Structural characterization reveals a novel bilobed architecture for the ectodomains of insect stage expressed Trypanosoma brucei PSSA-2 and Trypanosoma congolense ISA

Abstract

African trypanosomiasis, caused by parasites of the genus Trypanosoma, is a complex of devastating vector-borne diseases of humans and livestock in sub-Saharan Africa. Central to the pathogenesis of African trypanosomes is their transmission by the arthropod vector, Glossina spp. (tsetse fly). Intriguingly, the efficiency of parasite transmission through the vector is reduced following depletion of Trypanosoma brucei Procyclic-Specific Surface Antigen-2 (TbPSSA-2). To investigate the underlying molecular mechanism of TbPSSA-2, we determined the crystal structures of its ectodomain and that of its homolog T. congolense Insect Stage Antigen (TcISA) to resolutions of 1.65 Å and 2.45 Å, respectively using single wavelength anomalous dispersion. Both proteins adopt a novel bilobed architecture with the individual lobes displaying rotational flexibility around the central tether that suggest a potential mechanism for coordinating a binding partner. In support of this hypothesis, electron density consistent with a bound peptide was observed in the inter-lob cleft of a TcISA monomer. These first reported structures of insect stage transmembrane proteins expressed by African trypanosomes provide potentially valuable insight into the interface between parasite and tsetse vector.

Keywords: Trypanosoma brucei; Trypanosoma congolense; X-ray crystallography; bi-lobed architecture; conformational flexibility; ectodomain; tsetse.

Figure 1

TbPSSA‐2 and TcISA ectodomains adopt a novel architecture and exhibit conformational flexibility. (A) Schematic representation of TbPSSA‐2 and TcISA with predicted structural domains, TM regions and signal peptides. (B) Secondary structure depictions of TbPSSA‐2 (left) and TcISA (right) highlighting lobe 1 (green) and lobe 2 (blue). The beta strand and the loop connecting the two lobes are shown in red. Four disulfide bonds in lobe 2 are shown as yellow ball and sticks. (C) Topology diagram of TbPSSA‐2 and TcISA colored as in (B). Rotational flexibility between lobes L1 and L2 in (D) TbPSSA‐2 (chain A‐pink; B‐dark teal) and (E) TcISA (chain A‐orange; B‐cyan; C‐limegreen; D‐marine). An interactive view is available in the electronic version of the article.

Figure 2

Hinge region in TcISA reveals the potential for ligand binding. (A) TcISA chain A surface (grey) highlighting the bound peptide (pink). (B) Superimposition of TcISA monomers (left) with cleft indicated by black box. Middle panel: R127 and K130 in the hinge region of chain A coordinating a sulfate group which is depicted as stick model with a 2Fo‐Fc electron density map (green mesh) contoured at 1.5σ. Right panel: R128 and K130 of chains B, C, and D showing steric clash with overlaid peptide from chain A. An interactive view is available in the electronic version of the article.