal fragment of hCC for cathepsins. It was 9 of hCC has been determined and localized on also shown that Leu is the most discriminating chromosome 20 [61, 62]. Human cystatin Cis residue for selective binding of hCC to cathepsins present in all extracellular fluids. The highest con- B, H, L, and S [68]. Exchange of the absolutely 11 centration was found in seminal plasma (50 conserved Gly residue for other amino acids mg/L) [63], whereas normal blood plasma con- generally leads to a sharp decrease of the inhibi- tains 0.8–2.5 mg/L of hCC [64]. The level of se- tory potency [68], indicating that this residue may rum cystatin Cis used now as an endogenous function as a hinge between the conformationally marker of renal function [64, 65]. hCC is an effec- flexible N-terminal segment and the rest of the tive reversible inhibitor of cathepsins B, H, K, L molecule [69, 70]. and S [52]. The affinity of hCC for papain is too Structure–activity relationship for the remain- 55 59 high to be measured by equilibrium methods. ing two binding segments of hCC (Gln –Gly 105 106 Therefore, the dissociation constant, K = 1.1 ´ and Pro –Trp ) has been studied less exten- D 106 10–14 M (Table 1), for the hCC–papain complex sively. Substitution of Trp by Gly decreases Vol. 48 Structural studies of cysteine proteases and their inhibitors 7 the affinity for cathepsin B and H by approxi- centration range of 0.5–1 M Gdn×HCl, the same mately three orders of magnitude [68, 69]. The as that interpreted by Ekiel & Abrahamson [77] as Trp106 ® Gly106 substitution, when combined indicating the existence of dimeric cystatin Cin with a change in the N-terminal sequence of hCC, their NMR studies. Thus, it can be concluded that leads to a further sharp decrease of the inhibitory the intermediate detected in our measurements is potency [66]. also a dimer.

Leu68Gln mutation One point mutation with glutamine residue sub- stituting leucine at position 68 of hCC (Leu68Gln) is now recognized as a disease-causing disorder which leads to amyloid deposits in cerebral blood vessels [71–74]. This disorder known as heredi- tary cystatin C amyloid angiopathy (HCCAA) re- sults in paralysis and development of dementia due to multiple strokes and death [74]. Indeed, it was shown that under various conditions the Leu68Gln mutant displays much higher tendency to dimerize and aggregate than wild-type hCC Figure 4. Conformational changes of cystatin C mon- [75–77]. itored by CD and tryptophan fluorescence analysis. The fluorescence intensity was measured at 360 nm using an excitation wavelength of 295 nm. The ordinate shows Dimerization, oligomerization the fraction of the native state calculated according to the equation fN =(x–xD)/(xN–xD) where x is the value for spec- Early studies on thermal stability have shown troscopic parameter (ellipticity or fluorescence intensity) that human cystatin Creadily undergoes and xN and xD are the values for the native and denaturated states, respectively. dimerization with complete loss of its inhibitory activity [77]. At a temperature above 80°ChCCag- gregates, and consequently precipitates. Self-asso- In the first transition state we did not detect any ciation of hCC was further evident when the pro- changes in the tertiary structure of cystatin C. tein was treated with various denaturing agents Also very few changes were observed in the a-he- [76, 77]. NMR studies of human cystatin Chave lix content. The only secondary structure motif shown that it can form dimers through structural exhibiting conformational changes after dimer changes in its native fold [69, 77]. formation, was the b-sheet. The most probable ex- We have studied the structural changes of hCC planation of this fact is that b-strands participate occurring during both thermal and chemical dena- directly in the formation of the dimeric molecule. turation processes. Chemical denaturation (with After reverse conversion of the dimers into guanidine hydrochloride, Gdn×HCl) was exam- monomeric molecules, a dramatic loss of b-sheet ined by two spectroscopic methods: circular content connected with the changes in the second- dichroism (CD) and tryptophan fluorescence [78]. ary and tertiary structure of cystatin Coccurred. To observe protein unfolding induced by heating, At a concentration of 2.5–3 M Gdn×HCl the sec- Fourier-transform infrared spectroscopy (FT-IR) ond transition state, stabilized by partially recov- was applied. ered tertiary interactions, was detected (Fig. 4). The obtained results indicate that unfolding of However, it was only a temporary state preceding cystatin Ccaused by a denaturing agent is a com - complete unfolding of cystatin C. plex process, characterized by two transition To study thermal denaturation of cystatin C, states (Fig. 4). The first one appeared in the con- FT-IR spectroscopy was applied. The measure- 8 Z. Grzonka and others 2001 ments were performed for dry protein prepared actions with the target enzymes. Those regions in- by evaporation of a water solution. Oberg & Fink clude the N-terminal segment and two hairpin have reported [79] that solvent evaporation loops, L1 and L2. The general fold of protein in- should not change the protein structure in solu- hibitors belonging to the cystatin family has been tion. To confirm this statement, we carried out ex- defined by the crystal structure of chicken periments at 35°Cfor dry cystatin Cand cystatin cystatin [57]. Its canonical features include a long Cdissolved in water. The results reveal that at a1 helix running across a large, five-stranded 35°Cthe protein structure in both cases is almost antiparallel b sheet. The connectivity within the b the same (Table 3). However, at higher tempera- sheet is as follows: (N)-b1-(a1)-b2-L1-b3-(AS)-b4-

Table 3. Percentage contents of different secondary structure motifs in native cystatin C

b-sheet a-helix random coil turn 310-helix, open loop, turn Cystatin C in H2O 41.7 14.8 16.5 24.8 2.2 Solid cystatin C43.4 14.6 18.2 21.1 2.8

