Crystal structure of the parasite inhibitor chagasin in complex with papain allows identification of structural requirements for broad reactivity and specificity determinants for target proteases Izabela Redzynia1,*, Anna Ljunggren2,*, Anna Bujacz1, Magnus Abrahamson2, Mariusz Jaskolski3,4 and Grzegorz Bujacz1,4
1 Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Technical University of Lodz, Poland 2 Department of Laboratory Medicine, Division of Clinical Chemistry and Pharmacology, Lund University, Sweden 3 Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University, Poznan, Poland 4 Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland
Keywords A complex of chagasin, a protein inhibitor from Trypanosoma cruzi, and Chagas disease; cruzipain; cysteine papain, a classic family C1 cysteine protease, has been crystallized. Kinetic proteases; papain; protein inhibitors studies revealed that inactivation of papain by chagasin is very fast ) ) (k = 1.5 · 106 m 1Æs 1), and results in the formation of a very tight, Correspondence on m G. Bujacz, Institute of Technical Biochemis- reversible complex (Ki =36p ), with similar or better rate and equilib- try, Faculty of Biotechnology and Food rium constants than those for cathepsins L and B. The high-resolution Sciences, Technical University of Lodz, ul. crystal structure shows an inhibitory wedge comprising three loops, which Stefanowskiego 4/10, 90-924 Lodz, Poland forms a number of contacts responsible for the high-affinity binding. Com- Fax: +48 42 636 66 18 parison with the structure of papain in complex with human cystatin B Tel: +48 42 631 34 31 reveals that, despite entirely different folding, the two inhibitors utilize very E-mail: [email protected] similar atomic interactions, leading to essentially identical affinities for the M. Abrahamson, Department of Laboratory Medicine, Division of Clinical Chemistry and enzyme. Comparisons of the chagasin–papain complex with high-resolution Pharmacology, Lund University, University structures of chagasin in complexes with cathepsin L, cathepsin B and falci- Hospital, SE-221 85 Lund, Sweden pain allowed the creation of a consensus map of the structural features that Fax: +46 46 130064 are important for efficient inhibition of papain-like enzymes. The compari- Tel: +46 46 173445 sons also revealed a number of unique interactions that can be used to E-mail: [email protected] design enzyme-specific inhibitors. As papain exhibits high structural simi- larity to the catalytic domain of the T. cruzi enzyme cruzipain, the present *These authors contributed equally to this paper chagasin–papain complex provides a reliable model of chagasin–cruzipain interactions. Such information, coupled with our identification of specifi- Database city-conferring interactions, should be important for the development of Atomic coordinates, together with structure drugs for treatment of the devastating Chagas disease caused by this factors, have been deposited in the Protein parasite. Data Bank under the accession code 3E1Z
(Received 13 October 2008, revised 15 November 2008, accepted 1 December 2008) doi:10.1111/j.1742-4658.2008.06824.x
Papain (EC 3.4.22.2) from the latex of the papaya fruit studies in the 20th century peaked with efforts in the (Carica papaya) was one of the first known proteolytic 1960s, defining the chemistry of the enzymatic mecha- enzymes, and its digestive properties were already nism, delineating the concept of specificity for protein being utilized in the 19th century. Detailed biochemical substrate recognition [1–3], and with elucidation of the
FEBS Journal (2008) ª 2008 The Authors Journal compilation ª 2008 FEBS 1 Chagasin–papain complex structure I. Redzynia et al. crystal structure of the enzyme, one of the first protein binding epitope of chagasin consists of three loops structures to be determined [4]. Since then, papain has (L4, L2, L6) that together form a wedge-like enzyme- been used as a model protein in many studies, and is binding epitope. the founding member of the large C1 family of In this study, we present a high-quality crystal papain-like cysteine proteases [5]. Approximately 12 structure of chagasin in complex with papain, the mammalian cysteine proteases are evolutionarily clo- model C1 family cysteine protease and one of only sely related to papain and hence belong to this family two enzymes in the family for which structural infor- (e.g. cathepsins B, H, L, S and K). Enzymes from the mation for a cystatin complex is available [23,24]. C1 family generally function in every cell as compo- Based on the amino acid sequence and structure-based nents of the lysosomal degradation system, participat- alignment, papain has been shown to be a close ing in the turnover of proteins, but, in addition, have homolog of cruzipain [25]. Our results confirm map- been shown to participate in a number of specialized ping of the enzyme-binding epitope to the three loops, functions, such as proteolytic cleavages activating pro- as in chagasin complexes with mammalian enzymes, hormones, regulation of antigen presentation, etc. C1 and illustrate the degree of structural adjustments as family proteases are evolutionarily old, are found in well as precise atomic contacts formed during enzyme both prokaryotic and eukaryotic organisms, and in binding. Moreover, comparative analysis of several many cases show activity that is indispensable for the chagasin complexes has revealed a strikingly similar organism. The unicellular parasite Trypanosoma cruzi core structure involved in enzyme binding, which is an example of such an organism, in which the results in sub-nanomolar Ki values and rate constants papain-like