Crystal Structure of a Trypanosoma Brucei Metacaspase

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Crystal structure of a Trypanosoma brucei metacaspase

Karen McLuskeya,1, Jana Rudolfa, William R. Protoa, Neil W. Isaacsb, Graham H. Coombsc, Catherine X. Mossa, and Jeremy C. Mottrama,1

aWellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity, and Inﬂammation, College of Medical, Veterinary, and Life Sciences, University of Glasgow, Glasgow G12 8TA, United Kingdom; bSchool of Chemistry, College of Science and Engineering, University of Glasgow, Glasgow G12 8QQ, United Kingdom; and cStrathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow G4 0RE, United Kingdom

Edited by Robert Huber, Max Planck Institute for Biochemistry, Planegg-Martinsried, Germany, and approved March 28, 2012 (received for review January 23, 2012)

Metacaspases are distantly related caspase-family cysteine pepti- may reflect the distinct differences in substrate specificity and dases implicated in programmed cell death in plants and lower activation (11). For example, in yeast, the Yca1 metacaspase is eukaryotes. They differ significantly from caspases because they are required for clearance of cellular protein aggregates as well as calcium-activated, arginine-specific peptidases that do not require regulation of the timing of the cell cycle, a function that has also been identified in the parasitic protozoon Leishmania (9, 17–19). processing or dimerization for activity. To elucidate the basis of these fi differences and to determine the impact they might have on the Metacaspases have been classi ed as members of the clan CD (20) structural superfamily, which is represented by six peptidase control of cell death pathways in lower eukaryotes, the previously families: the caspases, legumains, separases, clostripains, gingi- undescribed crystal structure of a metacaspase, an inactive mutant of Trypanosoma brucei pains, and the repeats in toxin (RTX) self-cleaving toxins (cysteine metacaspase 2 (MCA2) from , has been deter- protease domain), all of which are predicted to share a core cas- mined to a resolution of 1.4 Å. The structure comprises a core caspase pase/hemoglobinase fold (CHF) containing the catalytic His/Cys fold, but with an unusual eight-stranded β-sheet that stabilizes the dyad. The core of the CHF domain is a simple α/β-fold (21) de- protein as a monomer. Essential aspartic acid residues, in the pre- fined by a four-stranded, parallel β-sheet and three conserved dicted S1 binding pocket, delineate the arginine-specificsubstrate α-helices. Currently, there are crystal structures available for the specificity. In addition, MCA2 possesses an unusual N terminus, caspases (1) [including paracaspase (22)], gingipain (23), and an which encircles the protein and traverses the catalytic dyad, with RTX toxin (24). Metacaspases are described as distant relatives of Y31 acting as a gatekeeper residue. The calcium-binding site is de- the caspases (4) and, despite low sequence identity with the cas- fined by samarium coordinated by four aspartic acid residues, pases, have been grouped [along with the Arg-specific para- MICROBIOLOGY whereas calcium binding itself induces an allosteric conformational caspases (4, 22)] into a structural subfamily of the caspase-type peptidases (family C14B). In the caspase structures, the funda- change that could stabilize the active site in a fashion analogous to β subunit processing in caspases. Collectively, these data give insights mental catalytic domain consists of a six-stranded -sheet, which into the mechanistic basis of substrate specificity and mode of acti- contains a large subunit (p20) and a small subunit (p10) (1) separated by an intersubunit linker between strands 4 and 5. Active vation of MCA2 and provide a detailed framework for understanding caspases are functional dimers. The recently identified structure of the role of metacaspases in cell death pathways of lower eukaryotes. the caspase domain of the mucosa-associated lymphoid tissue lymphoma translocation 1 (MALT1-C) paracaspase revealed an apoptosis | clan CD | parasite | X-ray crystallography identical topology (22). Caspases are activated by processing of the intersubunit linker or by dimerization (1, 25). Metacaspases rogrammed cell death (PCD) is essential for animal develop- are divided into two structural types based on their primary amino Pment and the maintenance of adult tissues. PCD itself is a reg- acid sequence (4, 11). Both types have been predicted to exhibit ulated