tures no conformational changes in solid cystatin L2-b5-(C), where AS is a broad “appending struc- Ccould be detected. The dry protein retained its ture”, rather unrelated to the compact core of the native state during the whole heating process. remaining part of the molecule and positioned on the opposite end of the b sheet relative to the Structural studies of hCC N-terminus and the two short loops L1 and L2. The latter three elements are aligned in a Our early molecular modeling studies on human wedge-like fashion in the inhibitory motif of cystatin C[80] have shown that the energy-opti- cystatins. Chicken cystatin shows 41% sequence mized structure of hCC is very close to the crystal- identity and 62.5% homology to hCC but the crys- lographic structure of chicken cystatin [57]. The tal structure corresponds to an N-truncated vari- results of fluorescence studies indicated that the ant [57]. On the other hand, the eleven N-terminal Trp106 residue is fully exposed to solvent. We amino-acid residues of hCC are important for its 106 found that, apart from Trp , the main contribu- very high-affinity binding to papain [52] (Ki 11 tion to fluorescence comes from Tyr62 and Tyr42. fM) and to other cysteine proteases [28]. It has The remaining tyrosine residues (Tyr34 and been shown that specific cleavage, by leukocyte 10 11 Tyr102) are efficiently quenched as a result of en- , of the single N-terminal Val –Gly ergy transfer to the Cys97–Cys112 disulfide bond of hCC results in seriously compromised af- bridge (Tyr34) and tryptophan (Tyr102) [80]. finities for such target enzymes as cathepsin B, H, Development of a second generation of more ef- and L [84]. fective, specific cysteine protease peptide inhibi- It is interesting to compare the topology of tors would be greatly facilitated by the knowledge cystatin with that of the intensely sweet plant pro- of three-dimensional structure of hCC. Similarly, tein, monellin. The structural similarity has been such a model is necessary for the elucidation of noted before in spite of the low sequence identity the pathophysiological background of the cerebral [48, 49]. However, natural monellin consists of hemorrhage produced by hCC, particularly its two protein chains: chain B, corresponding to he- L68Q variant. lix a1 and strands b1 and b2 (in the order Crystallographic and NMR studies of chicken b1-a1-b2), and chain A, corresponding to the re- cystatin [57, 81, 82], cystatin B in complex with maining, prominent part of the b sheet. The papain [58], cystatin A [60], and human N-terminus of chain A and C-terminus of chain B cystatin D [83], have shown a similar overall are close in space and seem to be the product of structure, with three regions implicated for inter- proteolytic cleavage of a single-chain protein [48] Vol. 48 Structural studies of cysteine proteases and their inhibitors 9 in a region that corresponds to cystatin loop L1 of dicative of structural disorder (N-terminus, ap- the inhibitory “wedge”. An artificial tethered B-A pending structure) and/or of lack of homogeneity protein retains the taste and conformation of nat- resulting from uncontrolled protein aggregation ural monellin [49]. (oligomerization) in the crystallization solutions There are two disulfide bonds in human cystatin and possibly also in the crystals. It should be C(Cys 73–Cys83,Cys97–Cys117) and in all other stressed, however, that hCC used for growing the proteins of family 2 and 3 cystatins [85]. Both are crystals represented pure monomeric protein ob- located within the b region of the chicken protein tained by gel filtration as the final isolation step. structure, in the C-terminal half of the molecule The Matthews volume [89] calculated for the two that would correspond to chain A in monellin. The forms of full-length hCC is indicative of the pres- conservation of these two S–S bridges in family 2 ence of multiple copies of the protein in the asym- and 3 cystatins [45, 86] may be interpreted as im- metric unit. The propensity of hCC to crystallize plicating their requirement for stable protein fold. with multiple copies of the molecule in the asym- However, there are no disulfide bridges in family metric unit, in combination with the additional 1 cystatins or in monellin. In the structure of possibilities offered by the point symmetry ele- chicken cystatin there are two b-bulges, in strands ments of the unit cells, may be also indicative of b2 (Arg46) and b5 (Leu111), of the b sheet. They the tendency of the protein to oligomerize. Such are preserved in the other structural models of oligomerization might reflect the amyloid-forming cystatins, and also in monellin. The “appending property of Leu68Gln cystatin C, as earlier obser- helix” of chicken cystatin is disputable. It is only vations demonstrate that both wild type and loosely connected with the molecular core and in Leu68Gln-substituted cystatin Care capable of the segment Cys71–Lys91 is very poorly defined. forming dimers [69, 74, 77]. In the tetragonal In particular, the Lys73–Leu78 fragment was form, as many as seven independent molecules weakly defined and tentatively placed, while in could be present. The cubic unit is likely to 85 91 3 electron density the Asp –Lys peptide was not contain two asymmetric copies (Vm 2.16 Å /Da), defined at all as it is completely disordered. In but one molecule and high solvent content (72%) spite of that, the Asp77–Asp85 fragment was mod- is also possible. To facilitate the solution of the eled as helix a2. This a helix is not seen in the pre- crystal structure of hCC, the full-length protein liminary structure of human cystatin D [83] or in was also produced in selenomethionyl form [87]. the structurally homologous monellin. Also, in the Electrospray mass spectrometry of the seleno- NMR studies of cystatins [69], no helical confor- methionyl protein confirmed that the three Met mation has been found for this fragment either in residues in the hCC sequence were fully substi- chicken or human cystatin C. It appears that this tuted by Se-Met. A successful Met®Se-Met substi- fragment must be rather disordered in solution. tution was additionally confirmed by analysis of Crystallization of human cystatin C has been a the amino-acid composition of the Se-Met protein challenge for a long time. Recently, formation of after acidic hydrolysis. The selenomethionyl pro- single crystals in several forms has been reported tein crystallized in the cubic form and X-ray ab- [87]. For the crystallization experiments, hCC was sorption spectra confirmed a significant content produced in its full-length form by recombinant of selenium in the crystals. Unfortunately, due to techniques in Escherichia coli [88]. This full-length weak diffraction, only multiwavelength anoma- wild-type protein crystallized in two forms, lous diffraction (MAD) data at 4.5 Å resolution tetragonal (P41212orP43212) and cubic (I432). could be measured for those crystals at the sele- Low-temperature synchrotron data are available nium absorption edge. for both forms at the originally reported resolu- Very recently, a new low-temperature data set tion of 3.0 and 3.1 Å, respectively [87]. The notori- was obtained for the cubic form of native ous poor quality and limited resolution of X-ray full-length hCC using synchrotron radiation diffraction by full-length hCC crystals, in spite of (R. Janowski, unpublished). This data set extends their perfect and beautiful appearance, may be in- to 2.5 Å resolution and is currently being used for 10 Z. Grzonka and others 2001 the determination of the structure of hCC. In ad- other hand, extention of the -Leu-Val-Gly- se- dition to the experiments involving full-length hu- quence by an Arg residue in Z-RLVG-DAM gave man cystatin C, preliminary crystallographic the most potent inhibitor of papain and cathepsin studies have also been reported for its B, with apparent second order rate constants N-terminally truncated variant [87]. hCC devoid (k+2’) of the same order of magnitude as those de- of ten N-terminal residues was obtained by incu- termined for E-64, which is used as standard in bation of recombinant wild type human cystatin C the inhibitory bio-assays of cysteine proteases with leukocyte elastase and isolated as described [91]. Addition of the next Pro7 residue in by Abrahamson et al. [84]. The protein could be Z-PRLVG-DAM decreased the activity. Pepti- crystallized in tetragonal form yielding crystals dyl-diazomethyl ketones with a free N-terminal that are very stable in the X-ray beam. Measure- amino group displayed a lower inhibitory potency. ment of diffraction data extending to 2.7 Å has None of the peptidyl-diazomethyl ketones de- been reported at room temperature, using con- signed after the N-terminal sequence of various ventional Cu Ka radiation [87]. Also in the case of cystatins had an inhibitory activity higher than N-truncated hCC, the asymmetric unit can be ex- that of hCC itself [91, 92]. These peptidyl-dia- pected to contain numerous (up to eleven) inde- zomethyl ketone inhibitors have been found to be pendent copies of the protein. Very recently, a very fast and irreversible inhibitors of cysteine new diffraction data set extending to 2.1 Å resolu- proteases. It should be noted that the reactivity of tion has been measured at low temperature using the diazomethyl ketone group with is gener- synchrotron radiation (R. Janowski, unpub- ally very low [93]. Modified neglect of diatomic lished). overlap (MNDO) studies of the mechanism of inhi- bition of cysteine proteases by diazomethyl ke- tones showed that the reaction is irreversible and LOW-MOLECULAR-MASS INHIBITORS OF leads to an a-thioketone derivative of the Cys25 CYSTEINE PROTEASES RELATED residue of papain [94]. Recently, we have shown TO THE STRUCTURE OF THE BINDING that Z-RLVG-DAM inhibits bone resorption in vi- CENTER OF CYSTATINS tro by a mechanism that seems primarily due to inhibition of bone matrix degradation via cysteine Peptidyl-diazomethyl ketones proteases [95].

