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Crystal Structure of a Trypanosoma Brucei Metacaspase

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Crystal Structure of a Trypanosoma Brucei Metacaspase 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) sep- arated 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 pro- cessed 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 dif- ferences between metacaspases and caspases, including the sub- strate 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 pre- viously 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).
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