Crystal Structures of TbCatB and rhodesain, potential chemotherapeutic targets and major cysteine proteases of Trypanosoma brucei

Abstract

Background: Trypanosoma brucei is the etiological agent of Human African Trypanosomiasis, an endemic parasitic disease of sub-Saharan Africa. TbCatB and rhodesain are the sole Clan CA papain-like cysteine proteases produced by the parasite during infection of the mammalian host and are implicated in the progression of disease. Of considerable interest is the exploration of these two enzymes as targets for cysteine protease inhibitors that are effective against T. brucei.

Methods and findings: We have determined, by X-ray crystallography, the first reported structure of TbCatB in complex with the cathepsin B selective inhibitor CA074. In addition we report the structure of rhodesain in complex with the vinyl-sulfone K11002.

Conclusions: The mature domain of our TbCat*CA074 structure contains unique features for a cathepsin B-like enzyme including an elongated N-terminus extending 16 residues past the predicted maturation cleavage site. N-terminal Edman sequencing reveals an even longer extension than is observed amongst the ordered portions of the crystal structure. The TbCat*CA074 structure confirms that the occluding loop, which is an essential part of the substrate-binding site, creates a larger prime side pocket in the active site cleft than is found in mammalian cathepsin B-small molecule structures. Our data further highlight enhanced flexibility in the occluding loop main chain and structural deviations from mammalian cathepsin B enzymes that may affect activity and inhibitor design. Comparisons with the rhodesain*K11002 structure highlight key differences that may impact the design of cysteine protease inhibitors as anti-trypanosomal drugs.

Figure 1. Structures of rhodesain and TbCatB.

Stereo pairs of (A) rhodesain and (B) TbCatB. The figure is annotated with the secondary structure and the L and R domains are colored green and grey respectively. The occluding loop and N-terminal extension in TbCatB are colored red.

Figure 2. Inhibitor binding in TbCatB and rhodesain.

Surface (left) and ball and stick (right) active site representations of (A) the rhodesain•K11002 complex and (B) the TbCatB•CA074 complex. Inhibitor molecules are colored grey and the unbiased mFo-DFc electron density for each is colored violet. Hydrophobic interactions with neutral/non-polar residues are mapped on the surface and colored purple.

Figure 3. The substrate binding sites of TbCatB and mammalian CatBs.

Surface and ribbon/ball and stick representations comparing the substrate binding sites of these two cysteine proteases. TbCatB (green), has a deep and spacious S2 pocket, with Gly328 at the bottom. Human CatB, a representative member of the mammalian homologs (1GMY - CA030 complex, yellow), has the larger Glu245 at this position and the pocket is shallower. In Human CatB•small molecule complexes, the occluding loop points into the substrate binding site. Conversely, the TbCatB occluding loop is pulled out of the active site. CA074 from the TbCatB crystal structure is shown in grey to orient the reader.

Figure 4. An acidic functionality around the S2 subsite of TbCatB.

Ribbon/ball and stick illustrating an acidic functionality in the S2 subsite of TbCatB. CA074 is colored in grey and a bound molecule of glycerol (from the crystal cryosolution) is colored in yellow.

Figure 5. The S2 subsites of TbCatB and rhodesain.

Superimposition of TbCatB•CA074 and rhodesain•K11002 reveals differences in the S2 subsites of the two enzymes. Rhodesain is colored yellow, TbCatB monomer A is blue and TbCatB monomer B (5A only) is pink. K11002 is included in grey to orient the reader w.r.t the active site. (A) The rhodesain pocket is partially restricted in comparison with TbCatB due to a Asp>Leu substitution (B) Ala208 at the bottom of the S2 subsite in rhodesain (Gly328 in TbCatB) makes the pocket shallower (C) The S2 subsite in TbCatB is further opened due to the conformation of the loop between strands β2 and β3. Glycine residues in the loop are colored purple.

Figure 6. Interactions stabilizing the occluding loop in cathepsin B-like enzymes.

Ribbon/ball and stick representations illustrating, important interactions that stabilize the occluding loop (pale cyan) of (A) Human cathepsin B (1GMY) and (B) TbCatB.