process, and in animals, apoptosis is controlled through the a caspase-like domain composed of p20 and p10 subunits (26) but action of caspases, aspartic acid-specific cysteine peptidases (1). differ in that only type I has a predicted N-terminal prodomain, PCD is also essential for plant development (2), and multiple whereas type II contains an additional linker between the subunits. markers for apoptosis have been described in yeast and a broad Plant genomes encode both types of metacaspases, whereas only range of protozoan parasites (3), yet these organisms do not en- type I has been reported in protozoa and fungi (11). code caspases in their genomes; thus, alternative pathways must The protozoan parasite Trypanosoma brucei is the causative have evolved for caspase-independent cell death to be regulated. agent of human African trypanosomiasis, and its genome encodes In recent years, attention has focused on the metacaspases, which five metacaspases (MCA1–MCA5). MCA2, MCA3, and MCA5 are a highly conserved group of caspase-like cysteine peptidases have the conserved catalytic residues (histidine/cysteine dyad) (27) found in plants, fungi, and protozoa but not in the metazoa (4). In and are collectively required for the bloodstream form of the plants, metacaspases are essential for embryogenesis (5, 6) and parasite (28). MCA2, which is an active peptidase, is located in have been shown to act in antagonistic relationships, functioning RAB11-positive endosomes and has roles in precytokinesis cell as both positive and negative regulators of PCD (7). In yeast and cycle control (28). T. brucei mutants lacking MCA2 have no ob- some protozoa, metacaspases have been implicated as mediators vious defect, indicating that functional redundancy exists between of cell death in response to oxidative stress and environmental MCA2, MCA3, and MCA5. MCA4 is an inactive pseudopeptidase change (3, 8–10). Various attempts have been made to draw parallels between caspases and metacaspases in respect to structure, activation, and Author contributions: K.M. and J.C.M. designed research; K.M., J.R., and C.X.M. performed function (11). However, two striking differences between meta- research; W.R.P. contributed new reagents/analytic tools; K.M., J.R., N.W.I., G.H.C., C.X.M., caspases and caspases are their substrate specificities and and J.C.M. analyzed data; and K.M., G.H.C., C.X.M., and J.C.M. wrote the paper. requirements for activation. Metacaspases have arginine/lysine The authors declare no conflict of interest. specificity, do not necessarily require processing or dimerization This article is a PNAS Direct Submission. for activity, and are activated by calcium (5, 12–15). There are few Freely available online through the PNAS open access option. known natural targets of the proteolytic activity of metacaspases. Data deposition: The atomic coordinates and structure factors reported in this paper have One, Tudor staphylococcal nuclease, a substrate for mcII-Pa been deposited in the Protein Data Bank in Europe, www.ebi.ac.uk/pdbe/ (PDB ID 4AF8, metacaspase in the Norway spruce, Picea abies, is also a substrate 4AFP, 4AFR, and 4AFV). for human caspase-3, and this has been taken as indicating a level 1To whom correspondence may be addressed. E-mail: [email protected] or of evolutionary conservation between PCD pathways (16). It is [email protected]. now apparent, however, that metacaspases have a variety of cel- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. lular functions that are distinct from those of the caspases, which 1073/pnas.1200885109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1200885109 PNAS Early Edition | 1of6 Downloaded by guest on September 23, 2021 because of a cysteine-to-serine substitution in the active site, yet it functions as a membrane-linked virulence factor (29). MCA4 is also bloodstream form-specific, released by the parasite and processed by MCA3 (29). This pseudopeptidase illustrates the di- versity of metacaspase function and the challenges faced in attempting to assign functional roles based on sequence alone. This study aimed to understand the structural basis for the differences between metacaspases and caspases, including the substrate recognition properties and mechanisms for activation of the metacaspases. To this end, we undertook structural and bio- chemical analysis of MCA2 from T. brucei, determining the previously undescribed 3D structure of a metacaspase and providing incisive insights into how this family of enzymes functions. Results