Structure of the Pf12 and Pf41 heterodimeric complex ofPlasmodium falciparum6-cysteine proteins

Abstract

During the different stages of the Plasmodium life cycle, surface-associated proteins establish key interactions with the host and play critical roles in parasite survival. The 6-cysteine (6-cys) protein family is one of the most abundant surface antigens and expressed throughout the Plasmodium falciparum life cycle. This protein family is conserved across Plasmodium species and plays critical roles in parasite transmission, evasion of the host immune response and host cell invasion. Several 6-cys proteins are present on the parasite surface as hetero-complexes but it is not known how two 6-cys proteins interact together. Here, we present a crystal structure of Pf12 bound to Pf41 at 2.85 Å resolution, two P. falciparum proteins usually found on the parasite surface of late schizonts and merozoites. Our structure revealed two critical interfaces required for complex formation with important implications on how different 6-cysteine proteins may interact with each other. Using structure-function analyses, we identified important residues for Pf12-Pf41 complex formation. In addition, we generated 16 nanobodies against Pf12 and Pf41 and showed that several Pf12-specific nanobodies inhibit Pf12-Pf41 complex formation. Using X-ray crystallography, we were able to describe the structural mechanism of an inhibitory nanobody in blocking Pf12-Pf41 complex formation. Future studies using these inhibitory nanobodies will be useful to determine the functional role of these two 6-cys proteins in malaria parasites.

Plasmodium 6-Cysteine Proteins: Functional Diversity, Transmission-Blocking Antibodies and Structural Scaffolds

“…Expression of recombinant Pf12 ( Tonkin et al., 2013 ), Pf12p ( Dietrich et al., 2021 ) and Pf41 ( Parker et al., 2015 ) via this system has yielded high resolution crystal structures of these proteins, as well as the Pf12-Pf41 complex ( Dietrich et al., 2022 ). Recombinant Pfs47 expressed using the baculovirus system was used to identify its binding partner, the mosquito receptor protein P47Rec, and elucidate the involvement of this interaction in evading the mosquito immune system ( Molina-Cruz et al., 2020 ).…”

“…While the overall fold and the spatial position of the disulfide bonds are similar in Pf12, Pf41 and Pf12p, their amino acid sequence identity is low (18-27%), and loops connecting β-strands vary especially in length and conformation. In the case of Pf41, a ~110 amino acid insertion, termed the inserted domain-like region (ID), connects the last two β-strands of the D1 domain and is critical for the interaction with Pf12 ( Parker et al., 2015 ; Dietrich et al., 2022 ).…”

“…Several members of the 6-cysteine protein family form hetero-complexes, such as Pfs230 and Pfs48/45, Pf12 and Pf41, and Pf36 and Pf52 ( Kumar, 1987 ; Kumar and Wizel, 1992 ; Taechalertpaisarn et al., 2012 ; Parker et al., 2015 ; Arredondo et al., 2018 ). Recently, the first crystal structure of a 6-cysteine hetero-complex of Pf12 and Pf41 was determined ( Figure 3A ) ( Dietrich et al., 2022 ). This Pf12-Pf41 structure identified two distinct binding sites and showed that the ID of Pf41 forms a 25 amino acid-long α-helix that binds to a concave surface of the Pf12 D2 domain.…”

“…Critical residues for complex formation have been identified on both proteins, suggesting that both binding sites are important for the interaction of Pf12 and Pf41. In addition, Pf12 specific nanobodies were able to inhibit complex formation between Pf12 and Pf41 ( Dietrich et al., 2022 ). The crystal structure of one of these nanobodies, Nb G7, in complex with recombinant Pf12 showed that Nb G7 bound to a hydrophobic groove on Pf12 that overlaps with the Pf41 ID binding site ( Figure 3B ).…”

“…Pf12 is expressed on the surface of schizonts and merozoites ( Taechalertpaisarn et al., 2012 ; Tonkin et al., 2013 ). Pf12 forms a heterodimer with Pf41 ( Taechalertpaisarn et al., 2012 ; Crosnier et al., 2013 ; Tonkin et al., 2013 ; Parker et al., 2015 ; Dietrich et al., 2022 ), another blood stage 6-cysteine protein with similar localization patterns ( Sanders et al., 2005 ). Pf41 has two 6-cysteine domains and no GPI anchor, likely being tethered to the membrane via its interaction with Pf12.…”