Crystal Structure and Activity Studies of the C11 Cysteine Peptidase from Parabacteroides merdae in the Human Gut Microbiome.

Clan CD cysteine peptidases, a structurally related group of peptidases that include mammalian caspases, exhibit a wide range of important functions, along with a variety of specificities and activation mechanisms. However, for the clostripain family (denoted C11), little is currently known. Here, we describe the first crystal structure of a C11 protein from the human gut bacterium, Parabacteroides merdae (PmC11), determined to 1.7-Å resolution. PmC11 is a monomeric cysteine peptidase that comprises an extended caspase-like α/ß/α sandwich and an unusual C-terminal domain. It shares core structural elements with clan CD cysteine peptidases but otherwise structurally differs from the other families in the clan. These studies also revealed a well ordered break in the polypeptide chain at Lys(147), resulting in a large conformational rearrangement close to the active site. Biochemical and kinetic analysis revealed Lys(147) to be an intramolecular processing site at which cleavage is required for full activation of the enzyme, suggesting an autoinhibitory mechanism for self-preservation. PmC11 has an acidic binding pocket and a preference for basic substrates, and accepts substrates with Arg and Lys in P1 and does not require Ca(2+) for activity. Collectively, these data provide insights into the mechanism and activity of PmC11 and a detailed framework for studies on C11 peptidases from other phylogenetic kingdoms.

PNT1 Is a C11 Cysteine Peptidase Essential for Replication of the Trypanosome Kinetoplast.

The structure of a C11 peptidase PmC11 from the gut bacterium, Parabacteroides merdae, has recently been determined, enabling the identification and characterization of a C11 orthologue, PNT1, in the parasitic protozoon Trypanosoma brucei. A phylogenetic analysis identified PmC11 orthologues in bacteria, archaea, Chromerids, Coccidia, and Kinetoplastida, the latter being the most divergent. A primary sequence alignment of PNT1 with clostripain and PmC11 revealed the position of the characteristic His-Cys catalytic dyad (His(99) and Cys(136)), and an Asp (Asp(134)) in the potential S1 binding site. Immunofluorescence and cryoelectron microscopy revealed that PNT1 localizes to the kinetoplast, an organelle containing the mitochondrial genome of the parasite (kDNA), with an accumulation of the protein at or near the antipodal sites. Depletion of PNT1 by RNAi in the T. brucei bloodstream form was lethal both in in vitro culture and in vivo in mice and the induced population accumulated cells lacking a kinetoplast. In contrast, overexpression of PNT1 led to cells having mislocated kinetoplasts. RNAi depletion of PNT1 in a kDNA independent cell line resulted in kinetoplast loss but was viable, indicating that PNT1 is required exclusively for kinetoplast maintenance. Expression of a recoded wild-type PNT1 allele, but not of an active site mutant restored parasite viability after induction in vitro and in vivo confirming that the peptidase activity of PNT1 is essential for parasite survival. These data provide evidence that PNT1 is a cysteine peptidase that is required exclusively for maintenance of the trypanosome kinetoplast.

Comparative structural analysis of the caspase family with other clan CD cysteine peptidases.

Clan CD forms a structural group of cysteine peptidases, containing seven individual families and two subfamilies of structurally related enzymes. Historically, it is most notable for containing the mammalian caspases, on which the structures of the clan were founded. Interestingly, the caspase family is split into two subfamilies: the caspases, and a second subfamily containing both the paracaspases and the metacaspases. Structural data are now available for both the paracaspases and the metacaspases, allowing a comprehensive structural analysis of the entire caspase family. In addition, a relative plethora of structural data has recently become available for many of the other families in the clan, allowing both the structures and the structure-function relationships of clan CD to be fully explored. The present review compares the enzymes in the caspase subfamilies with each other, together with a comprehensive comparison of all the structural families in clan CD. This reveals a diverse group of structures with highly conserved structural elements that provide the peptidases with a variety of substrate specificities and activation mechanisms. It also reveals conserved structural elements involved in substrate binding, and potential autoinhibitory functions, throughout the clan, and confirms that the metacaspases are structurally diverse from the caspases (and paracaspases), suggesting that they should form a distinct family of clan CD peptidases.

Purification, characterization, and crystallization of Trypanosoma metacaspases.

Metacaspases are cysteine peptidases found in trypanosomes but absent in mammals, and despite being distantly related to the mammalian caspases they show significant disparity in their cellular and enzymatic functions. The genome of the parasitic protozoa Trypanosoma brucei (the causative agent of African sleeping sickness) encodes five metacaspases: TbMCA1-TbMCA5. Of these TbMCA2, TbMCA3, and TbMCA5 are active cysteine peptidases expressed in the bloodstream form of the parasite. To investigate the structure-function relationship of the trypanosome metacaspases and the structural basis for their divergence from the caspases, paracaspases, and other Clan CD cysteine peptidases (or vice versa), we purified and characterized TbMCA2 and determined the three-dimensional structure of an inactive mutant using X-ray crystallography. The methods presented in this chapter describe the recombinant expression of active TbMCA2 and inactive TbMCA2(C213A). The protocols produce large amounts of recombinant protein for use in structural, biochemical, and kinetic studies and include detailed information on how to produce diffraction quality crystals of TbMCA2(C213A).

Substrate specificity and the effect of calcium on Trypanosoma brucei metacaspase 2.