Soon after the discovery that the N-terminal Oxirane-type inhibitors fragment: Arg8–Leu9–Val10–Gly11 of human cystatin Cinteracts with the S n subsites of E-64 [(2S,3S)-trans-epoxysuccinyl-L-leucyl-agma- cysteine proteases [53, 56], a series of tine] isolated from cultures of Aspergillus peptidyl-diazomethyl ketones based on the struc- japonicus is a very strong and irreversible inhibi- ture of this segment was synthesized. Preliminary tor of cysteine proteases [96, 97]. The first results showed that both Boc-Val-Gly-CHN2 oxirane-containing inhibitor, based on the struc- (Boc-VG-DAM) and Z-Leu-Val-Gly-CHN2 (Z-LVG- ture of the N-terminal segment of hCC designed DAM) inhibit papain, cathepsin B and streptococ- by us, Z-Leu-Val-NHCH2-CH(O)CH-CH2COOH, cal proteinase [56]. The latter compound was displayed only weak reversible inhibition [56]. tested for in vitro and in vivo antibacterial activity Therefore, taking into account the structure of against a large number of bacterial strains of dif- E-64 and its analogs, as well as our modeling stud- ferent species [90]. Mice injected with lethal doses ies, we have designed several new compounds of group A streptococci were cured by a single in- with more hydrophobic C-termini. Most of these jection of 0.2 mg of Z-LVG-DAM. Detailed struc- compounds displayed quite good inhibitory activi- ture-activity studies showed that the shortest ties towards papain and cathepsin B. However, among diazomethyl ketones, Z-Gly-CHN2, does the most striking result came from two oxirane- not inhibit cysteine proteases (Table 4). On the type compounds: Z-Arg-Leu-ValY[CH2NH]CO- Vol. 48 Structural studies of cysteine proteases and their inhibitors 11

CH(O)CH-C6H5 (Table 4, compound 16) and ible inhibitor of papain and cathepsin B, whereas Z-Arg-Leu-ValY[CH2NH]CO-CH(O)CH-COC6H5 compound 16, with the phenyl ring attached di- (Table 4, compound 17). Compound 17, with a rectly to the oxirane moiety, had no inhibitory po-

–1 –1 Table 4. Comparison of the inhibition rate constants (M s ) for inhibitors of cysteine proteases containing N-terminal binding segments of cystatins

Cysteine protease Streptococcus No. Compound Cathepsin B gr. A killing Papain (bovine) Other cathepsins effect –1 –1 Peptidyl-diazomethyl ketones k¢+2 [M s ] 1. Z-Gly-DAMa <102 inactive 2. Boc-Val-Gly-DAMa 1.66´104 1.21´03 3.57´104 Cath. L 3. Z-Leu-Val-Gly-DAMa 4.48´105 2.4´104 5.22´104 Cath. S +++ <102 Cath. H 4. H-Leu-Val-Gly-DAMa 2.26´104 1.5´103 5. Z-Arg-Leu-Val-Gly-DAMa 5.59´105 3.96´104 inactive 6. H-Arg-Leu-Val-Gly-DAMb 6.25´104 7. Z-Pro-Leu-Val-Gly-DAMb 4.22´105 8. Z-Ile-Val-Gly-DAMb 2.97´105 3.63´103 ++ 9. Z-Leu-Leu-Gly-DAMb 1.56´104 ++ 10. Z-Arg-Leu-Leu-Gly-DAMb 5.47´104 1.94´104 inactive 9.3´102 Cath. L 11. Z-Leu-Ala-Gly-DAMb 1.09´104 1.98´103 1.82´104 Cath. S ++ <102 Cath. H 12. Z-Ile-Pro-Gly-DAMb 3.52´103 inactive inactive 13. Z-Arg-Ile-Ile-Pro-Gly-DAMb 1.47´103 inactive inactive 14. Z-Arg-Thr-Leu-Ala-DAMb 2.62´104 inactive 15. Z-Phe-Leu-Gly-DAMb 1.16´104 3.50´103 ++ E-64a 8.13´105 5.35´104 –1 –1 Oxiranes k¢+2 [M s ]

16. Z-Arg-Leu-ValY[CH2NH]CO- inactive inactive b CH(O)CH-C6H5

17. Z-Arg-Leu-ValY[CH2NH]CO- 1.73´104 8.0´103 b CH(O)CH-CO-C6H5

18. Z-Arg-Leu-ValY[CH2NH]CO- 2.67´104 b CH(O)CH-COOCH3

19. dArg-Leu-ValY[CH2NH]CO- 3.06´103 5.43´103 b CH(O)CH-CO-C6H5

C H -CO-CH(O)CH-CO-Val- 20. 6 5 Ki= 16.3 nM Ki = 0.256 nM b Leu-NH(CH2)4NHC(NH)NH2 a [91]; b this paper. stronger electron-withdrawing benzoyl group at tency towards cysteine proteases. This discrep- the C-terminus, was found to be a good irrevers- ancy prompted us to undertake more detailed 12 Z. Grzonka and others 2001 structural studies using molecular modeling and covalently linked to the active-site Cys25 of the en- crystallographic methods. zyme. However, the maps calculated using the low-temperature data (data set II) clearly revealed Other inhibitors only a short stem of electron density near the ac- tive-site Cys25. An analysis of the shape of this Apart from diazomethyl ketone- and oxirane- electron density and of potential hydrogen bonds type inhibitors, we have designed several other strongly suggests that in these aged crystals of compounds containing the cystatin binding motif, the complex, the inhibitor that was originally at- as well as reactive groups for the thiol function of tached to the sulfhydryl group of Cys25 has been cysteine proteases [56, 98]. Good inhibitory po- replaced by a covalent hydroxyethyl substituent. tency was found for compounds containing an ac- The overall structure (room- and low-tempe- tivated olefinic double bond and compounds with rature models) of the enzyme is similar to other a C-terminal aldehyde group or chloro- and bromo- papain structures deposited in the PDB, and the methyl ketone groups. It was interesting to find r.m.s. deviation for Ca atoms of these two models that most of them displayed antibacterial activity is 0.24 Å. against seventeen clinically important bacterial The inhibitor moiety in the room-temperature species tested [98]. It should be mentioned that structure extends along the Sn subsites of the en- many cyclic peptides based on the N-terminal se- zyme (Fig. 5) and is stabilized in the active-site quence of cystatin Calso displayed antibacterial groove by a series of hydrogen bonds and hydro- properties. Recently, we have designed and syn- phobic interactions. The inhibitor forms hydro- thesized several azapeptides based on the binding gen bonds with Gly66, Asp158, and Gln19 as well sequence of cystatins, and some of them were as with two solvent molecules. Similar contacts found to be very selective inhibitors of different were also observed in the 2.1 Å resolution struc- cathepsins (E. Wieczerzak, unpublished). ture of a complex between papain and E-64c [99]. The hydrophobic interactions with the S subsite Crystallographic studies of papain–inhibitor 2 complexes characteristic for chloromethylketone inhibitors were not observed. The distances between the 133 157 Single crystals of the covalent complex papain— side chains of Val and Val (defining the en- Z-Arg-Leu-ValY[CH2NH]-CO-CH(O)CH-COC6H5 zyme’s S2 subsite) and the atoms of the Val resi- (Table 4, compound 17) were grown by the vapor due of the inhibitor, are longer than 6.0 Å. diffusion method at room temperature in hanging As a step towards understanding the specificity drops using a modification of the procedure de- of peptidic, covalent, irreversible inhibitors of scribed for the complex papain—E-64c [99]. De- papain, two peptidyl-diazomethyl ketone-type in- tailed crystallization conditions and the proce- hibitors: Z-Arg-Leu-Val-Gly-DAM and Z-Leu-Phe- dure for data collection at room temperature us- Gly-DAM (Table 4), with valine and phenylalanine ing freshly grown crystals (data set I — resolution residues in the P2 site, respectively, were synthe- 1.9 Å) were described previously [100]. Another, sized and reacted with the active site of papain. low-temperature data set was collected about 10 The complex between papain and the Z-Arg-Leu- months later using synchrotron radiation (resolu- Val-Gly-DAM has been characterized crystallo- tion 1.65 Å). The crystals used in those studies graphically (space group P21, 1.78 Å resolution, R correspond to the historically first crystal form of = 0.168). The side chain of Val from the Z-Arg-Leu- papain, form A, crystallized by Drenth & Janso- Val-Gly-DAM inhibitor molecule is rather far from nius [101], for which no crystal structure has yet the hydrophobic S2 pocket, the closest distances been reported. in this region being above 4.6 Å. Electron density Even preliminary difference electron density is clearly visible for the entire inhibitor moiety maps calculated using the room-temperature data with the exception of the benzyloxycarbonyl (Z) (data set I) clearly showed the inhibitor, which is group. The structure, therefore, demonstrates Vol. 48 Structural studies of cysteine proteases and their inhibitors 13 25 again no specific association between the S2 crystal forms the inhibitor is bound to the Cys pocket and the inhibitor’s P2 site, analogously to residue of the papain with a covalent bond formed the situation observed in the crystal structure of between the methylene group (DAM) of the inhibi- the Z-Arg-Leu-ValY[CH2NH]CO-CH(O)CH-CO- tor and the thiol group of the enzyme. The

