Báo cáo khoa học: 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 pptx

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 Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Technical University of Lodz, Poland Department of Laboratory Medicine, Division of Clinical Chemistry and Pharmacology, Lund University, Sweden Department of Crystallography, Faculty of Chemistry, A Mickiewicz University, Poznan, Poland Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland Keywords Chagas disease; cruzipain; cysteine proteases; papain; protein inhibitors Correspondence G Bujacz, Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Technical University of Lodz, ul Stefanowskiego 4/10, 90-924 Lodz, Poland Fax: +48 42 636 66 18 Tel: +48 42 631 34 31 E-mail: gdbujacz@p.lodz.pl M Abrahamson, Department of Laboratory Medicine, Division of Clinical Chemistry and Pharmacology, Lund University, University Hospital, SE-221 85 Lund, Sweden Fax: +46 46 130064 Tel: +46 46 173445 E-mail: magnus.abrahamson@med.lu.se *These authors contributed equally to this paper Database Atomic coordinates, together with structure factors, have been deposited in the Protein Data Bank under the accession code 3E1Z A complex of chagasin, a protein inhibitor from Trypanosoma cruzi, and papain, a classic family C1 cysteine protease, has been crystallized Kinetic studies revealed that inactivation of papain by chagasin is very fast (kon = 1.5 · 106 m)1Ỉs)1), and results in the formation of a very tight, reversible complex (Ki = 36 pm), with similar or better rate and equilibrium constants than those for cathepsins L and B The high-resolution crystal structure shows an inhibitory wedge comprising three loops, which forms a number of contacts responsible for the high-affinity binding Comparison with the structure of papain in complex with human cystatin B reveals that, despite entirely different folding, the two inhibitors utilize very similar atomic interactions, leading to essentially identical affinities for the enzyme Comparisons of the chagasin–papain complex with high-resolution structures of chagasin in complexes with cathepsin L, cathepsin B and falcipain allowed the creation of a consensus map of the structural features that are important for efficient inhibition of papain-like enzymes The comparisons also revealed a number of unique interactions that can be used to design enzyme-specific inhibitors As papain exhibits high structural similarity to the catalytic domain of the T cruzi enzyme cruzipain, the present chagasin–papain complex provides a reliable model of chagasin–cruzipain interactions Such information, coupled with our identification of specificity-conferring interactions, should be important for the development of drugs for treatment of the devastating Chagas disease caused by this parasite (Received 13 October 2008, revised 15 November 2008, accepted December 2008) doi:10.1111/j.1742-4658.2008.06824.x Papain (EC 3.4.22.2) from the latex of the papaya fruit (Carica papaya) was one of the first known proteolytic enzymes, and its digestive properties were already being utilized in the 19th century Detailed biochemical studies in the 20th century peaked with efforts in the 1960s, defining the chemistry of the enzymatic mechanism, delineating the concept of specificity for protein substrate recognition [1–3], and with elucidation of the FEBS Journal 276 (2009) 793–806 ª 2009 The Authors Journal compilation ª 2009 FEBS 793 Chagasin–papain complex structure I Redzynia et al crystal structure of the enzyme, one of the first protein structures to be determined [4] Since then, papain has been used as a model protein in many studies, and is the founding member of the large C1 family of papain-like cysteine proteases [5] Approximately 12 mammalian cysteine proteases are evolutionarily closely related to papain and hence belong to this family (e.g cathepsins B, H, L, S and K) Enzymes from the C1 family generally function in every cell as components of the lysosomal degradation system, participating in the turnover of proteins, but, in addition, have been shown to participate in a number of specialized functions, such as proteolytic cleavages activating prohormones, regulation of antigen presentation, etc C1 family proteases are evolutionarily old, are found in both prokaryotic and eukaryotic organisms, and in many cases show activity that is indispensable for the organism The unicellular parasite Trypanosoma cruzi is an example of such an organism, in which the papain-like enzyme, cruzipain, is essential for the lifecycle of the parasite and also acts as a virulence factor when the parasite infects its human host, causing the devastating Chagas disease [6,7] In a variety of species, from mammals, plants and insects to simpler eukaryotes such as the filarial parasites Onchocerca volvulus and Acanthocheilonema viteae, C1 family cysteine proteases are in equilibrium with protein inhibitors belonging to the cystatin family, I25 [5,8–10] Most cystatins, such as human cystatin B, are single-domain proteins of 100–120 residues with a characteristic wedge-like epitope consisting of the N-terminus and two b-hairpin loops, which blocks the active site cleft of the target enzyme, thereby inhibiting the activity in a reversible manner [11,12] Cystatins show high affinity for their target enzymes due to a large binding area, with dissociation constants (Ki) in the range 10)9–10)11 m In extreme cases, such as the human cystatin C–papain complex, Ki values as low as 10)14 m have been reported [13] Trypanosomatids, such as various Trypanosoma and Leishmania species, produce inhibitors of their own family C1 proteases [14] Chagasin, a tight-binding inhibitor of