and Discussion Metacaspase Structure. Attempts to crystallize active MCA2 (MCA2ac) were unsuccessful, most likely a result of the presence of mixed degradation products caused by in vitro autoprocessing of the full-length recombinant enzyme (14). Therefore, the crystal structures of two catalytically inactive forms of MCA2 [MCAC213G and MCAC213A (Table S1)] were determined to 1.4 and 1.6 Å, respectively (Tables S2 and S3). The MCAC213G and MCAC213A structures are essentially identical (SI Methods). The highest resolution structure of MCA2C213G is the most complete, and the term MCA2 is used to describe the overall structure, unless oth- erwise stated. The MCA2C213G structure consists of residues 3– 347, with the exception of two disordered loop regions consisting of residues 166–170 and 269–275 (denoted the 280-loop). MCA2 is monomeric and comprises an eight-stranded β-sheet (β1–β8) consisting of six parallel and two antiparallel strands with 2↑1↑3↑4↑7↑8↓5↑6↓ topology (30) (Figs. 1 and 2D). The first four parallel strands lie in the same plane, exhibiting only a very slight right-handed twist, whereas the second half of the sheet is more twisted, with the C terminus of β7 rotating out of the plane of the sheet and crossing under the N terminus of β8 (Fig. 1A). The β-sheet is surrounded by five α-helices (α1–α5) that lie approximately parallel to it. Helices α1, α4, and α5 are found on one side of the sheet, with α2 and α3 on the opposite side. At the C ter- β β β β minus of 3, a small section of antiparallel -sheet ( A- C) is Fig. 1. Structure and sequence of T. brucei MCA2C213G. General loop regions found with strands βB and βC, connected by a 10-residue linker are shown in gray, the N-terminal region in cyan, α-helices in blue, and containing a short section of α-helix (αA). This section of β-sheet is β-strands in red, apart from βA-βC, which is shown in purple. (A) Tertiary positioned at the N terminus of α2 and α3, capping the helices and structure of the MCA2 monomer with the position of the catalytic dyad in the space between them (Fig. 1). yellow. (B) MCA2 sequence with the assigned secondary structure. The cata- The residues that constitute the catalytic dyad (H158 and lytic dyad is highlighted in red, and residues involved in the S1 pocket and C213G/A) are found on loops situated at the C-terminal end of samarium binding are shown in orange and cyan, respectively. Known cleav- strands β3 and β4, respectively (Figs. 1 and 2D). The loop con- age sites (14) are highlighted (▼), and residues that are missing from the necting β4 and β5 comprising the catalytic cysteine (denoted the MCA2C213G electron density are shown in light gray and with a dashed line. catalytic loop) is 11 residues long (Figs. 1B and 2D) and shows a degree of flexibility (as defined by poorer electron density and higher B-factors in this region). One of the most striking features being much less compact than that of a caspase monomer of the MCA2 structure is the 70-residue N terminus preceding β1. (Fig. 2). MCA2 exists as an enzymatically active monomer (Fig. This domain is an ordered loop containing only a small region of S1), and the loop regions on its surface create an unfavorable 310-helix (α0′), two short β-strands (β0′ and β0), and a seven-res- environment for the type of β-sheet/β-sheet dimerization found in idue α-helix (α0) (Figs. 1 and 3A). It encircles the main body of the the caspases (Fig. 2). In addition, a structural overlay with the enzyme, traversing the top in the region of the catalytic dyad and caspase-7 dimer reveals that β5 from MCA2 overlays with β6from interacting with itself as it crosses underneath (Fig. 3A). In β ′ C213A the second caspase monomer ( 6 ), and thus occupies the space MCA2 , there are 129 residues contributing to the interface used in the caspase-dimer interface and renders it unavailable (Fig. between the N-terminal domain and the main body of the protein, 2 B and D). Hence, the larger eight-stranded β-sheet in MCA2 creating 32 hydrogen bonds and eight salt bridges. The interfacing prevents it from forming a dimer along the same interface as the residues include the catalytic dyad of H158 and C213A, which are caspases and stabilizes the protein as a monomer. 