Figure 5. Crystal structure of the papain–oxirane inhibitor (17) complex (M. Kozak, unpublished). The enzyme is shown as space-filling model viewed into the catalytic cleft from the outside. The inhibitor (green) is seen in the cleft in its fully extended conformation, with the Z group at the top and the “oxirane” moiety, now opened and covalently linked to the enzyme’s Cys25 Sg atom (yellow), at the bottom.

C6H5 complex [100], and in molecular dynamics phenylalanyl side chain is locked by hydrophobic 133 simulations [102]. This persistent lack of P2–S2 interactions (3.5–3.8 Å) with residues Val and 157 interactions in Z-Arg-Leu-Val-type inhibitors is in Val of the S2 pocket. The orientation of the contrast to the early findings by Drenth et al. phenyl ring is similar to that observed in the [103] that P2–S2 complementarity is essential for chloromethyl ketone complexes studied by Drenth productive inhibition and for enzyme specificity. et al. ( codes: 1pad, 5pad, 6pad) This evidence seems to indicate that, while it [103]. The inhibitor is stabilized by additional hy- might be important for efficient and precise dock- drogen bonds between its main chain and the resi- ing of the inhibitor in the active site, the S2 dues forming the catalytic cleft of the enzyme pocket does not play any significant role in the as- (highly conserved hydrogen bonds with Gln19 and sociation between the inhibitor and the enzyme Gly66). Electron density is very clear for the cova- once a covalent bond has been formed. lent bond connecting the inhibitor and the en- Two polymorphs of a complex between papain zyme as well as for the side chain of the and the Z-Leu-Phe-Gly-DAM inhibitor have been phenylalanyl residue. The N-terminal part of the crystallized. Diffraction data for crystal form I inhibitor, the benzyloxycarbonyl group (Z), has no (space group P21) were collected to 2.0 Å resolu- visible electron density in either of the crystal tion, and for crystal form II (space group forms. P212121) to 1.63 Å. Both structures were solved Based on the above structures and the struc- by molecular replacement using the 1ppn.pdb tures of other papain–inhibitor complexes one [104] model of papain as a probe. The final R fac- can conclude that, in covalent papain–inhibitor tors are 0.106 and 0.172, respectively. In both complexes, hydrophobicity of the P2 residue is not 14 Z. Grzonka and others 2001 sufficient for productive binding of the inhibitor namics runs indicated (Fig. 6) changes up to 5 Å in the S2 pocket and that its bulkiness is equally for some residues (C-terminus), but the overall C important. This does not preclude, however, that mobilities oscillated about 1 Å for InhA and 2 Å even a smaller residue, like valine, may be effec- for InhB. The average structures displayed simi- tive during recognition and docking prior to the larly significant changes in some flexible loops of formation of the covalent link. the enzyme. It should be stressed that the protein structures reproduced very well the mobility pat- Molecular modeling tern typical of the whole molecule as represented by the atomic displacement parameters (tempera- The initial models for papain—Z-Arg-Leu- ture factors) in the crystal structure of the ValY[CH2NH]-CH(O)CH-COC6H5 (Z-R-L-Nnv- papain–E-64c complex [100]. This result validated Oxi–papain; papain–InhA) and papain—Z-Arg- the use of the AMBER 5.0 force field as a suitable Leu-Val-Gly-CHN2 (Z-RLVG-CH2–papain; papain tool for scanning the conformational space of

Figure 6. Time-averaged resi- due-based deviations (mobilities) along the papain sequence dur- ing molecular dynamics (MD) runs. Symbols A1 and B1 correspond to InhA and InhB MD runs, respec- tively. The distribution of both quan- tities along the sequence is evident. It can be seen that some loops of the papain L lobe undergo high fluctua- tions during molecular dynamics runs.