cruzipain found in T cruzi [15], exhibits no sequence similarity with cystatins (GenBank ⁄ EMBL [16] accession number AJ299433), despite its similar size (110 residues) Molecular modeling studies predicted an immunoglobulin-like fold for chagasin [17], which was essentially confirmed by subsequent NMR [18] and crystallographic studies [19,20] Recently, crystal structures of chagasin in complex with human cathepsins L and B [20,21], and additionally with falcipain from the malaria parasite [22], have been determined The complex structures demonstrate that the enzyme794 binding epitope of chagasin consists of three loops (L4, L2, L6) that together form a wedge-like enzymebinding epitope In this study, we present a high-quality crystal structure of chagasin in complex with papain, the model C1 family cysteine protease and one of only two enzymes in the family for which structural information for a cystatin complex is available [23,24] Based on the amino acid sequence and structure-based alignment, papain has been shown to be a close homolog of cruzipain [25] Our results confirm mapping of the enzyme-binding epitope to the three loops, as in chagasin complexes with mammalian enzymes, and illustrate the degree of structural adjustments as well as precise atomic contacts formed during enzyme binding Moreover, comparative analysis of several chagasin complexes has revealed a strikingly similar core structure involved in enzyme binding, which results in sub-nanomolar Ki values and rate constants for inactivation in the 105–106 m)1Ỉs)1 range in all cases Additionally, several contacts unique to the individual enzyme complexes could be identified, raising the prospect of accurate structure-aided design of specific inhibitors of cruzipain and cathepsins Detailed knowledge of the structure and inhibition mode of chagasin should be valuable in guiding the development of drugs for the prevention and treatment of Chagas disease Results Function of chagasin as an inhibitor of papain Chagasin used in this study was expressed in Escherichia coli and purified to homogeneity as reported previously [20] The recombinant protein contains five extra N-terminal amino acid residues from the expression construct, and has a mass of 12 440 Da as expected [20] The protein shows almost 100% activity as a protease inhibitor based on titration of a papain solution with known activity, forms stoichiometric : complexes with cathepsin L or B, and is not cleaved by these proteases [20,21] Kinetic parameters for the interaction of chagasin with papain at pH 6.0 were determined in a continuousrate assay using the sensitive fluorogenic substrate carboxybenzoyl-Phe-Arg-7-(4-methyl)coumarylamide, with a sufficiently high inhibitor concentration for the binding reaction to be of pseudo-first order The kon value was determined to be 1.5 · 106 m)1Ỉs)1, very similar to that determined for cathepsin L and higher than that for cathepsin B under the same conditions (Table 1) The equilibrium constant for dissociation FEBS Journal 276 (2009) 793–806 ª 2009 The Authors Journal compilation ª 2009 FEBS I Redzynia et al Chagasin–papain complex structure Table Function of chagasin as an inhibitor of papain and other C1 family enzymes Equilibrium constants for dissociation (Ki) of chagasin–papain complexes were determined under steady-state conditions at pH 6.0 as described in Experimental procedures Corresponding values for the papain-like cysteine proteases cathepsin L, cathepsin B and falcipain, with known inhibitor complex structures [20–22], as well as for the papain complex with human cystatin B [12], are included for comparison The Ki values presented were corrected for substrate competition in the assays, as described in Experimental procedures ND, not determined kon (M)1Ỉs)1) Ki (nM) Enzyme Chagasin Cystatin B Chagasin Cathepsin L Cathepsin B H110A cathepsin B Papain Falcipain Cruzipain 0.039 0.93 0.35 0.036 1.7a 0.018b ND 16 ND 0.034 ND ND 2.5 · 8· 5· 1.5 · ND ND 106 104 105 106 a Determined under slightly different assay conditions than in the present study [22] b Determined for a recombinant variant of chagasin with a 16 residue N-terminal extension [15] (Ki) of the chagasin–papain complex was calculated from the results of similar assays, under conditions when steady-state enzyme rates could be determined before and after addition of chagasin to a specific concentration The Ki value for the papain–chagasin complex, corrected for substrate competition in the assays, was estimated as 36 pm, again similar to that of cathepsin L [20] and significantly lower than the values for wild-type cathepsin B or for a cathepsin B variant with an H110A substitution in the occluding loop, for which the structure of its chagasin complex is known [21] (Table 1) Crystallization and structure determination A complex between chagasin and papain was formed by incubating the proteins in a 1.3 : molar ratio for approximately h before setting up crystallization drops Single crystals of the chagasin–papain complex were obtained using Crystal Screen II in Hepes buffer at pH 7.5 without further optimization The crystal ˚ structure of the complex was solved to 1.86 A resolution by molecular replacement using the chagasin– cathepsin L model (PDB code 2NQD) [20] as a probe The initial atomic coordinates of the chagasin–papain complex were obtained by rigid-body substitution of cathepsin L by a papain model (PDB code 1KHQ) [26] After least-squares refinement, the main-chain traces of the chagasin and papain molecules were visible in 2Fo–Fc electron density maps without breaks at Table Data collection and structure refinement statistics Data collection Radiation source ˚ Wavelength (A) Temperature