100% and 50% buried by the N terminus, respectively. Recombinant MCA2ac undergoes autocatalytic processing at K55 in the N-terminal region and at K268 in the 280-loop (14) Comparison with Caspase-7. The overall fold of MCA2 is compa- SI Methods (Figs. 1B and 2D). However, cleavage does not generate separate rable to that of the mammalian caspases ( ). It is most β structurally similar to caspase-7, with 65% of its secondary subunits because the C-terminal region is embedded in the -sheet structural elements identified in the caspase-7 structure. How- and the N-terminal region remains associated with the main body ever, the structure of MCA2 reveals some striking architectural of the protein (Fig. S1). This processing has not been observed differences from the caspases, most notably in the length and in vivo (28) and is not required for the enzyme to be proteolytically arrangement of its β-sheet and in its extended N terminus (Fig. active (14). Consequently, the autoproteolytic processing of 2). The β-sheet in MCA2 is two strands longer than in caspase-7, recombinant MCA2ac in vitro may simply reflect the high con- which exhibits a six-stranded β-sheet with 2↑1↑3↑4↑5↑6↓ topology. centrations of the enzyme and the position of basic residues on These differences result in the monomeric structure of MCA2 solvent-exposed loops.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1200885109 McLuskey et al. Downloaded by guest on September 23, 2021 other clan CD peptidases (23, 32, 33) and the predicted direction for an MCA2 substrate. A 3D alignment of the structures of MCA2 and caspase-7 revealed that the position of the catalytic dyad is conserved between the two enzymes (Fig. 2 B and D), and this is also the case for clan CD members from other families: gingipain R (23) and the cysteine peptidase domain of the Vibrio cholerae RTX (24). However, in all the structures apart from MCA2, the dyad is found on the surface of the protein. In contrast, the N-terminal domain of MCA2 appears to block substrate access to the active site sterically, with the nature of the residues at the crossover preventing intramolecular autocleavage. Occlusion of the active site by an N-terminal domain is also observed in the proforms of the lysosomal cathepsins [clan CA, family C1 (20)], where proteolytic cleavage and removal of the prodomain are required for activation (34). However, in ca- thepsin B (35), the prodomain is only weakly associated with the enzyme, through ﬁve hydrogen bonds, and it crosses the active site in the opposite orientation to that observed in MCA2. Structural elucidation of a caspase prodomain is yet to be ach- ieved, but it has been reported to be ﬂexible, and hence disordered, in the crystal structures (36). In contrast, the N-terminal domain of MCA2 was both visible and highly ordered in our crystal structures, with its position clearly showing that it would be required to undergo a conformational shift to allow substrates access into the active site. Examining the structure of MCA2 without its N-terminal region reveals that the catalytic dyad forms part of a large acidic pocket (Fig. 3B), consistent with a binding site for the basic P1 amino acid of an MCA2 substrate. The functional groups of C92,

D95, S156, and D211 all point toward the surface of the S1 MICROBIOLOGY pocket, and the bottom is lined with three well-ordered water molecules (Fig. 3C), the locations of which were found to be conserved in all the MCA2 crystal structures. The N terminus of MCA2 crosses the S1 pocket and the catalytic dyad at residues Fig. 2. Comparison of MCA2 and caspase-7. Ribbon diagram of caspase-7 – (A) and metacaspase (C). General loops are colored gray/black, the main 30 36, with Y31 forming hydrogen bonds to D95 and S156, along β α with a water molecule, which is, in turn, coordinated to a second -sheet is colored red, surrounding -helices are shown in blue, and a small D section of the β-sheet at the C terminus of β3 is shown in green. In MCA2, water molecule and D211 (Fig. 3 ). There are no other hydro- secondary structural elements found on the N-terminal domain are colored gen bonds between the S1 pocket and the N-terminal domain. cyan. (B) Topology diagram of caspase-7. The 341-loop and the position of Residues C92, D95, S156, and D211 are conserved within the the catalytic dyad are highlighted, and the β-strands are numbered from the metacaspase family, whereas Y31 is conserved in the N-terminal N terminus. The intersubunit linker is drawn as a red line with the cleavage domain of T. brucei metacaspases (Fig. S2). In addition to their site highlighted (▲). (D) Topology diagram of MCA2, with the catalytic loop conservation, their location and orientation in the S1 pocket shown in red. The 280-loop affected during the Ca2+ activation of MCA2 and suggest that these