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Indeed, trypanosomatid metacaspases seemed to have evolved
alternative functions, e.g., as a negative regulator of amastigote
proliferation in Leishmania mexicana [ 3 ] and as a pseudopeptidase
virulence factor in T. brucei (TbMCA4) [ 4 ]. In addition, metacas-
pases from other species have also been implicated in a variety of
cellular functions including clearance of protein aggregates [ 5 ],
endoplasmic reticulum stress and cell proliferation (reviewed in
[ 6 ]), suggesting a signifi cant divergence from the cellular processes
defi ned for the caspases.
Along with their divergent roles in host cells, biochemical clas-
sifi cation of metacaspases and caspases has revealed that they form
two functionally distinct groups of cysteine peptidases, differing in
both their substrate specifi cities and modes of activation. Caspases
are homodimeric aspartic acid-specifi c peptidases for which dimer-
ization is critical, and proteolytic processing is often necessary for
activation [ 7 – 9 ]. Conversely, metacaspases are arginine/lysine-
specifi c fully functional monomers that do not always require
processing for activation but are activated by calcium [ 10 – 13 ].
Interestingly, the genome of T. brucei encodes fi ve metacaspases
(denoted TbMCA1–TbMCA5) which share between 40 and 89 %
primary sequence identity [ 14 ] and can have distinct in vivo func-
tions [ 4 , 15 ]. Metacaspases TbMCA2, TbMCA3, and TbMCA5
all encode proteins with the canonical His/Cys dyad and as a group
are thought to be essential for the bloodstream form of the parasite
[ 15 ]. TbMCA2 and TbMCA3 share 89 % sequence identity (dif-
fering only in their N-terminal regions), whereas TbMCA5 con-
tains a large (~16 kDa) C-terminal extension. Interestingly,
TbMCA1 and TbMCA4 contain amino acid substitutions in their
catalytic residues (His to Tyr and Cys to Ser, respectively). In
TbMCA4, even though the substitution results in a catalytically
inactive pseudopeptidase, it still functions as a membrane-linked
virulence factor, which is processed by TbMCA3 [ 4 ]. TbMCA4
provides an illustration of the functional diversity of the trypano-
some metacaspases and the challenges faced in deciphering indi-
vidual functions.
As part of a T. brucei drug discovery program we undertook to
further understand the structural and functional relationships of
trypanosome metacaspases, and to allow an accurate structural
comparison with the caspases, paracaspases, and other Clan CD
[ 16 ] cysteine peptidases. A large part of this work included deter-
mining the three dimensional crystal structure of a metacaspase
from T. brucei and to this end we successfully obtained the X-ray
crystal structure of an inactive mutant of TbMCA2 (TbMCA2 C213A )
to around 1.5 Å resolution ([ 17 ]; PDB ID: 4AFR). The structure
revealed a core caspase fold with an eight-stranded β-sheet that
stabilized the enzyme as a monomer and a well-ordered N-terminus,
which wrapped around the molecule covering the catalytic dyad.
In addition, biochemical and kinetic assays along with samariumKaren McLuskey et al.http://www.ebook3000.com