–InhB) complexes were prepared in the Sybyl pro- both ligands in the catalytic cleft of papain. gram [105] based on papain Protein Data Bank Detailed atomic-level analysis of the mobilities files 1pe6 and 1pad, respectively [106], in combi- of the inhibitor backbones reveals that the scatter nation with the respective ligands modeled as de- of the relaxed positions of the residues increases scribed elsewhere ([102] and P. Drabik, unpub- steeply towards the N-terminus of the inhibitors lished). The complexes were subsequently ana- (Fig. 7). Thus, the catalytic pocket S3, as defined lysed using the AMBER program [107]. New resi- by the pioneering studies of Schechter & Berger dues, absent in the original AMBER force field were parameterized according to standard proce- 5,0 dures [108]. 4,5 The initial structures of the papain–ligand com- 4,0 plexes were subjected to constrained simulated 3,5 annealing [102] enabling the simulation at very 3,0 y high, physically unrealistic temperature. The ad- 2,5 ditional kinetic energy enhanced the ability of the 2,0 system to explore the energy surface and to avoid 1,5 getting stuck in energetically unfavourable local 1,0 energy minima. Afterwards, the systems were 0,5 InhA 0,0 InhB subjected to 230 ps of unconstrained molecular P1-(OXI/GLM) P2-(NNV/VAL) P3-LEU P4-ARG P5-CBZ dynamics at 300 K. Time-averaged residue-based deviations as a Figure 7. Mobilities of inhibitor residues during mo- function of residue number for all molecular dy- lecular dynamics simulations. Vol. 48 Structural studies of cysteine proteases and their inhibitors 15 [6], appears rather elusive in view of the inhibitor 12. Afonso, S., Romagnano, L. & Babiarz, B. (1997) flexibility evident from the molecular dynamics The expression and function of cystatin Cand simulations and from the experimentally deter- cathepsin B and cathepsin L during mouse embryo mined structures of papain–inhibitor complexes implantation and placentation. Development 124, 3415–3425. [58, 99, 103, 109, 110]. The location and defini- tion of the substrate S4 is even more 13. Henskens, M.C., Veerman, E.C.I. & Amerongen, questionable. A.V.N. (1996) Cystatins in health and disease. Biol. Chem. Hoppe-Seyler 377, 71–86. 14. Grubb, A. (2000) Cystatin C — properties and use as REFERENCES diagnostic marker. Adv. Clin. Chem. 35, 63–99.