residues play a part in substrate recognition. To known autoprocessing sites (▲) (14) are shown. The CHF core secondary investigate their significance, single alanine point mutations in ac structural elements are highlighted (*) in B and D. MCA2 wereconstructed and their proteolytic activity was assessed using a fluorogenic peptide substrate (Z-GGR-AMC; Fig. S3). D95 is positioned at the back of the S1 pocket in close proximity Effector caspases, such as caspase-7, are activated by cleavage in to D211. Alanine mutations of these residues resulted in enzymes the intersubunit linker generating the p10 and p20 subunits (Fig. that exhibited no autoprocessing in vitro or any noteworthy ac 2B). This cleavage allows part of the intersubunit linker from one activity toward Z-GGR-AMC compared with MCA2 (0% for D95A D211A caspase monomer to interact with the neighboring monomer and MCA2 , <5% for MCA ) (Fig. 3 E and F). Consequently, helps to order the active site (31). MCA2 is very unusual in this these aspartic acid residues are aptly positioned to recognize and respect; the topology of the β-sheet is different, with β4 and β5 not coordinate the basic side chain of an arginine or lysine residue and ’ fi C92A being adjacent but, instead, separated by two antiparallel strands are involved in the enzyme s substrate speci city in P1. MCA2 (β7 and β8) (Fig. 2D), and the catalytic loop that connects these showed approximately half of the activity toward the substrate compared with MCA2ac, and the equivalent of C92 in Arabidopsis strands does not harbor any basic residues as potential cleavage thaliana sites (Fig. 1B), and, indeed, none have been identified (14). In metacaspase 9 (C29) has similarly been shown to be im- addition, this loop is much shorter than the intersubunit linker in portant for activity (37). Thus, C92 of MCA2 contributes to cor- rect formation of the substrate binding pocket. MCA2Y31A the caspases, suggesting that it does not have the same functional β exhibited a much higher degree of autoprocessing in vitro than relevance. Thus, the eight-stranded -sheet, the length and to- MCA2ac, or any of the other mutants (Fig. 3E), suggesting that pology of which preclude caspase-like dimerization and explain its Y31 controls access to the active site. MCA2Y31A had only 60% of monomeric peptidase activity, clearly distinguishes MCA2 from MCA2ac activity, probably attributable to the extensive autopro- other caspase family members. This is further supported by the cessing degrading the enzyme. In contrast MCA2S156A activity was lack of an intersubunit linker preventing the formation of two threefold higher than MCA2ac. We propose a model whereby Y31 distinct subunits or activation by cleavage in this region. Conse- acts as a gatekeeper by using S156 as a latch; when the latch is quently, the activation mechanism of MCA2 is quite distinct from released, substrate can access the active site. These data are that of the caspases. consistent with the N-terminal domain acting to regulate the activity of the enzyme by protecting the active site until it is activated N-Terminal Domain and Active Site. The catalytic dyad of MCA2 is by calcium (see below). buried beneath the N-terminal domain, which crosses the active The participation of D95, D211, and Y31 in S1 binding of site in the direction of the N terminus to the C terminus (Fig. 3B). MCA2 is further supported by analysis of substrate binding in This is the same direction as the peptide inhibitors bound to the caspase-7 and MALT1-C. In caspase-7, P1 substrate recognition is

McLuskey et al. PNAS Early Edition | 3of6 Downloaded by guest on September 23, 2021 Fig. 3. N-terminal domain of MCA2 crosses over the S1 binding pocket. (A) N-terminal domain (blue) encircles the main body of the enzyme (gray). (B) Electrostatic surface potential of MCA2C213A (rotated ∼90° from A), calculated without the N-terminal domain, where blue and red denote positively and negatively charged surface areas, respectively. The N-terminal domain is shown crossing the catalytic dyad as sticks with Y31 labeled. The water molecules that line the active site are shown as cyan spheres. (C) Close-up view of the S1 binding pocket. (D) Y31 interacts with residues in the S1 binding pocket. (E) SDS/ PAGE analysis of in vitro autoprocessing exhibited by MCA2 and mutants purified by Ni2+-affinity chromatography. (F) Activity of MCA2ac and mutants was compared by measuring the hydrolysis of the fluorogenic substrate Z-GGR-AMC (average of 3 experiments is shown ± SD).

