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.