of measurements (K) Space group ˚ Cell parameters (A) ˚ Resolution range (A) Reflections collected Unique reflections Completeness (%) Redundancy ⁄ Rintb Rpimc Refinement Number of reflections in the working ⁄ test sets Rd ⁄ Rfree (%) Number of atoms (protein ⁄ solvent ⁄ Zn ⁄ other) rms deviations from ideal ˚ Bond lengths (A) Bond angles (°) ˚ (A2) Residues in Ramachandran plot (%) Most favored regions Allowed regions PDB code X13, EMBL Hamburg 0.8086 100 I422 a = 99.1, c = 159.5 60.0–1.86 (1.93–1.86)a 350 034 33 263 98.4 (88.8) 10.5 (6.8) 22.2 (2.1) 0.090 (0.557) 0.028 (0.191) 31 568 ⁄ 1694 16.4 ⁄ 20.8 2561 ⁄ 298 ⁄ ⁄ 30 0.017 1.61 27.6 89.7 (98.1)e 10.3 3E1Z a Values in parentheses correspond to the last resolution shell P P P P Rint = h j | Ihj)| ⁄ h j Ihj, where Ihj is the intensity of P P observation j of reflection h c Rpim = h (1 ⁄ nh)1) j |Ihj)| ⁄ P P [42], calculated using SCALA [43] (from data h j P P processed using Denzo) d R = h | | Fo|)|Fc| | ⁄ for h |Fo| all reflections, where Fo and Fc are observed and calculated structure factors, respectively Rfree is calculated analogously for the test reflections, randomly selected and excluded from the refinement e Ramachandran ‘favored’ region, as defined by MolProbity [50] b the 1.7 r level, except for the N- and C-termini of the chagasin molecule All side chains, as well as both terminal segments, are clearly visible when the electron density maps are contoured at the 1.0 r level The GPLGS peptide introduced as an N-terminal extension of the recombinant chagasin is totally disordered and not visible in the electron density maps In addition to 298 water molecules, the model includes 10 formate ions from the crystallization buffer The refinement statistics are presented in Table The residues of the inhibitor are labeled without a chain designator The residues of the enzyme are marked ‘e’ When cystatin sequences are discussed in this paper, amino acid numbering according to the chicken cystatin sequence is used, as in the original papain–cystatin B structure [23] To convert to human cystatin C numbering, FEBS Journal 276 (2009) 793–806 ª 2009 The Authors Journal compilation ª 2009 FEBS 795 Chagasin–papain complex structure I Redzynia et al which is widely used, ‘2’ should be added to all residue numbers, so that G9 in cystatin B corresponds to G11 in cystatin C [27] A strong residual peak in the Fo–Fc electron density map, in close proximity to H72, H74, E23 and one of the formate ions, was interpreted as a zinc cation This interpretation is supported by the bond valence test [28,29] and by the tetrahedral coordination of this cation Although chagasin inhibition is not dependent on any cofactors, this site at the surface of the molecule may be of structural significance, as the same histidine residues in the cathepsin B complex were found to bind a phosphate ion [21] The chagasin–papain interface The papain chain in the present complex starts with residue I1e, which is well defined in the electron density map The last residue, N212e, is also clearly visible because the side chain is stabilized by hydrogen bonds with D108e and I148e, and the C-terminal carboxylate forms a salt bridge with R188e, the latter two interactions involving a symmetry-related molecule The enzyme used for crystallization was in an inactive form, with the catalytic C25e residue protected by carboxymethylation The blocking group is clearly visible in the electron density maps The overall conformation of the chagasin molecule in the present complex is similar to that found for free chagasin (PDB code 2NNR) [20] The C- and N-terminal residues of chagasin are somewhat flexible, but the contour level of r for the 2Fo–Fc electron density maps was sufficient for unambiguous modeling The first visible residue at the N-terminus is S2, which is anchored by a side-chain hydrogen bond to N64e from a symmetry-related molecule The C-terminal N110 residue points to a water channel In overall shape, the present complex is similar to the previously described complex structures of chagasin with cathepsins L and B [20,21], resembling an inverted mushroom, with the stalk formed by the cylindrical chagasin molecule and the cap by the globular papain (Fig 1) The C25e-H159e-N175e catalytic triad of papain is located at the bottom of a long cleft running across the width of the molecule, dividing it into the L and R domains [30] The binding region of chagasin formed by the loops L2 (N29–F34), L4 (P59–G68) and L6 (R91–S100) is docked very tightly to the papain molecule (Fig 2A) 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 310 tip of loop L2 inserted directly into the catalytic center Loops L4 and L6 embrace the enzyme molecule from both sides The interactions of each loop have different characteristics Loop L6 forms three types of interactions with the enzyme: hydrogen bonds (R91), hydrophobic contacts (P92) and p interactions (W93), which ‘probe’ different elements of the catalytic apparatus First, W93 interacts with a cluster of aromatic residues (F141e, W177e, W181e) that serve to position the N175e element of the catalytic triad (C25e-H159eN175e) through N-H p hydrogen bonds R91 assumes a fully extended conformation reaching to the catalytic site of the enzyme and loop L2 of chagasin The R91 guanidinium group forms two hydrogen bonds with the carbonyl group of T32 in loop L2, which is located next to the active-site-blocking residue, T31 [20,21] The other segment of the guanidinium group of R91 forms a pair of hydrogen bonds with the oxygen atom of the side-chain amide group of N18e It is interesting to note that the equivalent position in cruzipain is occupied by an aspartate, making the interaction