The Structure of Plasmodium falciparum Blood-Stage 6-Cys Protein Pf41 Reveals an Unexpected Intra-Domain Insertion Required for Pf12 Coordination

Abstract

Plasmodium falciparum is an apicomplexan parasite and the etiological agent of severe human malaria. The complex P. falciparum life cycle is supported by a diverse repertoire of surface proteins including the family of 6-Cys s48/45 antigens. Of these, Pf41 is localized to the surface of the blood-stage merozoite through its interaction with the glycophosphatidylinositol-anchored Pf12. Our recent structural characterization of Pf12 revealed two juxtaposed 6-Cys domains (D1 and D2). Pf41, however, contains an additional segment of 120 residues predicted to form a large spacer separating its two 6-Cys domains. To gain insight into the assembly mechanism and overall architecture of the Pf12-Pf41 complex, we first determined the 2.45 Å resolution crystal structure of Pf41 using zinc single-wavelength anomalous dispersion. Structural analysis revealed an unexpected domain organization where the Pf41 6-Cys domains are, in fact, intimately associated and the additional residues instead map predominately to an inserted domain-like region (ID) located between two β-strands in D1. Notably, the ID is largely proteolyzed in the final structure suggesting inherent flexibility. To assess the contribution of the ID to complex formation, we engineered a form of Pf41 where the ID was replaced by a short glycine-serine linker and showed by isothermal titration calorimetry that binding to Pf12 was abrogated. Finally, protease protection assays showed that the proteolytic susceptibility of the ID was significantly reduced in the complex, consistent with the Pf41 ID directly engaging Pf12. Collectively, these data establish the architectural organization of Pf41 and define an essential role for the Pf41 ID in promoting assembly of the Pf12-Pf41 heterodimeric complex.

Structural and Immunological Characterization of Recombinant 6-Cysteine Domains of the Plasmodium falciparum Sexual Stage Protein Pfs230

“…In addition, the two disulfide bonds within Pfs230D1M were mapped and assigned as C1 to C2 and C3 to C4, which was in agreement with the NMR analysis of recombinant Pfs12, another malaria protein with a comparable 6-cysteine-rich motif (29). It is interesting to note that the latter disulfide is homologous to the C3-C6 disulfide pairing observed for Pf12 and Pf41 (29,30).…”

Structural delineation of potent transmission-blocking epitope I on malaria antigen Pfs48/45

“…Disulfide bonds are indicated by red lines. c Secondary structure cartoon representation of Pfs48/45 6C in comparison to known structures of highest homology (PDB IDs: 2YMO [ https://www.rcsb.org/structure/2YMO ] 55 , 4YS4 [ https://www.rcsb.org/structure/4YS4 ] 56 , 2WNK [ https://www.rcsb.org/structure/2WNK ] 57 ). Disulfide bonds are represented as red sticks.…”

Plasmodium P36 determines host cell receptor usage during sporozoite invasion

“…In P. falciparum blood stages, the two 6-cys proteins P41 and P12 interact to form stable heterodimers on the surface of merozoites (Tonkin et al, 2013; Taechalertpaisarn et al, 2012; Parker et al, 2015). Whilst P12 is predicted to be GPI-anchored, P41 lacks a membrane-binding domain, similarly to P36.…”