enzyme, cruzipain, is essential for the life- for inactivation in the 105–106 m)1Æs)1 range in all cycle of the parasite and also acts as a virulence factor cases. Additionally, several contacts unique to the when the parasite infects its human host, causing the individual enzyme complexes could be identified, rais- devastating Chagas disease [6,7]. ing the prospect of accurate structure-aided design of In a variety of species, from mammals, plants and specific inhibitors of cruzipain and cathepsins. insects to simpler eukaryotes such as the filarial Detailed knowledge of the structure and inhibition parasites Onchocerca volvulus and Acanthocheilonema mode of chagasin should be valuable in guiding the viteae, C1 family cysteine proteases are in equilibrium development of drugs for the prevention and treat- with protein inhibitors belonging to the cystatin fam- ment of Chagas disease. ily, I25 [5,8–10]. Most cystatins, such as human cysta- tin B, are single-domain proteins of 100–120 residues Results with a characteristic wedge-like epitope consisting of the N-terminus and two b-hairpin loops, which blocks Function of chagasin as an inhibitor of papain the active site cleft of the target enzyme, thereby inhib- iting the activity in a reversible manner [11,12]. Cysta- Chagasin used in this study was expressed in Escheri- tins show high affinity for their target enzymes due to chia coli and purified to homogeneity as reported a large binding area, with dissociation constants (Ki)in previously [20]. The recombinant protein contains five the range 10)9–10)11 m. In extreme cases, such as the extra N-terminal amino acid residues from the expres- human cystatin C–papain complex, Ki values as low as sion construct, and has a mass of 12 440 Da as 10)14 m have been reported [13]. expected [20]. The protein shows almost 100% activity Trypanosomatids, such as various Trypanosoma and as a protease inhibitor based on titration of a papain Leishmania species, produce inhibitors of their own solution with known activity, forms stoichiometric family C1 proteases [14]. Chagasin, a tight-binding 1 : 1 complexes with cathepsin L or B, and is not inhibitor of cruzipain found in T. cruzi [15], exhibits cleaved by these proteases [20,21]. no sequence similarity with cystatins (GenBank ⁄ EMBL Kinetic parameters for the interaction of chagasin [16] accession number AJ299433), despite its similar with papain at pH 6.0 were determined in a continuous- size (110 residues). Molecular modeling studies pre- rate assay using the sensitive fluorogenic substrate car- dicted an immunoglobulin-like fold for chagasin [17], boxybenzoyl-Phe-Arg-7-(4-methyl)coumarylamide, with which was essentially confirmed by subsequent NMR a sufficiently high inhibitor concentration for the
[18] and crystallographic studies [19,20]. Recently, crys- binding reaction to be of pseudo-first order. The kon ) ) tal structures of chagasin in complex with human cath- value was determined to be 1.5 · 106 m 1Æs 1, very simi- epsins L and B [20,21], and additionally with falcipain lar to that determined for cathepsin L and higher than from the malaria parasite [22], have been determined. that for cathepsin B under the same conditions The complex structures demonstrate that the enzyme- (Table 1). The equilibrium constant for dissociation
2 FEBS Journal (2008) ª 2008 The Authors Journal compilation ª 2008 FEBS I. Redzynia et al. Chagasin–papain complex structure
Table 1. Function of chagasin as an inhibitor of papain and other Table 2. Data collection and structure refinement statistics.
C1 family enzymes. Equilibrium constants for dissociation (Ki)of chagasin–papain complexes were determined under steady-state Data collection conditions at pH 6.0 as described in Experimental procedures. Cor- Radiation source X13, EMBL Hamburg ˚ responding values for the papain-like cysteine proteases cathep- Wavelength (A) 0.8086 sin L, cathepsin B and falcipain, with known inhibitor complex Temperature of measurements (K) 100 structures [20–22], as well as for the papain complex with human Space group I422 Cell parameters (A˚ ) a = 99.1, c = 159.5 cystatin B [12], are included for comparison. The Ki values pre- ˚ a sented were corrected for substrate competition in the assays, as Resolution range (A) 60.0–1.86 (1.93–1.86) described in Experimental procedures. ND, not determined. Reflections collected 350 034 Unique reflections 33 263 )1 )1 Ki (nM) kon (M Æs ) Completeness (%) 98.4 (88.8) Redundancy 10.5 (6.8) Enzyme Chagasin Cystatin B Chagasin ⁄
Crystallization and structure determination the 1.7 r level, except for the N- and C-termini of the A complex between chagasin and papain was formed chagasin molecule. All side chains, as well as both ter- by incubating the proteins in a 1.3 : 1 molar ratio for minal segments, are clearly visible when the electron approximately 4 h before setting up crystallization density maps are contoured at the 1.0 r level. The drops. Single crystals of the chagasin–papain complex GPLGS peptide introduced as an N-terminal extension were obtained using Crystal Screen II in Hepes buffer of the recombinant chagasin is totally disordered and at pH 7.5 without further optimization. The crystal not visible in the electron density maps. In addition to structure of the complex was solved to 1.86 A˚resolu- 298 water molecules, the model includes 10 formate tion by molecular replacement using the chagasin– ions from the crystallization buffer. The refinement cathepsin L model (PDB code 2NQD) [20] as a probe. statistics are presented in Table 2. The residues of the The initial atomic coordinates of the chagasin–papain inhibitor are labeled without a chain designator. The complex were obtained by rigid-body substitution of residues of the enzyme are marked ‘e’. When cystatin cathepsin L by a papain model (PDB code 1KHQ) sequences are discussed in this paper, amino acid num- [26]. After least-squares refinement, the main-chain bering according to the chicken cystatin sequence is traces of the chagasin and papain molecules were visi- used, as in the original papain–cystatin B structure ble in 2Fo–Fc electron density maps without breaks at [23]. To convert to human cystatin C numbering,
