Structure, interdomain dynamics and pH-dependent autoactivation of pro-rhodesain, the main lysosomal cysteine protease from African trypanosomes

Abstract

Rhodesain is the lysosomal cathepsin L-like cysteine protease of T. brucei rhodesiense, the causative agent of Human African Trypanosomiasis. The enzyme is essential for the proliferation and pathogenicity of the parasite as well as its ability to overcome the blood-brain barrier of the host. Lysosomal cathepsins are expressed as zymogens with an inactivating pro-domain that is cleaved under acidic conditions. A structure of the uncleaved maturation intermediate from a trypanosomal cathepsin L-like protease is currently not available. We thus established the heterologous expression of T. brucei rhodesiense pro-rhodesain in E. coli and determined its crystal structure. The trypanosomal pro-domain differs from non-parasitic pro-cathepsins by a unique, extended α-helix that blocks the active site and whose interactions resemble that of the antiprotozoal inhibitor K11777. Interdomain dynamics between pro- and core protease domain as observed by photoinduced electron transfer fluorescence correlation spectroscopy increase at low pH, where pro-rhodesain also undergoes autocleavage. Using the crystal structure, molecular dynamics simulations and mutagenesis, we identify a conserved interdomain salt bridge that prevents premature intramolecular cleavage at higher pH values and may thus present a control switch for the observed pH-sensitivity of pro-enzyme cleavage in (trypanosomal) CathL-like proteases.

Two Tags in One Probe: Combining Fluorescence‐ and Biotin‐based Detection of the Trypanosomal Cysteine Protease Rhodesain

“…Biology : The zymogen of T. b. rhodesiense rhodesain (Uniprot Q95PM0) was heterologously expressed without its C‐terminal domain in Pichia pastoris as described elsewhere [3,4] . Autoactivated rhodesain from the Pichia culture supernatants was purified on a phenyl sepharose column.…”

“…Rhodesain, a papain‐like cysteine protease of Trypanosoma brucei spp., also known as TbCatL ( T. brucei cathepsin L), has been identified as a target enzyme for the development of inhibitors to combat acute and chronic infections. The lysosomal protease is activated by either inter‐ or intramolecular catalysis, preferably at low pH [3] . However, in contrast to related human cathepsins, rhodesain also shows activity up to pH 8.0, consistent with a possible function in the host's blood stream [4] .…”