dependent on the three highly conserved residues, R87, Q184, and (Fig. 4A and SI Methods). Varying concentrations of CaCl2 were R233 (1), and in MALT1-C, three negatively charged residues, added to crystallization trials in an attempt to identify calcium- D365, D462, and E500, were found to coordinate the P1 Arg of the binding sites in the protein. However, even concentrations as low as inhibitor (22). The structural overlay revealed that the secondary 10 μM inhibited crystal formation. Lanthanides are often used to structure of MCA2 is conserved with caspase-7 residues R87 and define calcium-binding sites in proteins (38); consequently, samar- Q184, overlaying with residues L89 and D211, respectively. L89 ium was tested as a heavy atom derivative for the structure solution. would not be important in recognizing a basic metacaspase sub- A samarium-derivatized crystal (MCA2C213G-Sm) was obtained strate; however, D95, which sits nearby on the N-terminal end of that revealed a single, fully occupied Sm3+-binding site on the α1 and overlays with D365 in MALT1-C, was shown to be essential surface of the molecule (Fig. 4B). The samarium ion exhibits a co- for MCA2 activity. R233 of caspase-7, which lies on a loop (termed ordination number of 7 from two water molecules and four highly the 341-loop; ref. 1, Fig. 2B) at the C terminus of β5 and undergoes conserved aspartic acid residues: D173, D189, D190, and D220. significant conformational changes during activation (33), has no These residues are well conserved in metacaspases from a diverse ordered equivalent in MCA2. In the structure of MALT1-C, this range of organisms (Fig. S2) but are not found in the caspases or loop contains the P1 binding residue E500. In the MCA2 structure, paracaspases, which correlates with their lack of calcium-dependent this corresponds to the disordered loop region at the C terminus of activation. The structures of MCA2C213G and MCA2C213G-Sm are β7 (denoted the 280-loop; Figs. 1B and 2D), suggesting that this essentially identical, with the exception that MCA2C213G-Sm lacks loop could play a role in substrate binding of MCA2. In addition, electron density for several additional residues in the 280-loop Y31 from the MCA2 N-terminal domain occupies the same posi- (266–279 compared with 269–275 for MCA2C213G; Table S3), tion as R233 in caspase-7 (and E500 in MALT1-C), implying that showing an increased degree of flexibility in this region. Y31 would be involved in binding a substrate at the P1 position. To investigate the location of the calcium-binding site further, Despite the distinct structural and functional differences be- a double mutant, MCA2D189A/D190A, was made. In the presence of tween the caspases and metacaspases, MCA2 uses residues at calcium, this mutant had no detectable activity against Z-GGR- similar structural positions in its S1 binding pocket to other cas- AMC (specific activity <0.14 mU/mg), nor did it autoprocess (Fig. pase family members to determine its substrate specificity. Al- 4C). Furthermore, CD spectra of MCA2D189A/D190A confirmed though two of the three proposed MCA2 P1 binding residues do that the mutation had not adversely affected the overall structure not share conserved secondary structure with the caspases, the of the protein (Fig. S4). This confirms the role of these aspartic position of the residues in the S1 binding pocket remains pre- acid residues in the putative calcium-binding site and shows they served. This suggests a common substrate recognition mechanism are essential for MCA2 activity. To gain additional information on for caspases, paracaspases, and metacaspases. how calcium influences MCA2 activity, various CaCl2 soaks were carried out on MCA2C213A crystals (MCA2C213A-Ca). Two data- Calcium Binding/Activation. MCA2 and other metacaspases are de- sets were collected from each crystal soak at wavelengths of 0.97 Å pendent on calcium for activation (5, 12–15).Furthermore,the and 1.7 Å, but Ca2+ could not be detected in any of these datasets peptide-based inhibitor Z-VRPR-FMK inhibits the autoprocessing (SI Methods). However, structures determined from calcium- activity of MCA2ac, and calcium is required for inhibitor binding soaked crystals revealed a large Ca2+-induced conformational