FEBS Journal (2008) ª 2008 The Authors Journal compilation ª 2008 FEBS 3 Chagasin–papain complex structure I. Redzynia et al. which is widely used, ‘2’ should be added to all residue In overall shape, the present complex is similar to numbers, so that G9 in cystatin B corresponds to G11 the previously described complex structures of chaga- in cystatin C [27]. sin with cathepsins L and B [20,21], resembling an
A strong residual peak in the Fo–Fc electron density inverted mushroom, with the stalk formed by the map, in close proximity to H72, H74, E23 and one of cylindrical chagasin molecule and the cap by the glob- the formate ions, was interpreted as a zinc cation. This ular papain (Fig. 1). The C25e-H159e-N175e catalytic interpretation is supported by the bond valence test triad of papain is located at the bottom of a long cleft [28,29] and by the tetrahedral coordination of this running across the width of the molecule, dividing it cation. Although chagasin inhibition is not dependent into the L and R domains [30]. on any cofactors, this site at the surface of the mole- The binding region of chagasin formed by the loops cule may be of structural significance, as the same his- L2 (N29–F34), L4 (P59–G68) and L6 (R91–S100) is tidine residues in the cathepsin B complex were found docked very tightly to the papain molecule (Fig. 2A). to bind a phosphate ion [21]. The main hydrogen bonds between chagasin and papain observed in the complex are listed in Table S1. All three loops are located in the catalytic groove, with The chagasin–papain interface the 310 tip of loop L2 inserted directly into the cata- The papain chain in the present complex starts with lytic center. Loops L4 and L6 embrace the enzyme residue I1e, which is well defined in the electron den- molecule from both sides. sity map. The last residue, N212e, is also clearly visible The interactions of each loop have different charac- because the side chain is stabilized by hydrogen bonds teristics. Loop L6 forms three types of interactions with D108e and I148e, and the C-terminal carboxylate with the enzyme: hydrogen bonds (R91), hydrophobic forms a salt bridge with R188e, the latter two inter- contacts (P92) and p interactions (W93), which ‘probe’ actions involving a symmetry-related molecule. The different elements of the catalytic apparatus. First, enzyme used for crystallization was in an inactive W93 interacts with a cluster of aromatic residues form, with the catalytic C25e residue protected by (F141e, W177e, W181e) that serve to position the carboxymethylation. The blocking group is clearly N175e element of the catalytic triad (C25e-H159e- visible in the electron density maps. N175e) through N-H...p hydrogen bonds. R91 assumes The overall conformation of the chagasin molecule a fully extended conformation reaching to the catalytic in the present complex is similar to that found for free site of the enzyme and loop L2 of chagasin. The R91 chagasin (PDB code 2NNR) [20]. The C- and N-termi- guanidinium group forms two hydrogen bonds with nal residues of chagasin are somewhat flexible, but the the carbonyl group of T32 in loop L2, which is located contour level of 1 r for the 2Fo–Fc electron density next to the active-site-blocking residue, T31 [20,21]. maps was sufficient for unambiguous modeling. The The other segment of the guanidinium group of R91 first visible residue at the N-terminus is S2, which is forms a pair of hydrogen bonds with the oxygen atom anchored by a side-chain hydrogen bond to N64e from of the side-chain amide group of N18e. It is interesting a symmetry-related molecule. The C-terminal N110 to note that the equivalent position in cruzipain is residue points to a water channel. occupied by an aspartate, making the interaction with
Fig. 1. Stereoview of the chagasin–papain complex. The chagasin molecule is colored green and papain is colored pink. The surfaces of both proteins are marked correspondingly. The view is along the catalytic cleft of papain and corresponds to the standard orientation used for cysteine proteases, with the L and R lobes on the left and right, respectively.
4 FEBS Journal (2008) ª 2008 The Authors Journal compilation ª 2008 FEBS I. Redzynia et al. Chagasin–papain complex structure
A B
C
D
Fig. 2. Interactions of chagasin and cystatin B with papain. Color coding: chagasin (green)–papain (pink); cystatin B (brown)–papain (gray). (A) Stereoview of aligned molecules created by superposition of the Ca atoms of papain from the crystal structures of its complexes with cystatin B (PDB code 1STF) and chagasin (this work). The upper panel emphasizes the different angle of approach of the two inhibitors in the standard orientation of papain. The lower panel, rotated by 90 (papain R domain at the front) emphasizes the similar shape of inhibitory elements (loops and the cystatin B N-terminus). (B) Zoom-in view of the interactions of papain with loop L6 of chagasin and loop L2 of cysta- tin B. (C) Zoom-in view of the interactions of papain with loop L4 of chagasin and the N-terminal segment of cystatin B. (D) Zoom-in view of the interactions of papain with loop L2 of chagasin and loop L1 of cystatin B.