with Fig 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 796 FEBS Journal 276 (2009) 793–806 ª 2009 The Authors Journal compilation ª 2009 FEBS I Redzynia et al Chagasin–papain complex structure B A C D Fig 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 cystatin 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 R91 is also hydrogen-bonded to the carbonyl group of G20e The third element of L6, P92, shapes the loop for optimal interactions with the enzyme by forming hydrophobic contacts with the side chain of L143e (Fig 2B) In addition to the direct interactions of loop L6 described above, there are also contacts mediated by water molecules The interactions of loop L4 with the enzyme are based on formation of an antiparallel intermolecular b-sheet Two residues from chagasin, G66 and L65, interact with the papain main-chain atoms N64e–G66e (Fig 2C) In addition, the side-chain carbonyl Od1 atom of N64e forms a water-mediated contact with the main-chain N atom of G68, and the main-chain nitrogen of G66 of chagasin forms a water-mediated FEBS Journal 276 (2009) 793–806 ª 2009 The Authors Journal compilation ª 2009 FEBS 797 Chagasin–papain complex structure I Redzynia et al contact with the main-chain carbonyl group of D158e from papain Compared to the very strong and extended interactions of loops L4 and L6, the interactions of loop L2 are very limited A repulsive contact is seen between the carbonyl O atom of T31 and the Nd1 atom of the imidazole ring of the catalytic H159e residue A much longer, attractive contact exists between the same T31 carbonyl and the Ne1 atom of W177e (Table S1) The hydroxyl group of T31 interacts with the main-chain carbonyl of D158e (Fig 2D) The four additional atoms of the carboxymethyl modification of the catalytic C25e residue are easily accommodated at the inhibitor–enzyme interface The oxygen atoms of the carboxymethyl block form contacts with both the enzyme (main-chain N of C25e and side chain of Q19e) and the inhibitor (OH group of T31) The inhibition mode of chagasin The best-studied group of cysteine protease inhibitors are the cystatins, which are small proteins with a molecular mass of 11–14 kDa [27] The structure of papain in complex with cystatin B (PDB code 1STF) [23] offers an excellent opportunity for comparison of the mode of interaction of the two very different inhibitors with the same target enzyme Although chagasin and cystatin B show essentially identical affinity for papain (Table 1), superposition of the two complexes based on Ca alignment [31] of the enzyme portions shows a completely different fold for the two inhibitors (Fig 2A) The characteristic b-sheet grip around a long a-helix, characteristic of cystatins, contrasts with the all-b structure of chagasin However, despite their different overall fold, the epitope presented by both inhibitors to the enzyme is arranged similarly The L4–L2–L6 wedge of chagasin overlaps with a similar wedge of cystatin B formed by 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 approach the enzyme from a different angle We have defined the angle of approach, s (Table 3), as the dihedral angle between two planes, one (a) dividing the Table 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 crystallographic 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 A2) 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 Cruzipain Chagasin A B Chagasin–cathepsin B Form I 0.44 (98) 0.37 (98) Chagasin–cathepsin L Chagasin–papain Chagasin–falcipain 1.46 (197) [27.8%] 0.81 (190) [43.4%] 1.07 (191) [35.8%] 1.10 (192) [37.7%] 0.55 (101) 0.54 (105) 0.48 (100) 0.52 (103) c 1.16 (348) e 0.54 (233) ch 0.46 (100) 1.38 (198) Form II c 1.25 (301) e 1.28 (198) ch 0.43 (102) c 1.55 (300) e 1.31 (196) ch 0.55 (107) [28.2%] Form II Chagasin–cathepsin L 0.44 (92) 0.35 (91) c 1.15 (287) e 1.32 (192) ch 0.43 (96) c 1.10 (278) e 1.42 (191) ch 0.42 (101) [29.7%] c 1.18 (297) e 0.79 (188) ch 0.61 (101) [40.6%] Chagasin–papain Contact area Angle of approach s 798 1221 7.2 1373 2.3 972 11.3 922 5.8 0.38 (99) 0.37 (102) c 1.15 (294) e 1.30 (193) ch 0.37 (101) c 1.19 (293) e 1.35 (190) ch 0.52 (107) [24.1%] c 1.19 (300) e 1.00 (189) ch 0.44 (105) [35.9%] c 0.87 (274) e 1.18 (187) ch 0.34 (93) [37.7%] 984 5.4 FEBS Journal 276 (2009) 793–806 ª 2009 The Authors Journal compilation ª 2009 FEBS I Redzynia et al enzyme into the R and L lobes along the catalytic groove, defined by the Ca atoms of three papain residues, I40e, Y67e and W177e (or their equivalents in other enzymes), and the other (b) created by three Ca atoms defining the inhibitor framework and passing along the inhibitory wedge In the case of chagasin, plane (b) is triangulated by the tips of the peripheral loops L4, L6 and the C-terminus, or specifically by the Ca atoms of G66, R91 and A109 The corresponding Ca atoms of cystatin B are located in residues G9 (in the N-terminal binding segment, according to chicken cystatin numbering [23]), D68 (a loop from the opposite pole) and L102 (loop L2) The s angle in the chagasin–papain complex is 5.8°, indicating that the chagasin molecule is slanting towards the R domain The angle of approach of cystatin B is )12.7°, and the inhibitor molecule is inclined towards the L domain of the enzyme The difference in the angles of approach between chagasin and cystatin B is 18.5° It is also of note that the sequential epitope of cystatins corresponds to a non-sequential binding site of chagasin The contact area [32] is similar for both complexes, ˚ and is 853 and 922 A2 for the cystatin B–papain and chagasin–papain complexes, respectively The three crucial residues of loop L6 of chagasin (R91, P92 and W93) correspond to L102, P103 and H104, respectively, in