The first cysteine protease structure to be determined was that of papain from Carica papaya
. Since its discovery, many ‘papain-like’ proteases, also referred to as thiol or sulfhydryl peptidases, have been characterized and are classified as clan CA proteases. The cysteine proteases are grouped into seven clans defined according to the linear organization of catalytic residues in the sequence, e.g.
clan CA has the catalytic residues Cys, His and Asn or Asp ordered in sequence, clan CD presents two catalytic residues, His and Cys, in sequence, clan CE has a triad formed by His, Glu or Asp and Cys at the C-terminus, clan CF also presents a catalytic triad, but ordered as Glu, Cys and His, clan CG has a dyad of two cysteine residues and clan CH presents a Cys, Thr and His triad with the catalytic cysteine at the N-terminus (Rawlings et al.
). Additionally, clan membership depends upon specificity, with clan CA proteases characterized by sensitivity to the inhibitor E64 [l
-epoxysuccinyl-leucyl-amido-(4-guanidino)butane] and by having substrate specificity defined by the S2 pocket (Sajid & McKerrow, 2002
). The majority of protozoan parasite cysteine proteases belong to clan CA family C1 papain-like proteases. This family of parasite-derived cysteine peptidases are critical to the life cycle or pathogenicity of many parasites, where they contribute key roles in immunoevasion, enzyme activation, pathogenesis, virulence and tissue and cellular invasion as well as excystment, hatching and moulting, and are considered to be promising chemotherapeutic targets (Sajid & McKerrow, 2002
; Mottram et al.
The actions of mammalian cysteine proteases are controlled in part by endogenous tight-binding inhibitors from the cystatin superfamily (Grzonka et al.
; Abrahamson et al.
). The Leishmania
genome lacks genes encoding cystatins. However, in Trypanosoma cruzi
a potent inhibitor of the parasite’s own cysteine protease cruzipain was identified and called chagasin (Besteiro et al.
). Subsequently, several homologues of these inhibitors of cysteine proteases (ICPs) were identified in the parasitic protozoa T. brucei
, L. major
and L. mexicana
and the bacterium Pseudomonas aeruginosa
(Sanderson et al.
). ICPs inhibit clan CA family C1 cysteine proteases with varying specificities. The molar ratio of inhibition is 1:1 and inhibition is competitive. The ICP of T. brucei
ICP) appears to be more potent than the L. mexicana
ICP and displays low nanomolar K
values against the clan CA family. Whilst ICPs share low sequence homologies and no significant identity with cystatins or other cysteine protease inhibitors, their functional homology implies a common evolutionary origin between bacterial and protozoal proteins (Sanderson et al.
We set out to cocrystallize the TbICP–papain complex, seeking to generate structural data on an ICP and to understand the mode of inhibition. Here, we report the resulting papain structure with ICP-derived peptide fragments bound within the active-site cleft.