R91 even stronger. Finally, the guanidinium group of The interactions of loop L4 with the enzyme are R91 is also hydrogen-bonded to the carbonyl group of based on formation of an antiparallel intermolecular G20e. The third element of L6, P92, shapes the loop b-sheet. Two residues from chagasin, G66 and L65, for optimal interactions with the enzyme by forming interact with the papain main-chain atoms N64e–G66e hydrophobic contacts with the side chain of L143e (Fig. 2C). In addition, the side-chain carbonyl Od1 (Fig. 2B). In addition to the direct interactions of loop atom of N64e forms a water-mediated contact with L6 described above, there are also contacts mediated the main-chain N atom of G68, and the main-chain by water molecules. nitrogen of G66 of chagasin forms a water-mediated
FEBS Journal (2008) ª 2008 The Authors Journal compilation ª 2008 FEBS 5 Chagasin–papain complex structure I. Redzynia et al. contact with the main-chain carbonyl group of D158e molecular mass of 11–14 kDa [27]. The structure of from papain. papain in complex with cystatin B (PDB code 1STF) Compared to the very strong and extended interac- [23] offers an excellent opportunity for comparison of tions of loops L4 and L6, the interactions of loop L2 the mode of interaction of the two very different inhib- are very limited. A repulsive contact is seen between itors with the same target enzyme. the carbonyl O atom of T31 and the Nd1 atom of the Although chagasin and cystatin B show essentially imidazole ring of the catalytic H159e residue. A much identical affinity for papain (Table 1), superposition of longer, attractive contact exists between the same T31 the two complexes based on Ca alignment [31] of the carbonyl and the Ne1 atom of W177e (Table S1). The enzyme portions shows a completely different fold for hydroxyl group of T31 interacts with the main-chain the two inhibitors (Fig. 2A). The characteristic b-sheet carbonyl of D158e (Fig. 2D). The four additional grip around a long a-helix, characteristic of cystatins, atoms of the carboxymethyl modification of the cata- contrasts with the all-b structure of chagasin. lytic C25e residue are easily accommodated at the However, despite their different overall fold, the inhibitor–enzyme interface. The oxygen atoms of epitope presented by both inhibitors to the enzyme is the carboxymethyl block form contacts with both the arranged similarly. The L4–L2–L6 wedge of chagasin enzyme (main-chain N of C25e and side chain of overlaps with a similar wedge of cystatin B formed by Q19e) and the inhibitor (OH group of T31). the N-terminal segment and two b-hairpin loops, L1 and L2 (Fig. 2A–D). This similarity does not extend beyond the active site, and, in fact, the two molecules The inhibition mode of chagasin approach the enzyme from a different angle. We have The best-studied group of cysteine protease inhibitors defined the angle of approach, s (Table 3), as the are the cystatins, which are small proteins with a dihedral angle between two planes, one (a) dividing the
Table 3. Comparison of various enzyme complexes of chagasin. The superpositions of Ca atoms were calculated using ALIGN [31] for the entire complex (c), for the enzyme molecule only (e), and for the chagasin molecule only (ch). Each superposition is characterized by the root mean square (rms) deviation in A˚ and the number of aligned Ca atoms (in parentheses). For comparison, superpositions with the crystallo- graphic models of cruzipain and free chagasin (molecules A and B) are also included. Where appropriate, a number in square brackets shows the level of sequence identity (%) between the compared enzymes. The last two rows characterize the chagasin complexes by giving the contact area (in A˚ 2) calculated using Areaimol [32] and by specifying the angle of inhibitor approach s (in degrees) relative to the enzyme framework (see definition in the text).
Chagasin–cathepsin B
Form I Form II Chagasin–cathepsin L Chagasin–papain Chagasin–falcipain
Cruzipain 1.38 (198) 1.46 (197) 0.81 (190) 1.07 (191) 1.10 (192) [27.8%] [43.4%] [35.8%] [37.7%] Chagasin A 0.44 (98) 0.55 (101) 0.48 (100) 0.44 (92) 0.38 (99) B 0.37 (98) 0.54 (105) 0.52 (103) 0.35 (91) 0.37 (102) Chagasin–cathepsin B Form I c 1.16 (348) c 1.25 (301) c 1.15 (287) c 1.15 (294) e 0.54 (233) e 1.28 (198) e 1.32 (192) e 1.30 (193) ch 0.46 (100) ch 0.43 (102) ch 0.43 (96) ch 0.37 (101) Form II c 1.55 (300) c 1.10 (278) c 1.19 (293) e 1.31 (196) e 1.42 (191) e 1.35 (190) ch 0.55 (107) ch 0.42 (101) ch 0.52 (107) [28.2%] [29.7%] [24.1%] Chagasin–cathepsin L c 1.18 (297) c 1.19 (300) e 0.79 (188) e 1.00 (189) ch 0.61 (101) ch 0.44 (105) [40.6%] [35.9%] Chagasin–papain c 0.87 (274) e 1.18 (187) ch 0.34 (93) [37.7%] Contact area 1221 1373 972 922 984 Angle of approach s 7.2 2.3 11.3 5.8 5.4
6 FEBS Journal (2008) ª 2008 The Authors Journal compilation ª 2008 FEBS I. Redzynia et al. Chagasin–papain complex structure enzyme into the R and L lobes along the catalytic The interaction of loop L4 of chagasin with papain groove, defined by the Ca atoms of three papain resi- is based on formation of an intermolecular b-sheet dues, I40e, Y67e and W177e (or their equivalents in (Fig. 2C). There is an analogous interaction between other enzymes), and the other (b) created by three Ca the N-terminus of cystatin B and papain. G9 from the atoms defining the inhibitor framework and passing N-terminal cystatin B segment and G66 from loop L4 along the inhibitory wedge. In the case of chagasin, of chagasin provide a degree of flexibility, thus allow- plane (b) is triangulated by the tips of the peripheral ing optimal interactions between the two main chains. loops L4, L6 and the C-terminus, or specifically by the The same role is played by G65e of papain. The short Ca atoms of G66, R91 and A109. The corresponding antiparallel b-sheet interaction is formed by only one Ca atoms of cystatin B are located in residues G9 (in residue, G66e, of papain with L65 or S66 of chagasin the N-terminal binding segment, according to chicken or cystatin B, respectively. This antiparallel interaction cystatin numbering [23]), D68 (a loop from the oppo- is supported by a water molecule linking the N atom site pole) and L102 (loop L2). The s angle in the of G66 of chagasin and the main-chain O atom of chagasin–papain complex is 5.8 , indicating that the D158e of papain. In the cystatin B complex, an equiv- chagasin molecule is slanting towards the R domain. alent