the cystatin B molecule (Fig 3A) It is noteworthy that the pattern Pro–aromatic residue is conserved in chagasin-like inhibitors and in cystatins (where it is predominantly PW), despite the lack of overall sequence similarity The role of the proline residue appears to be to maintain the specific shape of the loop The aromatic residue, on the other hand, interacts with the aromatic cluster of the enzyme (Fig 2B) The residue preceding the Pro–aromatic motif, which is invariably an arginine in chagasin-type inhibitors of protozoan origin, is replaced by an aliphatic residue in cystatins (Fig 3A) This difference may be an important element regulating the enzyme specificity of these two groups of inhibitors The R91 residue of chagasin provides direct communication between loops L6 and L2, and also interacts with the crucial D18e ⁄ N18e residues of cruzipain ⁄ papain The role of the L102 residue of cystatin B is different, and supports interaction with the aromatic cluster of the enzyme (Fig 2B) An additional interaction between loops L2 and L6 of chagasin is provided by the carbonyl group of the main chain of M90 and the nitrogen atom of A35 A similar stabilizing contact between cystatin B loops L2 and L1 is formed by the mainchain carbonyl of Q101 and the peptide nitrogen atom of T58 Chagasin–papain complex structure The interaction of loop L4 of chagasin with papain is based on formation of an intermolecular b-sheet (Fig 2C) There is an analogous interaction between the N-terminus of cystatin B and papain G9 from the N-terminal cystatin B segment and G66 from loop L4 of chagasin provide a degree of flexibility, thus allowing optimal interactions between the two main chains The same role is played by G65e of papain The short antiparallel b-sheet interaction is formed by only one residue, G66e, of papain with L65 or S66 of chagasin or cystatin B, respectively This antiparallel interaction is supported by a water molecule linking the N atom of G66 of chagasin and the main-chain O atom of D158e of papain In the cystatin B complex, an equivalent carbonyl is involved in a water-mediated interaction with the N atom of A10 The L2 loop of chagasin and the corresponding loop L1 of cystatin B interact with the catalytic center of papain (Fig 2D) Our structural alignment (Fig 3A) shows that loop L2 of chagasin is one residue longer, and thus T31 has no equivalent in loop L1 of cystatin B Loop L2 of chagasin not only interacts with loop L6 but also with loop L4, by forming a hydrogen bond between the side-chain amide of N29 and mainchain carbonyl of G66 A similar interaction is observed in cystatin B, where the side-chain amide of Q53 forms a hydrogen bond with the main-chain carbonyl of G9, stabilizing the interaction between loop L1 and the N-terminus Although the conformation of these two loops is somewhat different, in both cases they have the same, substrate-like, polarity There is a surprisingly small number of specific interactions with the catalytic residues of the enzyme for both chagasin loop L2 and cystatin loop L1, which explains why chagasin (and also cystatins) can bind with high affinity to cysteine proteases with the catalytic -SH group protected by a carboxymethyl group The repulsive interactions between the chagasin loop L2 and the catalytic site of papain, described above, are reproduced in the cystatin complex Discussion Comparison of the existing structures of chagasin complexes with cysteine proteases In addition to the chagasin–papain complex presented in this paper, four additional crystal structures of chagasin complexes with other cysteine proteases are available in the Protein Data Bank The target enzymes for chagasin in these complexes are cathepsin L (PDB code 2NQD) [20], cathepsin B in two crystal forms (PDB codes 3CBJ and 3CBK) [21] and FEBS Journal 276 (2009) 793–806 ª 2009 The Authors Journal compilation ª 2009 FEBS 799 Chagasin–papain complex structure I Redzynia et al A B Fig Structure-based sequence alignment of the interacting residues of cysteine proteases 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 cathepsin L (X12451), H sapiens cathepsin B (BC010240), Plasmodium falciparum falcipain (AAF97809), T cruzi cruzipain (X54414) (B) Alignment of structurally equivalent residues 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 form an excellent platform for comparison of the interactions between chagasin and the targeted proteolytic enzymes The residues from the catalytic cleft of various cysteine proteases that interact with chagasin are structurally aligned (Fig 3B) The inhibitor binds papain and cathepsin L with essentially the same, very high, affinity (Ki approximately 0.03 nm); the affinity for cathepsin B is approximately one order of magni800 tude lower, and that for falcipain is yet another order of magnitude lower, although still in the nanomolar range (Table 1) The contact surface area for chagasin–cysteine pro˚ tease complexes varies between 922 and 1373 A2 (Table 3), and does not directly correlate with the efficiency of inhibition The extra contact area found in both crystal forms of the chagasin–cathepsin B complex is created by the additional and unique occluding FEBS Journal 276 (2009) 793–806 ª 2009 The Authors Journal compilation ª 2009 FEBS I Redzynia et al Chagasin–papain complex structure A L6 L6 L2 L2 Fig Stereoview of aligned chagasin complexes (A) Superposition of the Ca atoms of the chagasin molecules from the complexes 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 (midgreen) (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 darkgreen (chain A) and purple (chain B) L4 L4 B loop of this enzyme The inhibition of cathepsin B by chagasin is relatively