Metacaspases are cysteine peptidases found only in yeast, plants and lower eukaryotes, including the protozoa. To investigate the extended substrate specificity and effects of Ca(2+) on the activation of these enzymes, detailed kinetic, biochemical and structural analyses were carried out on metacaspase 2 from Trypanosoma brucei (TbMCA2). These results reveal that TbMCA2 has an unambiguous preference for basic amino acids at the P1 position of peptide substrates and that this is most probably a result of hydrogen bonding from the P1 residue to Asp95 and Asp211 in TbMCA2. In addition, TbMCA2 also has a preference for charged residues at the P2 and P3 positions and for small residues at the prime side of a peptide substrate. Studies into the effects of Ca(2+) on the enzyme revealed the presence of two Ca(2+) binding sites and a reversible structural modification of the enzyme upon Ca(2+) binding. In addition, the concentration of Ca(2+) used for activation of TbMCA2 was found to produce a differential effect on the activity of TbMCA2, but only when a series of peptides that differed in P2 were examined, suggesting that Ca(2+) activation of TbMCA2 has a structural effect on the enzyme in the vicinity of the S2 binding pocket. Collectively, these data give new insights into the substrate specificity and Ca(2+) activation of TbMCA2. This provides important functional details and leads to a better understanding of metacaspases, which are known to play an important role in trypanosomes and make attractive drug targets due to their absence in humans.

Crystal structure of a Trypanosoma brucei metacaspase.

Metacaspases are distantly related caspase-family cysteine peptidases implicated in programmed cell death in plants and lower eukaryotes. They differ significantly from caspases because they are calcium-activated, arginine-specific peptidases that do not require processing or dimerization for activity. To elucidate the basis of these differences and to determine the impact they might have on the control of cell death pathways in lower eukaryotes, the previously undescribed crystal structure of a metacaspase, an inactive mutant of metacaspase 2 (MCA2) from Trypanosoma brucei, has been determined to a resolution of 1.4 Å. The structure comprises a core caspase fold, but with an unusual eight-stranded ß-sheet that stabilizes the protein as a monomer. Essential aspartic acid residues, in the predicted S1 binding pocket, delineate the arginine-specific substrate specificity. In addition, MCA2 possesses an unusual N terminus, which encircles the protein and traverses the catalytic dyad, with Y31 acting as a gatekeeper residue. The calcium-binding site is defined by samarium coordinated by four aspartic acid residues, whereas calcium binding itself induces an allosteric conformational change that could stabilize the active site in a fashion analogous to subunit processing in caspases. Collectively, these data give insights into the mechanistic basis of substrate specificity and mode of activation of MCA2 and provide a detailed framework for understanding the role of metacaspases in cell death pathways of lower eukaryotes.

Oligopeptidase B deficient mutants of Leishmania major.

Oligopeptidase B is a clan SC, family S9 serine peptidase found in gram positive bacteria, plants and trypanosomatids. Evidence suggests it is a virulence factor and thus therapeutic target in both Trypanosoma cruzi and T. brucei, but little is known about its function in Leishmania. In this study L. major OPB-deficient mutants (Δopb) were created. These grew normally as promastigotes, had a small deficiency in their ability to undergo differentiation to metacyclic promastigotes, were significantly less able to infect and survive within macrophages in vitro, but were virulent to mice. These data suggest that L. major OPB itself is not an important virulence factor, indicating functional differences between trypanosomes and Leishmania in their interaction with the mammalian host. The possibility that an OPB-like enzyme (designated OPB2) in L. major might compensate for the loss of OPB in Δopb was investigated via by mapping its sequence onto the 1.6Å structure of L. major OPB. This suggested that the residues involved in the S1 and S2 subsites of OPB2 are identical to OPB and hence the substrate specificity would be similar. Consequently there may be redundancy between the two enzymes.

Structure and reactivity of Trypanosoma brucei pteridine reductase: inhibition by the archetypal antifolate methotrexate.

The protozoan Trypanosoma brucei has a functional pteridine reductase (TbPTR1), an NADPH-dependent short-chain reductase that participates in the salvage of pterins, which are essential for parasite growth. PTR1 displays broad-spectrum activity with pterins and folates, provides a metabolic bypass for inhibition of the trypanosomatid dihydrofolate reductase and therefore compromises the use of antifolates for treatment of trypanosomiasis. Catalytic properties of recombinant TbPTR1 and inhibition by the archetypal antifolate methotrexate have been characterized and the crystal structure of the ternary complex with cofactor NADP+ and the inhibitor determined at 2.2 A resolution. This enzyme shares 50% amino acid sequence identity with Leishmania major PTR1 (LmPTR1) and comparisons show that the architecture of the cofactor binding site, and the catalytic centre are highly conserved, as are most interactions with the inhibitor. However, specific amino acid differences, in particular the placement of Trp221 at the side of the active site, and adjustment of the beta6-alpha6 loop and alpha6 helix at one side of the substrate-binding cleft significantly reduce the size of the substrate binding site of TbPTR1 and alter the chemical properties compared with LmPTR1. A reactive Cys168, within the active site cleft, in conjunction with the C-terminus carboxyl group and His267 of a partner subunit forms a triad similar to the catalytic component of cysteine proteases. TbPTR1 therefore offers novel structural features to exploit in the search for inhibitors of therapeutic value against African trypanosomiasis.