1. Barrett, A.J. (1994) Classification of peptidases. 15. Warwas, M. & Haczyñska, H. (1998) Rola cystatyn Methods Enzymol. 244, 1–15. w procesie nowotworowym i jego diagnostyce. Post. Hig. Med. Doœw. 52, 515–526 (in Polish). 2. Hartley, B.S. (1960) Proteolytic enzymes. Annu. Rev. Biochem. 9, 45–72. 16. Machleidt, W., Borchart, U., Fritz, H., Brzin, J., Ritonja, A. & Turk, V. (1983) Protein inhibitors of 3. Otto, H.-H. & Schirmeister, T. (1997) Cysteine pro- cysteine proteinases: II. Primary structure of teases and their inhibitors. Chem. Rev. 97, stefin, a cytosolic protein inhibitor of cysteine pro- 133–171. teinases from human polymorphonuclear granulo- 4. Rawlings, N.D. & Barrett, A.J. (1999) MEROPS: cytes. Hoppe-Seyler’s Physiol. Chem. 364, 1481– The peptidase database. Nucleic Acids Res. 27, 1486. 325–331. 17. Green, G.D.J., Kembhavi, A.A., Davies, M.E. & 5. McGrath, M.E. (1999) The lysosomal cysteine pro- Barrett, A.J. (1984) Cystatin-like proteinase inhibi- teases. Annu. Rev. Biophys. Biomol. Struct. 28, tors from human liver. Biochem. J. 218, 939–946. 181–204. 18. Ritonja, A., Machleidt, W. & Barrett, A.J. (1985) 6. Schechter, I. & Berger, A. (1967) On the size of the sequence of the intracellular cysteine active site in proteases. I. Papain. Biochem. proteinase inhibitor cystatin B from human liver. Biophys. Res. Commun. 27, 157–162. Biochem. Biophys. Res. Commun. 131, 1187–1192. 7. Turk, D., Gunèar, G., Podobnik, M. & Turk, B. 19. Takeda, A., Kobayashi, S. & Samejima, T. (1983) (1998) Revised definition of substrate sites of Isolation and characterization of two thiol papain-like cysteine proteases. Biol. Chem. 379, proteinase inhibitors of low molecular weight from 137–147. newborn rat epidermis. J. Biochem. 94, 811–820. 8. Berti, P.J. & Storer, A.C. (1995) Alignment/phylog- 20. Turk, B., Kri[aj, I. & Turk, V. (1992) Isolation and eny of the papain superfamily of cysteine proteas- characterization of bovine stefin B. Biol. Chem. es. J. Mol. Biol. 246, 273–283. Hoppe-Seyler 373, 441–446. 9. Turk, B., Turk, V. & Turk, D. (1997) Structural and 21. Turk, B., Ritonja, A., Björk, I., Stoka, V., Dolenc, I. functional aspects of papain-like cysteine protein- & Turk, V. (1995) Identification of bovine stefin A, ases and their protein inhibitors. Biol. Chem. 378, a novel inhibitor of cysteine proteinases. FEBS 141–150. Lett. 360, 101–105. 10. Storer, A.C. & Menard, R. (1994) Catalytic mecha- 22. Lenarèiè, B., Ritonja, A., Dolenc, I., Stoka, V., nism in papain family of cysteine peptidases. Berbic, S., Pungercar, J., Strukelj, P. & Turk, V. Methods Enzymol. 244, 486–500. (1993) Pig leukocyte cysteine proteinase inhibitor (PLCPI), a new member of stefin family. FEBS 11. Kirschke, H., Barrett, A.J. & Rawlings, N.D. (1995) Lett. 336, 289–292. Proteinases 1: Lysosomal cysteine proteinases; in Proteine Profile 2 (Sheterline, P., ed.) pp. 23. Kondo, H., Ijjri, S., Abe, K., Maeda, H. & Arai, S. 1587–1643, Oxford University Press. (1992) Inhibitory effect of oryzacystatins and a truncation mutant on the replication of poliovirus in infected Vero cells. FEBS Lett. 299, 48–50. 16 Z. Grzonka and others 2001 24. Lenarèiè, B., Kri[aj, I., Zunec, P. & Turk, V. (1996) chromosome 20p11.21 by in situ hybridization. Differences in specificity for the interactions of Cytogen. Cell. Genet. 62, 29–31. stefins A, B and D with cysteine proteinases. FEBS 35. Isemura, S., Saitoh, E., Sanada, K. & Minekata, K. Lett. 395, 113–118. (1991) Identification of full-sized forms of salivary 25. Rawlings, N.D. & Barrett, A.J. (1990) Evolution of (S-type) cystatins (cystatin SN, cystatin SA, proteins of the cystatin superfamily. J. Mol. Evol. cystatin S, and two phosphorylated forms of 30, 60–71. cystatin S) in human whole saliva and determina- tion of phosphorylation sites of cystatin S. J. 26. Lenarèiè, B., Ritonja, A., Sali, A., Kotnik, M., Turk, Biochem. (Tokyo) 110, 648–654. V. & Machleidt, W. (1986) Properties and structure of human spleen stefin B — a low molecular weight 36. Balbin, B., Freije, J.P., Abrahamson, M., Velasco, protein inhibitor of cysteine proteinases; in G., Grubb, A. & Lopez-Otin, C. (1993) A sequence Cysteine Proteinases and their Inhibitors (Turk, V., variation in the human cystatin D gene resulting in ed.) pp. 473–487, Walter de Gruyter. an amino acid (Cys/Arg) polymorphism at the pro- tein level. Hum. Genet. 90, 668–669. 27. Katunuma, N. & Kominami, E. (1986) Distribution and localization of lysosomal cysteine proteinases 37. Freije, J.P., Balbin, M., Abrahamson, M., Velasco, and cystatins; in Cysteine Proteinases and their In- G., Dalbøge, M., Grubb, A. & Lopez-Otin, C. (1993) hibitors (Turk, V., ed.) pp. 219–227, Walter de Human cystatin D. cDNA cloning, characterization Gruyter. of the Escherichia coli expressed inhibitor, and identification of the native protein in saliva. J. Biol. 28. Abrahamson, M. (1993) Cystatins — protein inhibi- Chem. 268, 15737–15744. tors of papain-like cysteine proteinases. Ciencia e Cultura 45, 229–304. 38. Ni, J., Abrahamson, M., Zhang, M., Alvarez-Fer- nandez, M., Grubb, A., Su, J., Yu, G.-L., Li, Y., 29. Hsieh, W.-T., Fong, D., Sloane, B.F., Golembieski, Parmelee, D., Xing, L., Coleman, T.A., Gentz, S., W. & Smith, D.I. (1991) Mapping of the gene for hu- Thotakura, R., Nguyen, N., Hesselberg, M. & man cysteine proteinase inhibitor stefin A, STFI, to Gentz, R. (1997) Cystatin E is a novel human chromosome 3cen-q21. Genomics 9, 207–209. cysteine proteinase inhibitor with structural re- 30. Pennacchio, L.A., Lahesjoki, A.E., Stone, N.E., semblance to family 2 cystatins. J. Biol. Chem. 272, Willour, V.L., Virtaneva, K., Miao, J., D’Amato, E., 10853–10858. Ramirez, L., Faham, M., Koskiniemi, M., 39. Sotiropoulou, G., Anisonowicz, A. & Sager, R. Warrington, J.A., Norio, R., de la Chapelle, A., Cox, (1997) Identification, cloning, and characterization D.R. & Myers, R.M. (1996) Mutations in the gene of cystatin M, a novel cysteine proteinase inhibitor, encoding cystatin B in progressive myoclonus epi- down-regulated in breast cancer. J. Biol. Chem. lepsy (EPM1). Science 271, 1731–1734. 272, 903–910. 31. Fossum, K. & Whitaker, J.R. (1968) Ficin and 40. Ni, J., Fernandez, M.A., Danielsson, L., Chillakuru, papain inhibitor from chicken egg white. Arch. R.A., Zhong, J., Grubb, A., Su, J., Gentz, R. & Biochem. Biophys. 125, 367–375. Abrahamson, M. (1998) Cystatin F is a glycosylated 32. Sen, L.C. & Whitaker, J.R. (1973) Some properties human low molecular weight cysteine proteinase of a ficin-papain inhibitor from avian egg white. inhibitor. J. Biol. Chem. 273, 24797–24804. Arch. Biochem. Biophys. 158, 623–632. 41. Halfon, S., Ford, J., Foster, J., Dowling, L., Lucian, 33. Saitoh, E., Sabatini, L., Eddy, R.L., Shows, T.B., L., Sterling, M., Xu, Y., Weiss, M., Ikeda, M., Azen, S., Isemura, S. & Sanada, K. (1989) The hu- Liggett, D., Helm, A., Caux, C., Lebecques, S., man cystatin Cgene (CST3)is a member of the Hannum, C., Menon, S., Mc Clanahan, T., Gorman, cystatin gene family which is localized on chromo- D. & Zurawski, G. (1998) Leukocystatin, a new some 20. Biochem. Biophys. Res. Commun. 162, class II cystatin expressed selectively by hemato- 1324–1331. poietic cells. J. Biol. Chem. 273, 16400–16408. 