carbonyl is involved in a water-mediated interac- The angle of approach of cystatin B is )12.7 , and the tion with the N atom of A10. inhibitor molecule is inclined towards the L domain of The L2 loop of chagasin and the corresponding loop the enzyme. The difference in the angles of approach L1 of cystatin B interact with the catalytic center of between chagasin and cystatin B is 18.5 . It is also of papain (Fig. 2D). Our structural alignment (Fig. 3A) note that the sequential epitope of cystatins corre- shows that loop L2 of chagasin is one residue longer, sponds to a non-sequential binding site of chagasin. and thus T31 has no equivalent in loop L1 of cysta- The contact area [32] is similar for both complexes, tin B. Loop L2 of chagasin not only interacts with and is 853 and 922 A˚2 for the cystatin B–papain and loop L6 but also with loop L4, by forming a hydrogen chagasin–papain complexes, respectively. bond between the side-chain amide of N29 and main- The three crucial residues of loop L6 of chagasin chain carbonyl of G66. A similar interaction is (R91, P92 and W93) correspond to L102, P103 and observed in cystatin B, where the side-chain amide of H104, respectively, in the cystatin B molecule Q53 forms a hydrogen bond with the main-chain car- (Fig. 3A). It is noteworthy that the pattern Pro–aro- bonyl of G9, stabilizing the interaction between loop matic residue is conserved in chagasin-like inhibitors L1 and the N-terminus. Although the conformation of and in cystatins (where it is predominantly PW), these two loops is somewhat different, in both cases despite the lack of overall sequence similarity. The they have the same, substrate-like, polarity. There is a role of the proline residue appears to be to maintain surprisingly small number of specific interactions with the specific shape of the loop. The aromatic residue, the catalytic residues of the enzyme for both chagasin on the other hand, interacts with the aromatic clus- loop L2 and cystatin loop L1, which explains why ter of the enzyme (Fig. 2B). The residue preceding chagasin (and also cystatins) can bind with high affin- the Pro–aromatic motif, which is invariably an argi- ity to cysteine proteases with the catalytic -SH group nine in chagasin-type inhibitors of protozoan origin, protected by a carboxymethyl group. The repulsive is replaced by an aliphatic residue in cystatins interactions between the chagasin loop L2 and the cat- (Fig. 3A). This difference may be an important ele- alytic site of papain, described above, are reproduced ment regulating the enzyme specificity of these two in the cystatin complex. groups of inhibitors. The R91 residue of chagasin provides direct communication between loops L6 and Discussion L2, and also interacts with the crucial D18e ⁄ N18e residues of cruzipain ⁄ papain. The role of the L102 Comparison of the existing structures of residue of cystatin B is different, and supports inter- chagasin complexes with cysteine proteases action with the aromatic cluster of the enzyme (Fig. 2B). An additional interaction between loops In addition to the chagasin–papain complex presented L2 and L6 of chagasin is provided by the carbonyl in this paper, four additional crystal structures of group of the main chain of M90 and the nitrogen chagasin complexes with other cysteine proteases are atom of A35. A similar stabilizing contact between available in the Protein Data Bank. The target cystatin B loops L2 and L1 is formed by the main- enzymes for chagasin in these complexes are cath- chain carbonyl of Q101 and the peptide nitrogen epsin L (PDB code 2NQD) [20], cathepsin B in two atom of T58. crystal forms (PDB codes 3CBJ and 3CBK) [21] and
FEBS Journal (2008) ª 2008 The Authors Journal compilation ª 2008 FEBS 7 Chagasin–papain complex structure I. Redzynia et al.
A
B
Fig. 3. Structure-based sequence alignment of the interacting residues of cysteine prote- ases and their inhibitors. (A) Alignment of structurally equivalent residues forming the enzyme-binding epitopes of chagasin-like (L4, L2 and L6) and cystatin-like inhibitors (N-terminus, L1 and L2). The following protein sequences have been used: inhibitors, Trypanosoma cruzi (GenBank accession number AJ299433), Trypanosoma brucei (AJ548777), Leishmania mexicana (AJ548776), Leishmania major (AJ548878), Entamoeba histolytica (AJ634054), Pseudomonas aeruginosa (AAG04167) [53], Gallus gallus cystatin (J05077), Homo sapiens cystatin B (BC010532), H. sapiens cystatin C (BC110305); proteases, Carica papaya papain (M15203), H. sapiens cathep- sin L (X12451), H. sapiens cathepsin B (BC010240), Plasmodium falciparum falci- pain (AAF97809), T. cruzi cruzipain (X54414). (B) Alignment of structurally equivalent resi- dues from the catalytic groove of various cysteine proteases, based on the crystal structures of their complexes with chagasin, except for cruzipain, for which a complex with a small-molecule inhibitor (PDB code 1ME3) is used. The residues crucial for interactions with chagasin are color-coded as yellow (catalytic triad), red (aromatic cluster), green (residues forming hydrogen bonds) and blue (hydrophobic contacts). falcipain (PDB code 2OUL) [22]. These structural data tude lower, and that for falcipain is yet another order form an excellent platform for comparison of the inter- of magnitude lower, although still in the nanomolar actions between chagasin and the targeted proteolytic range (Table 1). enzymes. The residues from the catalytic cleft of vari- The contact surface area for chagasin–cysteine pro- ous cysteine proteases that interact with chagasin are tease complexes varies between 922 and 1373 A˚2 structurally aligned (Fig. 3B). The inhibitor binds (Table 3), and does not directly correlate with the effi- papain and cathepsin L with essentially the same, very ciency of inhibition. The extra contact area found in high, affinity (Ki approximately 0.03 nm); the affinity both crystal forms of the chagasin–cathepsin B com- for cathepsin B is approximately one order of magni- plex is created by the additional and unique occluding
8 FEBS Journal (2008) ª 2008 The Authors Journal compilation ª 2008 FEBS I. Redzynia et al. Chagasin–papain complex structure
A