weak, which may be due to the fact that some of the binding energy has to be invested in pushing the occluding loop out of the catalytic cleft The angle of approach, calculated in the way described above, has the lowest value for the tetragonal form of the chagasin–cathepsin B complex and the highest for the chagasin–cathepsin L complex (Table 3) The difference of 9° between these complexes may be correlated with the variation of the rate of binding and affinity for chagasin of these enzymes On the other hand, in the two crystal forms of the chagasin–cathepsin B complex, the difference is 5°, showing that there is some degree of variability in inhibitor–enzyme docking, resulting either from inherent freedom of movement or adaptability to environmental factors, such as crystal packing interactions The rms deviations for the four enzyme-bound ˚ chagasin molecules are 0.34–0.61 A, a range that is similar to that for comparisons of the two crystal ˚ structures of native chagasin (0.35–0.55 A) These results show that the chagasin molecule has a rigid conformation and does not change upon complex formation This contradicts the conclusions drawn from an NMR study that predicted a high level of flexibility of the chagasin molecule [18] A superposition of all the chagasin molecules from the complex and native structures is shown in Fig 4A A different conformation is only visible for a few N-terminal residues Additionally, a small difference between native and complexed chagasin is observed at loop L4, which is rich in Gly residues, where a conformational change is responsible for adjustment of the inhibitor to the enzyme in the b-sheet-forming motif The C-terminus has a relatively stable conformation, although the last two residues protrude from the protein surface The C-terminal end of chagasin is a good marker of the variable angle of approach of the inhibitor relative to the enzyme, as illustrated in Fig 4B FEBS Journal 276 (2009) 793–806 ª 2009 The Authors Journal compilation ª 2009 FEBS 801 Chagasin–papain complex structure I Redzynia et al Considering the stability of the chagasin structure, the similarity or dissimilarity of its complexes with various enzymes may be regarded as the result of two factors: (a) the overall similarity of the enzymatic part, and (b) the variability of the angle of approach of the inhibitor relative to the catalytic cleft of the enzyme The latter factor may reflect not so much the geometry of the catalytic site itself, which is highly conserved, but rather the general shape of the peripheral regions surrounding the active site of the enzymes, which may guide the inhibitor molecule during its docking The data in Table show that the Ca traces of cathepsin L, papain, falcipain and cruzipain have rms deviations ˚ in the range 0.79–1.18 A Much higher deviations are ˚ observed for cathepsin B (1.28–1.42 A), in agreement with the view that it is the most unique member of this group of enzymes Although the complexes include a variety of enzyme sequences and differ in the angle of inhibitor approach, they have a relatively similar shape; the rms ˚ deviations for the entire complexes range from 0.87 A for the chagasin–papain ⁄ chagasin–falcipain pair, to ˚ 1.55 A for the superposition of chagasin complexes with cathepsins L and B tion profile of chagasin The crucial aromatic W ⁄ F residue at position 93 of chagasin is conserved as W ⁄ H in cystatins This residue interacts with the aromatic cluster that is present in all cysteine proteases as an extension of the catalytic triad The proline residue at position 92 in chagasin, which is responsible for the shape of the L6 loop, is also conserved in cystatins The shape of loop L4 is very similar in all complex structures Conserved interactions formed by loop L4 include those of residues L64–A67, which participate in both the antiparallel intermolecular b-sheet and hydrophobic contacts with Y67e and P68e (Fig 3B) Although papain, cathepsin L and cruzipain show moderate sequence identity (36–43%), the residues responsible for the interaction with chagasin in the catalytic groove are conserved Chagasin thus utilizes a few conserved residues in the active site cleft of C1 family enzymes to become a broadly-reactive inhibitor with quite similar affinity for all these enzymes From a biological perspective, it appears that these residues in C1 family enzymes have been conserved to allow binding by chagasin- or cystatin-type inhibitors, resulting 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, and inhibits all the investigated C1 family proteases This efficient binding is achieved despite some differences in the architecture of the active site clefts of the enzymes, which are especially evident for cathepsin B [21] What are the principal elements utilized by chagasin that enable it to become such a broadly-reactive inhibitor? Correct identification of these core elements would be useful for guiding the rational design of efficient cysteine protease inhibitors In all the presented structures, the inhibitory loops creating the enzyme-binding epitope have the same architecture except for loop L6 in the C-terminal fragment from the chagasin–papain complex, which adopts a slightly different conformation in comparison with the other structures The different shape of this loop is caused by formation of a hydrogen bond between the side chain of D99 and the main-chain N atom of S21e (Fig 2B) Residue R91 of loop L6 forms important hydrogen bonds in all chagasin complexes, both with the N ⁄ D residue at position 18e (papain numbering) and the G ⁄ K residue at position 20e (Fig 3B), and is an important core elements explaining the broad inhibi- Chagasin also utilizes some