34. Freije, J.P., Pendas, A.M., Velasco, G., Roca, A., 42. Grubb, A. & Lofberg, H. (1982) Human g-trace, a Abrahamson, M. & Lopez-Otin, C. (1993) Localiza- basic microprotein: Amino acid sequence and pres- tion of the human cystatin D gene (CST5) to human Vol. 48 Structural studies of cysteine proteases and their inhibitors 17 ence in the adenophysis. Proc. Natl. Acad. Sci. cysteine proteinases papain and actinidin. Bio- U.S.A. 79, 3024–3027. chem. J. 28, 49–55. 43. Abrahamson, M., Grubb, A., Olafsson, I. & Lund- 54. Müller-Esterl, W., Fritz, H., Machleidt, W., Ritonja, wall, A. (1987) Molecular cloning and sequence A., Brzin, J., Kotnik, M., Turk, V., Kellermann, J. & analysis of cDNA coding for the precursor of the Lottspeich, F. (1985) Human plasma kininogens human cysteine proteinase inhibitor cystatin C. are identical with a-cysteine proteinase inhibitors. FEBS Lett. 216, 229–233. FEBS Lett. 182, 310–314. 44. Abrahamson, M., Ritonja, A., Brown, M.A., Grubb, 55. Barrett, A.J., Rawlings, N.D., Davies, M.E., A., Machleidt, W. & Barrett, A.J. (1987) Identifica- Machleidt, W., Salvesen, G. & Turk, V. (1986) tion of the probable inhibitory reactive sites of the Cysteine proteinase inhibitors of the cystatin cysteine proteinase inhibitors human cystatin C superfamily; in Proteinase Inhibitors (Barrett, A.J. and chicken cystatin. J. Biol. Chem. 262, 9688– & Salvesen, G., eds.) pp. 515–569, Springer-Verlag. 9694. 56. Grubb, A., Abrahamson, M., Olafsson, I., Trojnar, 45. Abrahamson, M., Barrett, A.J., Salvessen, G. & J., Kasprzykowska, R., Kasprzykowski, F. & Grubb, A. (1986) Isolation of six cysteine Grzonka, Z. (1990) Synthesis of cysteine protein- proteinase inhibitors from human urine. Their ase inhibitors structurally based on the proteinase physicochemical and enzyme kinetic properties interacting N-terminal region of human cystatin C. and concentrations in biological fluids. J. Biol. Biol. Chem. Hoppe-Seyler 371 (Suppl.), 137–144. Chem. 261, 11282–11289. 57. Bode, W., Engh, R., Musil, D., Thiele, U., Huber, R., 46. DeLa Cadena, R.A. & Colman, R.N. (1991) Struc- Karshnikov, A., Brzin, J., Kos, J. & Turk, V. (1988) ture and functions of human kininogens. Trends The 2.0 Å X-ray crystal structure of chicken egg Pharmacol. Sci. 12, 272–275. white cystatin and its possible mode of interaction with cysteine proteinases. EMBO J. 7, 2593–2599. 47. Brown, W.M. & Dziegielewska, K.M. (1997) Friends and relations of the cystatin superfamily – 58. Stubbs, M.T., Laber, B., Bode, W., Huber, R., New members and their evolution. Protein Sci. 6, Jerala, R., Lenarèiè, B. & Turk, V. (1990) The re- 5–12. fined 2.4 Å X-ray crystal structure of the recombi- nant human stefin B in complex with the cysteine 48. Murzin, A. (1993) Sweet-tasting protein monellin is proteinase papain: A novel type of proteinase inhib- related to the cystatin family of thiol proteinase in- itor interaction. EMBO J. 9, 1939–1947. hibitors. J. Mol. Biol. 230, 680–694. 59. Martin, J.R., Jerala, R., Kroon-Zitko, L., [erovnik, 49. Somoza, J.R., Jiang, F., Tong, L., Kang, C.-H., Cho, E., Turk, V. & Waltho, J.P. (1994) Structural char- J.M. & Kim, S.-H. (1993) The crystal structures of a acterization of human stefin A in solution and im- potently sweet protein. Natural monellin at 2.75 Å plications for binding to cysteine proteinases. Eur. resolution and a single-chain monellin at 1.7 Å res- J. Biochem. 225, 1181–1194. olution. J. Mol. Biol. 234, 390–404. 60. Martin, J.R., Craven, C.J., Jerala, R., Kroon-Zitko, 50. Bode, W., Engh, R., Musil, D., Laber, B., Stubbs, L., [erovnik, E., Turk, V. & Waltho, J.P. (1995) The M., Huber, R. & Turk, V. (1990) Mechanism of in- three-dimensional solution structure of human teraction of cysteine proteinases and their protein stefin A. J. Mol. Biol. 246, 331–343. inhibitors as compared to the serine proteinase– inhibitor interaction. Biol. Chem. Hoppe-Seyler 61. Abrahamson, M., Olafsson, I., Palsdottir, A. 371, 111–118. Ulvsbäck, M., Lundwall, Å., Jensson, O. & Grubb, A. (1990) Structure and expression of the human 51. Björk, I. & Ylinenjärvi, K. (1989) Interaction of cystatin Cgene. Biochem. J. 268, 287–294. chicken cystatin with inactivated papains. Bio- chem. J. 260, 61–68. 62. Schnittger, S., Gopal Rao, V.V.N., Abrahamson, M. & Hansmann, L. (1993) Cystatin C (CST3), the can- 52. Abrahamson, M. (1994) Cystatins. Methods Enzy- didate gene for hereditary cystatin Camyloid mol. 244, 685–700. angiopathy (HCCAA), and other members of the 53. Lindahl, P., Abrahamson, M. & Björk, I. (1992) In- cystatin gene family are clustered on chromosome teraction of recombinant human cystatin Cwith 20p11.2. Genomics, 16, 50–55. 18 Z. Grzonka and others 2001 63. Grubb, A., Weiber, H. & Löfberg, H. (1983) The 73. Abrahamson, M. & Grubb, A. (1994) Increased g-trace concentration of normal human seminal body temperature accelerates aggregation of the plasma is thirty-six times that of normal human Leu®Gln mutant cystatin C, the amyloid-forming blood plasma. Scand. J. Clin. Lab. 43, 421–425. protein in hereditary cystatin Camyloid angiopathy. Proc. Natl. Acad. Sci. U.S.A. 91, 64. Grubb, A. (1992) Diagnostic value of analysis of 1416–1420. cystatin Cand protein HCin biological fluids. Clin. Nephrol. 38 (Suppl. 1), S20–S27. 74. Olafsson, I. & Grubb, A. (2000) Hereditary cystatin Camyloid angiopathy. J. Exp. Clin. Invest. 7, 65. Randers, E. & Erlandsen, E.J. (1999) Serum 70–79. cystatin Cas an endogenous marker of the renal function — a review. Clin. Chem. Lab. Med. 37, 75. Gerhartz, B., Ekiel, I. & Abrahamson, M. (1998) 389–395. Two stable unfolding intermediates of the dis- ease-causing L68Q variant of human cystatin C. 66. Hall, A., Håkansson, K., Mason, R.W., Grubb, A. & 37, 17309–17317. Abrahamson, M. (1995) Structural basis for the bi- ological specificity of cystatin C. Identification of 76. [erovnik, E., Cimerman, N., Kos, J., Turk, V. & leucine 9 in the N-terminal binding region as a se- Lohner, K. (1997) Thermal denaturation of human lectivity-conferring residue in the inhibition of cystatin Cand two of its variants; comparison to mammalian cysteine peptidases. J. Biol. Chem. chicken cystatin. Biol. Chem. Hoppe-Seyler 378, 270, 5115–5121. 1199–1203. 67. Mason, R.W., Sol-Church, K. & Abrahamson, M. 77. Ekiel, I. & Abrahamson, M. (1996) Folding-related (1998) Amino acid substitutions in the N-terminal dimerization of human cystatin C. J. Biol. Chem. segment of cystatin Ccreate selective protein inhib- 271, 1314–1321. itors of lysosomal cysteine proteinases. Biochem. J. 78. Jankowska, E., Banecki, B., Wiczk, W. & Grzonka, 330, 833–838. Z. (1999) Self-association of cystatin; in Peptide Sci- 68. Hall, A., Dalbøge, H., Grubb, A. & Abrahamson, M. ence — Present and Future (Shomonishi, Y., ed.), pp. (1993) Importance of the evolutionary conserved 654–656, Kluwer Academic Publishers, Dordrecht. glycine residue in the N-terminal region of human 79. Oberg, K.A. & Fink, A.L. (1998) A new attenuated cystatin C. Biochem. J. 291, 123–129. total reflectance Fourrier transform infrared spec- 69. Ekiel, I., Abrahamson, M., Fulton, D.B., Lindahl, troscopy method for the study of protein in solu- P., Storer, A.C., Levadoux, W., Lafrance, M., tion. Anal. Biochem. 