L6 L6 L2 L2 L4 L4 Fig. 4. Stereoview of aligned chagasin com- plexes. (A) Superposition of the Ca atoms of the chagasin molecules from the complexes B of papain, cathepsins L and B, and falcipain with native chagasin. (B) Alignment of all above chagasin complexes based on superposition of the Ca atoms of the enzyme components. Color code: chagasin (green)–papain (pink) (this work), chagasin (gold)–cathepsin L (dark blue) (PDB code 2NQD), chagasin (orange)–cathepsin B (mid- green) (monoclinic form, 3CBJ), chagasin (yellow)–cathepsin B (lime green) (tetragonal form, 3CBK), chagasin (light blue)–falcipain (gray) (2OUL). The additional two molecules of native chagasin (2NNR) are colored dark- green (chain A) and purple (chain B). loop of this enzyme. The inhibition of cathepsin B by structures of native chagasin (0.35–0.55 A˚). These chagasin is relatively weak, which may be due to the results show that the chagasin molecule has a rigid fact that some of the binding energy has to be invested conformation and does not change upon complex for- in pushing the occluding loop out of the catalytic cleft. mation. This contradicts the conclusions drawn from The angle of approach, calculated in the way described an NMR study that predicted a high level of flexibil- above, has the lowest value for the tetragonal form of ity of the chagasin molecule [18]. A superposition of the chagasin–cathepsin B complex and the highest for all the chagasin molecules from the complex and the chagasin–cathepsin L complex (Table 3). The dif- native structures is shown in Fig. 4A. A different ference of 9 between these complexes may be corre- conformation is only visible for a few N-terminal resi- lated with the variation of the rate of binding and dues. Additionally, a small difference between native affinity for chagasin of these enzymes. On the other and complexed chagasin is observed at loop L4, hand, in the two crystal forms of the chagasin–cathep- which is rich in Gly residues, where a conformational sin B complex, the difference is 5 , showing that there change is responsible for adjustment of the inhibitor is some degree of variability in inhibitor–enzyme dock- to the enzyme in the b-sheet-forming motif. The ing, resulting either from inherent freedom of move- C-terminus has a relatively stable conformation, ment or adaptability to environmental factors, such as although the last two residues protrude from the pro- crystal packing interactions. tein surface. The C-terminal end of chagasin is a The rms deviations for the four enzyme-bound good marker of the variable angle of approach of the chagasin molecules are 0.34–0.61 A˚, a range that is inhibitor relative to the enzyme, as illustrated in similar to that for comparisons of the two crystal Fig. 4B.
FEBS Journal (2008) ª 2008 The Authors Journal compilation ª 2008 FEBS 9 Chagasin–papain complex structure I. Redzynia et al.
Considering the stability of the chagasin structure, tion profile of chagasin. The crucial aromatic W ⁄ F the similarity or dissimilarity of its complexes with residue at position 93 of chagasin is conserved as various enzymes may be regarded as the result of W ⁄ H in cystatins. This residue interacts with the two factors: (a) the overall similarity of the enzy- aromatic cluster that is present in all cysteine prote- matic part, and (b) the variability of the angle of ases as an extension of the catalytic triad. The pro- approach of the inhibitor relative to the catalytic line residue at position 92 in chagasin, which is cleft of the enzyme. The latter factor may reflect not responsible for the shape of the L6 loop, is also con- so much the geometry of the catalytic site itself, served in cystatins. which is highly conserved, but rather the general The shape of loop L4 is very similar in all com- shape of the peripheral regions surrounding the plex structures. Conserved interactions formed by active site of the enzymes, which may guide the loop L4 include those of residues L64–A67, which inhibitor molecule during its docking. The data in participate in both the antiparallel intermolecular Table 3 show that the Ca traces of cathepsin L, b-sheet and hydrophobic contacts with Y67e and papain, falcipain and cruzipain have rms deviations P68e (Fig. 3B). in the range 0.79–1.18 A˚. Much higher deviations are Although papain, cathepsin L and cruzipain show observed for cathepsin B (1.28–1.42 A˚), in agreement moderate sequence identity (36–43%), the residues with the view that it is the most unique member of responsible for the interaction with chagasin in the cat- this group of enzymes. alytic groove are conserved. Chagasin thus utilizes a Although the complexes include a variety of enzyme few conserved residues in the active site cleft of C1 sequences and differ in the angle of inhibitor family enzymes to become a broadly-reactive inhibitor approach, they have a relatively similar shape; the rms with quite similar affinity for all these enzymes. From deviations for the entire complexes range from 0.87 A˚ a biological perspective, it appears that these residues for the chagasin–papain ⁄ chagasin–falcipain pair, to in C1 family enzymes have been conserved to allow 1.55 A˚ for the superposition of chagasin complexes binding by chagasin- or cystatin-type inhibitors, result- with cathepsins L and B. ing in a means by which the organism can regulate cysteine protease activity as required. Core structural elements explaining the broad inhibition profile of chagasin Enzyme-specific interactions of chagasin Chagasin displays a broadly-reactive inhibition profile, Chagasin also utilizes some enzyme-specific interac- and inhibits all the investigated C1 family proteases. tions, explaining why it binds more tightly to papain, This efficient binding is achieved despite some differ- cathepsin L and cruzipain than to cathepsin B and ences in the architecture of the active site clefts of the falcipain. Identification of these interactions is now enzymes, which are especially evident for cathepsin B possible based on structural and functional data. [21]. What are the principal elements utilized by chaga- Detailed comparison of the various complexes reveals sin that enable it to become such a broadly-reactive a few contacts, all positioned close to the consensus inhibitor? Correct identification of these core elements elements of the L4–L2–L6 loops, that are unique to would be useful for guiding the rational design of each of the papain, cathepsin L and cathepsin B efficient cysteine protease inhibitors. complexes (Fig. 3B). For papain, this is the contact In all the presented structures, the inhibitory loops of S21e, for cathepsin L the contacts of Y72e and creating the enzyme-binding