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1200885109 McLuskey et al. Downloaded by guest on September 23, 2021 change in the position of the 280-loop. This loop changes direction catalytic dyad (Fig. 4E). Because the 280-loop is structurally ho- at the Cα position of A280, resulting in a 5.6-Å, 110° shift in the mologous to the 341-loop in the caspases (Fig. 2 B and D), it positions of the Cα positions of G279 between MCA2C213A-Ca appears that in MCA2, the 280-loop moves to stabilize substrates and MCA2C213A (Fig. 4, inset). This movement also resulted in the in the active site in response to calcium binding. We also found disorder of the ﬁrst 15 residues of the N-terminal domain of that calcium binding alone does not induce a conformational shift MCA2C213A-Ca, which does not appear to be functional but, in- in the N-terminal region crossing the active site suggesting that the stead, is a direct result of the 280-loop movement in the crystals. presence of a substrate is also required to trigger the release of the In both MCA2C213A and MCA2C213A-Ca, residues 280 and 281 N-terminal latch. form a section of β-sheet with N-terminal residues 32 and 31, respectively. In MCA2C213A, this loop then moves away from the N- Conclusions. The 1.4-Å structure of T. brucei MCA2 has given terminal domain and crosses over a short loop between α5 and β8. insights into the substrate speciﬁcity and mode of activation of In MCA2C213A-Ca, the 280-loop continues to run parallel, forming MCA2. It has revealed that MCA2 possesses an unusual N-ter- a section of β-sheet with the N-terminal domain (Fig. 4E). In minal domain, which likely serves to regulate substrate access to contrast to MCA2C213A, the 280-loop in MCA2C213A-Ca is in line the active site until the enzyme is activated by calcium in the with the wall of the S1 binding pocket, sitting directly above the presence of a substrate. In MCA2, calcium is required for both MICROBIOLOGY

Fig. 4. Allosteric binding of calcium induces a conformational shift in MCA2C213A.(A) Inhibitor Z-VRPR-FMK binds to MCA2ac on the addition of Ca2+, causing a small size shift on SDS/PAGE, and protects MCA2ac from autoproteolysis in the presence of Ca2+.(B) Samarium ion (Sm3+) binds to the surface of MCA2C213G via four aspartic acid residues and two water molecules (cyan spheres), deﬁning the Ca2+-binding site. (C) SDS/PAGE analysis of in vitro autoprocessing exhibited by MCA2ac and MCA2D189A/D190A.(D) Composite diagram shows the structure of MCA2C213A-Ca (gray) and its 280-loop (+Ca2+, red) with the catalytic dyad highlighted in orange and the N-terminal domain shown in green. Addition of the 280-loop from the MCA2C213A structure (APO, blue) and the bound Sm3+ ion from the MCA2C213G-Sm structure shows the 280-loop movement with respect to the position of the calcium-binding site. Missing residues from the 280-loop regions are represented by dotted lines. (Inset) Structure of MCA2C213A-Ca shows an approximate 5.6-Å, 110° shift in the position of the MCA2C213A 280-loop. (E)Inthe structure of MCA2C213A-Ca, the 280-loop (red) forms a section of the β-sheet with the N-terminal domain (green) that sits above the catalytic dyad (orange).