256, 92–106. Labelle, S., Pomerleau, Y., Groleau, D., LeSauteur, 80. Grzonka, Z., Kasprzykowski, F., Oldziej, S., Wiczk, L. & Gehring, K. (1997) NMR structural studies of W. & Grubb, A. (1993) Recent developments in the human cystatin Cdimers and monomers. J. Mol. chemistry and biology of cystatins; in Peptide Biol. 271, 266–277. Chemistry 1992 (Yanaihara, N., ed.) pp. 243–246, 70. Hall, A., Ekiel, I., Mason, R.W., Kasprzykowski, F., ESCOM, Leiden. Grubb, A. & Abrahamson, M. (1998) Structural ba- 81. Dieckmann, T., Mitschang, L., Hofmann, M., Kos, sis for different inhibitory specificities of human J., Turk, V., Auerswald, E.A., Jaenicke, R. & cystatins Cand D. Biochemistry 37, 4071–4079. Oschkinat, H. (1993) The structures of native 71. Gudmundsson, G., Hallgrimsson, J., Jonasson, phosphorylated chicken cystatin and of recombi- T.A. & Bjarnason, O. (1972) Hereditary cerebral nant unphosphorylated variant in solution. J. Mol. hemorrhage with amyloidosis. Brain 95, 387–404. Biol. 234, 1048–1059. 72. Löfberg, H., Grubb, A., Nilsson, E.K., Jensson, O., 82. Engh, R.A., Dieckmann, T., Bode, W., Auerswald, Gudmundsson, G., Blondal, H., Arnason, A. & E.A., Turk, V., Huber, R. & Oschkinat, H. (1993) Thorsteinsson, L. (1987) Immunohistochemical Conformational variability of chicken cystatin. characterization of the amyloid deposits and quan- Comparison of structures determined by X-ray dif- tification of pertinent cerebrospinal proteins in he- fraction and NMR spectroscopy. J. Mol. Biol. 234, reditary cerebral hemorrhage with amyloidosis. 1060–1069. Stroke 18, 431–440. Vol. 48 Structural studies of cysteine proteases and their inhibitors 19 83. Alvarez-Fernadez, M., Abrahamson, M. & Su, X.-D. 93. Leary, R., Larsen, D., Watanabe, H. & Shaw, E. (2000) Crystal structure of cystatin D, an inhibitor (1977) Diazomethyl ketone substrate derivatives as of papain-like cysteine proteinases; in Max-lab Ac- active-site-directed inhibitors of thiol proteases. tivity Report 1999 (Andersen, J.N., Nyholm, S.L. & Papain. Biochemistry 16, 5857–5861. Sorensen, H., eds.) pp. 224–225. 94. Tarnowska, M., O³dziej, S., Liwo, A., Kania, P., 84. Abrahamson, M., Mason, R.W., Hansson, H., Kasprzykowski, F. & Grzonka, Z. (1992) MNDO Buttle, D.J., Grubb, A. & Ohlsson, K. (1991) Hu- study of the mechanism of the inhibition of man cystatin C. Role of the N-terminal segment in cysteine proteinases by diazomethyl ketones. Eur. the inhibition of human cysteine proteinases and in Biophys. J. 21, 217–222. its inactivation by leucocyte elastase. Biochem. J. 95. Johansson, L., Grubb, A., Abrahamson, M., 273, 621–626. Kasprzykowski, F., Kasprzykowska, R., Grzonka, 85. Grubb, A., Löfberg, H. & Barrett, A.J. (1984) The Z. & Lerner, U.H. (2000) A peptidyl derivative disuphide bridges of human cystatin C( g-trace) and structurally based on the inhibitory center of chicken cystatin. FEBS Lett. 170, 370–374. cystatin Cinhibits bone resorption in vitro. Bone 26, 451–459. 86. Barret, A.J. (1987) The cystatins: A new class of peptidase inhibitors. Trends Biochem. Sci. 12, 96. Barrett, A.J., Kembhavi, A., Brown, M.A., 193–196. Kirschke, H., Knight, C.G., Tamai, M. & Hanada, K. (1982) L-trans-epoxysuccinyl-leucylamido(4-gua- 87. Kozak, M., Jankowska, E., Janowski, R., Grzonka, nidino)butane (E-64) and its analogues as inhibi- Z., Grubb, A., Alvarez-Fernadez, M., Abrahamson, tors of cysteine proteinases including cathepsins B, M. & Jaskólski, M. (1999) Expression of a H and L. Biochem. J. 201, 189–198. selonomethionyl derivative and preliminary crys- tallographic studies of human cystatin C. Acta 97. Matsumoto, K., Mizoue, K., Kitamura, K., Tse, Crystallogr. D55, 1939–1942. W.C., Huber, C.P. & Ishida, T. (1999) Structural ba- sis of inhibition of cysteine proteases by E-64 and 88. Abrahamson, M., Dalbøge, H., Olafsson, I., its derivatives. Biopolymers 51, 99–107. Carlsen, S. & Grubb, A. (1988) Efficient production of native, biologically active human cystatin Cby 98. Kasprzykowski, F., Schalen, C., Kasprzykowska, Escherichia coli. FEBS Lett. 236, 14–18. R., Jastrzêbska, B. & Grubb, A. (2000) Synthesis and antibacterial properties of peptidyl derivatives 89. Matthews, B.W. (1968) Solvent content in protein and cyclopeptides structurally based upon the in- crystals. J. Mol. Biol. 33, 491–497. hibitory center of human cystatin C. APMIS 108, 90. Björck, L., Åkesson, P., Bohus, M., Trojnar, J., 471–481. Abrahamson, M., Olafsson, I. & Grubb, A. (1989) 99. Yamamoto, D., Matsumoto, K., Ohishi, H., Inoue, Bacterial growth blocked by a synthetic peptide M., Kitamura, K. & Mizuno, H. (1991) Refined based on the structure of a human proteinase inhib- X-ray structure of papain E-64 complex at 2.1 Å res- itor. Nature 337, 385–386. olution. J. Biol. Chem. 266, 14771–14777. 91. Hall, A., Abrahamson, M., Grubb, A., Trojnar, J., 100.Kozak, M., Kozian, E., Grzonka, Z. & Jaskólski, M. Kania, P., Kasprzykowska, R. & Kasprzykowski, F. (1997) Crystalization and preliminary crystallo- (1992) Cystatin C based peptidyl diazomethanes as graphic studies of a covalent complex between cysteine proteinase inhibitors: Influence of the papain an oxirane-based inhibitor; in Peptides peptidyl chain length. J. Enzyme. Inhib. 6, 113– 1996 (Ramage, R. & Epton, E., eds.) pp. 551–552, 123. European Peptide Society. 92. Kasprzykowski, F., Kasprzykowska, R., Pluciñska, 101. Drenth, J. & Jansonius, J.N. (1959) The unit cell of K., Kania, P., Grubb, A., Abrahamson, M. & mercuripapain crystals. Nature 28, 1718–1719. Schalen, C. (2001) Peptidyl diazomethylketones as cysteine protease inhibitors structurally based 102.Czaplewski, C., Grzonka, Z., Jaskólski, M., upon the inhibitory centers of cystatins. Pol. J. Kasprzykowski, F., Kozak, M., Politowska, E. & Chem. (in press). Ciarkowski, J. (1999) Binding modes of a new epoxysuccinyl-peptide inhibitor of cysteine pro- teases. Where and how do cysteine proteases ex- 20 Z. Grzonka and others 2001 press their selectivity? Biochim. Biophys. Acta K.M., Stanton, R.V., Cheng, A., Vincent, J.J., 1431, 290–305. Crowley, M., Ferguson, D.M., Radmer, R., Seibel, G.L., Singh, U.C., Wiener, P. & Kollman, P.A. 103.Drenth, J., Kalk, K.H. & Swen, H.M. (1976) Bind- (1997) Amber 5.0, University of California, San ing of chloromethyl ketone substrate analogues to Francisco. crystalline papain. Biochemistry 15, 3731–3738. 108.Bayly, C.I., Cieplak, P., Cornell, W.D. & Kollman, 104. Pickersgill, R.W., Harris, G.W. & Garman, E. P.A. (1993) A well-behaved electrostatic potential (1992) Structure of monoclinic papain at 1.6 Å based method using charge restraints for deriving resolution. Acta Crystallogr. B48, 59–67. atomic charges: The RESP model. J. Phys. Chem. 105.SYBYL 6.1. (1994) Tripos Inc., 1699 S. Manley 97, 10269–10280. Rd., St. Louis, MO 63144, U.S.A. 109.Schroder, E., Phillips, C., Garman, E., Harlos, K. 106.Bernstein, F.C., Koetzle, T.F., Williams, G.J.B., & Crawford, C. (1993) X-Ray crystallographic Meyer, E.F., Jr., Brice, M.D., Rodgers, J.R., structure of a papain- complex. FEBS Kennard, O., Shimanouchi, T. & Tasami, M. Lett. 315, 38–42. (1977) The protein data bank: A computer-based 110. Varughese, K.I., Ahmed, F.R., Carey, P.R., archive file for macromolecular structures. J. Mol. Hasnain, S., Huber, C.P. & Storer, A.C. (1989) Biol. 112, 535–542. Crystal structure of a papain-E-64 complex. Bio- 107. Case, D.A., Pearlman, D.A., Caldwell, J.W., chemistry 28, 1330–1332. Cheatham III, T.E., Ross, W.S., Darden, T., Merz,