epitope have the same E141e, and for cathepsin B the contacts of E194e architecture except for loop L6 in the C-terminal frag- and D224e with chagasin residues boxed in Fig. 3B. ment from the chagasin–papain complex, which adopts The role of the conserved residues indicated by our a slightly different conformation in comparison with structural data is consistent with published mutagene- the other structures. The different shape of this loop is sis studies [33]. The identified characteristic interac- caused by formation of a hydrogen bond between the tions appear promising for use when designing side chain of D99 and the main-chain N atom of S21e specific inhibitors to a particular enzyme. A struc- (Fig. 2B). ture-based sequence alignment of the residues from Residue R91 of loop L6 forms important hydrogen the catalytic groove of cysteine proteases that inter- bonds in all chagasin complexes, both with the N ⁄ D act with chagasin is shown in Fig. 3B. These residues residue at position 18e (papain numbering) and the are also preserved in cruzipain, which justifies our G ⁄ K residue at position 20e (Fig. 3B), and is an suggestion that these interactions are also maintained important core elements explaining the broad inhibi- in a chagasin–cruzipain complex.
10 FEBS Journal (2008) ª 2008 The Authors Journal compilation ª 2008 FEBS I. Redzynia et al. Chagasin–papain complex structure
papain providing an illustrative example. A compari- Interaction of chagasin with cruzipain son of the present chagasin–papain complex with the The crystal structure of the possibly most physiologi- structure of papain in complex with the human inhibi- cally relevant chagasin complex, that with the T. cruzi tor cystatin B reveals that, despite their entirely differ- enzyme cruzipain, has not yet been presented. How- ent folds, the two inhibitors utilize very similar atomic ever, the available models can be used to predict the interactions for papain binding, leading to essentially interactions of the parasite protease–inhibitor pair. The identical affinities for the enzyme. As papain shows a rms deviations for the superimposed known structures high structural similarity among the C1 family prote- are presented in Table 3. Additionally, Table 3 com- ases to the catalytic domain of the T. cruzi enzyme cru- pares the inhibitor part of each complex with unbound zipain, the present chagasin–papain complex structure chagasin (PDB code 2NNR) [20] and cruzipain (PDB provides a reliable starting point for a model of the code 1ME3) [34] with the enzyme portions of the com- interactions of the two T. cruzi proteins, which, plexes. A graphical illustration of the aligned complexes together with our identification of the specificity-con- based on enzyme superposition is presented in Fig. 4B. ferring interactions, will be important for development Three recently published studies [18,20,21] have of drugs for the prevention and treatment of Chagas attempted to model the interactions between chagasin disease. and cruzipain so as to elucidate the binding interactions utilized in order to achieve efficient Experimental procedures enzyme inhibition. These previous conclusions are corroborated by superposition of the cruzipain model Expression, purification and characterization of on the papain portion of the complex described in the recombinant chagasin present study. Chagasin was produced using a glutathione S-transferase gene fusion protein system with the vector pGEX-6P-1 Conclusions (Amersham Biosciences, Uppsala, Sweden), as described The chagasin–papain complex presented here is the previously [20]. Following purification to homogeneity, the third high-resolution structure for the T. cruzi inhibitor chagasin solution was concentrated using a Vivaspin col- chagasin in a cysteine protease complex, completing a umn with a cut-off limit of 5 kDa (Vivascience, Lincoln, )1 series that started with the human C1 family enzymes UK) to a final concentration of 5 mgÆmL . The protein cathepsins L and B [20,21]. Together with detailed was characterized as correctly expressed GLPGS-chagasin functional studies of chagasin binding to the three by mass spectrometry, N-terminal protein sequencing and target enzymes, the results provide a platform for a electrophoretic analyses [20]. thorough understanding of the basic elements required for efficient inhibition by this parasite protease inhibi- Preparation of papain tor and explain its broadly-reactive properties. The Active papain was prepared from the commercial papaya core determinants of the broad reactivity of chagasin latex enzyme (Sigma-Aldrich, St Louis, MO, USA) through appear to be based on a rigid protein structure, opti- affinity purification on Sepharose 4B (GE Healthcare Bio- mally presenting an epitope formed by three loops, L4, Sciences AB, Uppsala, Sweden), to which Gly-Gly-Tyr-Arg L2 and L6, which precisely match their interaction had been covalently coupled, as described previously [36,37]. partners in the substrate-binding cleft of the C1 family Purified this way, the enzyme could be activated to at least enzymes. Smaller adaptations, connected with adjust- 65% of its original activity after storage at )80 C for up to ment of the angle of chagasin approach during enzyme 3 months. docking, optimize the interactions of loops L4 and L6 at the periphery of the enzyme-binding epitope with the distal elements in the enzyme’s active-site clefts Protein analyses ˚2 [21,35], to create a large contact area (922 A ), result- Protein concentration was estimated using a Coomassie ing in sub-nanomolar Ki values and rate constants for protein assay (Pierce Biotechnology Inc., Rockford, IL, 5 6 )1 )1 inactivation in the 10 –10 m Æs range. A few unique USA). N-terminal sequencing was performed after electro- interatomic interactions in each complex could phoresis in agarose gels, blotting to a poly(vinylidene diflu- additionally be identified and are interpreted as further oride) membrane (Millipore, Bedford, MA, USA) and improving the binding efficiency to papain and cathep- staining with 0.05% Coomassie blue. Edman degradation sin L versus the other enzymes, with the hydrogen was performed using 470A gas–liquid solid-phase seq- bond between D99 in loop L6 of chagasin and S21e of uencer (Applied Biosystems, Foster City, CA, USA) at the
FEBS Journal (2008) ª 2008 The Authors Journal compilation ª 2008 FEBS 11 Chagasin–papain complex structure I. Redzynia et al.