enzyme-specific interactions, explaining why it binds more tightly to papain, cathepsin L and cruzipain than to cathepsin B and falcipain Identification of these interactions is now possible based on structural and functional data Detailed comparison of the various complexes reveals a few contacts, all positioned close to the consensus elements of the L4–L2–L6 loops, that are unique to each of the papain, cathepsin L and cathepsin B complexes (Fig 3B) For papain, this is the contact of S21e, for cathepsin L the contacts of Y72e and E141e, and for cathepsin B the contacts of E194e and D224e with chagasin residues boxed in Fig 3B The role of the conserved residues indicated by our structural data is consistent with published mutagenesis studies [33] The identified characteristic interactions appear promising for use when designing specific inhibitors to a particular enzyme A structure-based sequence alignment of the residues from the catalytic groove of cysteine proteases that interact with chagasin is shown in Fig 3B These residues are also preserved in cruzipain, which justifies our suggestion that these interactions are also maintained in a chagasin–cruzipain complex 802 FEBS Journal 276 (2009) 793–806 ª 2009 The Authors Journal compilation ª 2009 FEBS I Redzynia et al Interaction of chagasin with cruzipain The crystal structure of the possibly most physiologically relevant chagasin complex, that with the T cruzi enzyme cruzipain, has not yet been presented However, the available models can be used to predict the interactions of the parasite protease–inhibitor pair The rms deviations for the superimposed known structures are presented in Table Additionally, Table compares the inhibitor part of each complex with unbound chagasin (PDB code 2NNR) [20] and cruzipain (PDB code 1ME3) [34] with the enzyme portions of the complexes A graphical illustration of the aligned complexes based on enzyme superposition is presented in Fig 4B Three recently published studies [18,20,21] have attempted to model the interactions between chagasin and cruzipain so as to elucidate the binding interactions utilized in order to achieve efficient enzyme inhibition These previous conclusions are corroborated by superposition of the cruzipain model on the papain portion of the complex described in the present study Conclusions The chagasin–papain complex presented here is the third high-resolution structure for the T cruzi inhibitor chagasin in a cysteine protease complex, completing a series that started with the human C1 family enzymes cathepsins L and B [20,21] Together with detailed functional studies of chagasin binding to the three target enzymes, the results provide a platform for a thorough understanding of the basic elements required for efficient inhibition by this parasite protease inhibitor and explain its broadly-reactive properties The core determinants of the broad reactivity of chagasin appear to be based on a rigid protein structure, optimally presenting an epitope formed by three loops, L4, L2 and L6, which precisely match their interaction partners in the substrate-binding cleft of the C1 family enzymes Smaller adaptations, connected with adjustment of the angle of chagasin approach during enzyme 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 ˚ [21,35], to create a large contact area (922 A2), resulting in sub-nanomolar Ki values and rate constants for inactivation in the 105–106 m)1Ỉs)1 range A few unique interatomic interactions in each complex could additionally be identified and are interpreted as further improving the binding efficiency to papain and cathepsin L versus the other enzymes, with the hydrogen bond between D99 in loop L6 of chagasin and S21e of Chagasin–papain complex structure papain providing an illustrative example A comparison of the present chagasin–papain complex with the structure of papain in complex with the human inhibitor cystatin B reveals that, despite their entirely different folds, the two inhibitors utilize very similar atomic interactions for papain binding, leading to essentially identical affinities for the enzyme As papain shows a high structural similarity among the C1 family proteases to the catalytic domain of the T cruzi enzyme cruzipain, the present chagasin–papain complex structure provides a reliable starting point for a model of the interactions of the two T cruzi proteins, which, together with our identification of the specificity-conferring interactions, will be important for development of drugs for the prevention and treatment of Chagas disease Experimental procedures Expression, purification and characterization of recombinant chagasin Chagasin was produced using a glutathione S-transferase gene fusion protein system with the vector pGEX-6P-1 (Amersham Biosciences, Uppsala, Sweden), as described previously [20] Following purification to homogeneity, the chagasin solution was concentrated using a Vivaspin column with a cut-off limit of kDa (Vivascience, Lincoln, UK) to a final concentration of mgỈmL)1 The protein was characterized as correctly expressed GLPGS-chagasin by mass spectrometry, N-terminal protein sequencing and electrophoretic analyses [20] Preparation of papain Active papain was prepared from the commercial papaya latex enzyme (Sigma-Aldrich, St Louis, MO, USA) through affinity purification on Sepharose 4B (GE Healthcare BioSciences AB, Uppsala, Sweden), to which Gly-Gly-Tyr-Arg had been covalently coupled, as described previously [36,37] Purified this way, the enzyme could be activated to at least 65% of its original activity after storage at )80 °C for up to months Protein analyses Protein concentration was estimated using a Coomassie protein assay (Pierce