McLuskey et al. PNAS Early Edition | 5of6 Downloaded by guest on September 23, 2021 activation of the enzyme and binding of the peptide inhibitor Z- Methods VRPR-FMK, and it was found to induce an allosteric conforma- The construction of plasmids, purification of protein, and enzyme assays were tional change in the 280-loop, bringing it closer to the active site. carried out as described previously (14) and in SI Methods. Initial crystals of This suggests that MCA2 requires this calcium-induced loop MCA2C213G or MCA2C213A were grown at 4 °C using vapor diffusion techni- movement to stabilize substrate binding, and hence to regulate ques in a 96-well sitting drop plate (Innovadyne) containing 500 nL of pro- activity. Little is known about Ca2+ concentrations and/or Ca2+ signaling in African trypanosomes, and nothing is known about tein and 500 nL of reservoir against a reservoir of 50 mM Hepes (pH 7.0), Ca2+ in the RAB11-positive recycling endosome compartment in 0.1% tryptone, and 20% (wt/vol) PEG3350 (PEG/Ion screen; Hampton). These which MCA2 primarily resides. However, endoplasmic reticulum crystals were composed of multiple plates and were optimized in 24-well stress-induced cell death in Leishmania is Ca2+-dependent (39); sitting drop plates (Cryschem; Hampton) using microseeding techniques. thus, it will be interesting to investigate whether there is any re- Crystals were flash-frozen in liquid nitrogen using the reservoir solution plus lationship between such cell death and the Ca2+ activation central 20% (vol/vol) 2-methyl-2,4-pentanediol (MPD) as a cryoprotectant before to metacaspase function. data collection. A samarium-derivatized crystal (MCA2C213G-Sm) was pre- The structural similarity of MCA2 to the caspases suggests that pared by soaking an MCA2C213G crystal in artificial mother liquor [AML; 50 they evolved from a common ancestor. However, the length and mM Hepes (pH 7.0), 0.1% tryptone, 20% PEG3350] containing 60 mM sa- construction of their β-sheets, and the differences identified in the marium acetate for 2 min. In addition, an MCA2C213A crystal soaked in 5 mM C213A intersubunit linker and domain structure, show that they are ar- CaCl2 for 1 h (MCA2 -Ca) was prepared and cryoprotected in AML con-

chitecturally very distinct. Furthermore, the lack of structurally taining 20% MPD and 1 mM CaCl2. All native and multiwavelength anom- conserved autocatalytic cleavage sites and dimerization interfaces alous dispersion diffraction data were collected at Diamond Light Source in MCA2 is consistent with MCA2 having a mode of activation (DLS; Didcot, United Kingdom) on beamlines I03 and/or I04 and processed different from that of the caspases. These findings, together with ’ fi with Fast_dp at DLS. Details of data collection, structure determination, and metacaspase s occluded active site, distinct substrate speci city, structural analyses are described in SI Methods. and dependence on calcium for activation (14), show that MCA2 activity is regulated very differently from that of the caspases. ACKNOWLEDGMENTS. We thank E. Brown for help with cloning, A. Scott Thus, caspases and metacaspases may have evolved independently for protein purification, T. Z. Murray for inhibitor assays, S. M. Kelly for CD from an ancestral metacaspase-like peptidase, with each family of analysis, M. Gabrielsen for helpful discussion, and DLS for access (Proposal enzymes evolving distinct activation mechanisms to regulate cell Mx6683). This work was supported by Wellcome Trust Grant 091790 and death pathways. Overall, this work provides a context for the de- Medical Research Council Grant 0700127. The Wellcome Trust Centre for sign of metacaspase-specific inhibitors that can potentially be used Molecular Parasitology is supported by core funding from Wellcome Trust for the development of novel antiparasite drugs (40). Grant 085349.

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