Department of Clinical Chemistry, Malmo¨University Hos- X-ray data collection and analysis pital, Sweden. MALDI-TOF mass spectrometry analysis using a Reflex III mass spectrometer (Bruker Daltonics X-ray diffraction experiments were performed at 100 K Inc., Billerica, MA, USA) was used to verify the correct using the X13 EMBL beamline of the DESY synchrotron mass of recombinant chagasin as described previously [38]. (Hamburg, Germany). Diffraction data extending to 1.86 A˚ resolution were indexed, integrated and scaled using Denzo and Scalepack from the HKL program package [41]. Addi- Enzyme inhibition assays tionally, Rpim [42] was calculated using SCALA [43]. Active-site titration of papain [with E-64 (l-trans-epoxy- Table 2 shows the data collection and processing statistics. succinyl-leucylamido-(4-guanidino)butane), using Bz-dl- Arg-NHPhNO (a-N-benzoyl-dl-arginine p-nitroanilide) as 2 Structure solution and refinement substrate; Bachem Feinchemikalien, Bubendorf, Switzerland] and titration of the molar papain-inhibitory concentration The structure of the complex was solved by molecular of chagasin were accomplished as described previously for replacement. An initial molecular-replacement solution was cystatin analysis [39]. Active inhibitor concentrations deter- obtained using the chagasin–cathepsin L complex (PDB mined in this way were used for calculation of the Ki values. code 2NQD) as the search model in MolRep [44]. The The fluorogenic substrate used for determination of equilib- complete model of the chagasin–papain complex was rium constants for dissociation (Ki) of complexes between obtained by superposition of papain (PDB code 2KHQ) on chagasin and papain or other family C1 cysteine peptidases the oriented cathepsin L portion as the target. Manual was carboxybenzoyl-Phe-Arg-7-(4-methyl)coumarylamide model rebuilding was subsequently performed using coot (10 mm; Bachem) and the assay medium was 100 mm sodium [45]. refmac5 [46] was used for structure refinement with phosphate buffer, pH 6.0, containing 1 mm dithiothreitol TLS (translation, libration, screw motion – parameters and 1 mm EDTA. Steady-state velocities were measured describing anisotropic motion of rigid body molecules) [47] before and after addition of varying amounts of chagasin, parameters defined separately for papain and chagasin. and the Ki values were calculated as described previously Water molecules were introduced manually using coot [45]. [40]. Corrections for substrate competition were made using Rfree [48] was monitored using a randomly chosen subset of Km values determined for the substrate batch used, under the reflections comprising 5% of the unique data set. Side assay conditions employed (60, 55, 3.2 and 1.9 lm for chains of a number of residues were modeled in two con- papain, cathepsin B, cathepsin L and cruzipain, respec- formations. The quality of the final structure was assessed tively). To determine the association rate constant for the using procheck [49] and molprobity [50]. The final refine- chagasin–papain interaction, the pseudo-first-order rate ment statistics are shown in Table 2. All crystallographic constants (kobs) in continuous-rate assays with various con- calculations were performed using the CCP4 suite of pro- centrations of chagasin were determined by non-linear grams [51]. Molecular illustrations were prepared using regression. The association rate constant (kon) was then pymol [52]. calculated from the slope of a plot of kobs versus inhibitor concentration. Acknowledgements This work was supported in part by grant N-N404- Crystallization 2348-33 from the Polish Ministry of Science and All crystallization experiments were performed at 18 C Higher Education to G. B. The work in Lund was sup- using the hanging-drop vapor diffusion method and Crystal ported by grants to M. A. from the Crafoord and Screens I and II and PEG ⁄ Ion Screen from Hampton A. O¨sterlund Foundations, the Swedish Research Research (Aliso Viejo, CA, USA). The crystals of the Council (project number 09915), the Swedish Cancer chagasin–papain complex used for diffraction experiments Society (project number 07 0030) and the Research )1 were grown by mixing chagasin at 4 mgÆmL concentra- School in Pharmaceutical Chemistry (‘FLA¨K’), Lund )1 tion with papain at 0.7 mgÆmL concentration in a 1.3 : 1 University, Sweden. This paper is dedicated to Profes- molar ratio. The complex was incubated for approximately sor Zofia Kosturkiewicz on the occasion of her 80th 4 h. Drops from a 1.5 lL protein solution and 1 lL of pre- birthday. cipitant solution containing 2.0 m ammonium formate and 0.1 m Hepes, pH 7.5, were mixed. Single crystals of the complex in the form of thin square plates reached maxi- References mum dimensions of 0.16 · 0.16 · 0.01 mm in approxi- 1 Lowe G & Williams A (1965) Direct evidence for an mately 30 days. 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12 FEBS Journal (2008) ª 2008 The Authors Journal compilation ª 2008 FEBS I. Redzynia et al. Chagasin–papain complex structure
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