Biotechnology Inc., Rockford, IL, USA) N-terminal sequencing was performed after electrophoresis in agarose gels, blotting to a poly(vinylidene difluoride) membrane (Millipore, Bedford, MA, USA) and staining with 0.05% Coomassie blue Edman degradation was performed using 470A gas–liquid solid-phase sequencer (Applied Biosystems, Foster City, CA, USA) at the FEBS Journal 276 (2009) 793–806 ª 2009 The Authors Journal compilation ª 2009 FEBS 803 Chagasin–papain complex structure I Redzynia et al Department of Clinical Chemistry, Malmo University Hosă pital, Sweden MALDI-TOF mass spectrometry analysis using a Reflex III mass spectrometer (Bruker Daltonics Inc., Billerica, MA, USA) was used to verify the correct mass of recombinant chagasin as described previously [38] Enzyme inhibition assays Active-site titration of papain [with E-64 (l-trans-epoxysuccinyl-leucylamido-(4-guanidino)butane), using Bz-dlArg-NHPhNO2(a-N-benzoyl-dl-arginine p-nitroanilide) as substrate; Bachem Feinchemikalien, Bubendorf, Switzerland] and titration of the molar papain-inhibitory concentration of chagasin were accomplished as described previously for cystatin analysis [39] Active inhibitor concentrations determined in this way were used for calculation of the Ki values The fluorogenic substrate used for determination of equilibrium constants for dissociation (Ki) of complexes between chagasin and papain or other family C1 cysteine peptidases was carboxybenzoyl-Phe-Arg-7-(4-methyl)coumarylamide (10 mm; Bachem) and the assay medium was 100 mm sodium phosphate buffer, pH 6.0, containing mm dithiothreitol and mm EDTA Steady-state velocities were measured before and after addition of varying amounts of chagasin, and the Ki values were calculated as described previously [40] Corrections for substrate competition were made using Km values determined for the substrate batch used, under the assay conditions employed (60, 55, 3.2 and 1.9 lm for papain, cathepsin B, cathepsin L and cruzipain, respectively) To determine the association rate constant for the chagasin–papain interaction, the pseudo-first-order rate constants (kobs) in continuous-rate assays with various concentrations of chagasin were determined by non-linear regression The association rate constant (kon) was then calculated from the slope of a plot of kobs versus inhibitor concentration Crystallization All crystallization experiments were performed at 18 °C using the hanging-drop vapor diffusion method and Crystal Screens I and II and PEG ⁄ Ion Screen from Hampton Research (Aliso Viejo, CA, USA) The crystals of the chagasin–papain complex used for diffraction experiments were grown by mixing chagasin at mgỈmL)1 concentration with papain at 0.7 mgỈmL)1 concentration in a 1.3 : molar ratio The complex was incubated for approximately h Drops from a 1.5 lL protein solution and lL of precipitant 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 maximum dimensions of 0.16 · 0.16 · 0.01 mm in approximately 30 days A mixture of the well solution with 50% v ⁄ v poly(ethylene glycol) (PEG 400) in a : volume ratio was used as cryoprotectant 804 X-ray data collection and analysis X-ray diffraction experiments were performed at 100 K using the X13 EMBL beamline of the DESY synchrotron ˚ (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] Additionally, Rpim [42] was calculated using SCALA [43] Table shows the data collection and processing statistics Structure solution and refinement The structure of the complex was solved by molecular replacement An initial molecular-replacement solution was obtained using the chagasin–cathepsin L complex (PDB code 2NQD) as the search model in MolRep [44] The complete model of the chagasin–papain complex was obtained by superposition of papain (PDB code 2KHQ) on the oriented cathepsin L portion as the target Manual model rebuilding was subsequently performed using coot [45] refmac5 [46] was used for structure refinement with TLS (translation, libration, screw motion – parameters describing anisotropic motion of rigid body molecules) [47] parameters defined separately for papain and chagasin Water molecules were introduced manually using coot [45] Rfree [48] was monitored using a randomly chosen subset of reflections comprising 5% of the unique data set Side chains of a number of residues were modeled in two conformations The quality of the final structure was assessed using procheck [49] and molprobity [50] The final refinement statistics are shown in Table All crystallographic calculations were performed using the CCP4 suite of programs [51] Molecular illustrations were prepared using pymol [52] Acknowledgements This work was supported in part by grant N-N4042348-33 from the Polish Ministry of Science and Higher Education to G B The work in Lund was supported by grants to M A from the Crafoord and ă A Osterlund Foundations, the Swedish Research Council (project number 09915), the Swedish Cancer Society (project number 07 0030) and the Research ă School in Pharmaceutical Chemistry (FLAK), Lund University, Sweden This 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N-terminus) (B) Zoom -in view of the interactions of papain with loop L6 of chagasin and loop L2 of cystatin B (C) Zoom -in view of the interactions of papain with loop L4 of chagasin and the N-terminal... 3) The difference of 9° between these complexes may be correlated with the variation of the rate of binding and affinity for chagasin of these enzymes On the other hand, in the two crystal forms... high-quality crystal structure of chagasin in complex with papain, the model C1 family cysteine protease and one of only two enzymes in the family for which structural information for a cystatin complex