Abstract
Surface-associated proteins play critical roles in the Plasmodium parasite life cycle and are major targets for vaccine development. The 6-cysteine (6-cys) protein family is expressed in a stage-specific manner throughout Plasmodium falciparum life cycle and characterized by the presence of 6-cys domains, which are β-sandwich domains with conserved sets of disulfide bonds. Although several 6-cys family members have been implicated to play a role in sexual stages, mosquito transmission, evasion of the host immune response and host cell invasion, the precise function of many family members is still unknown and structural information is only available for four 6-cys proteins. Here, we present to the best of our knowledge, the first crystal structure of the 6-cys protein Pf12p determined at 2.8 Å resolution. The monomeric molecule folds into two domains, D1 and D2, both of which adopt the canonical 6-cys domain fold. Although the structural fold is similar to that of Pf12, its paralog in P. falciparum, we show that Pf12p does not complex with Pf41, which is a known interaction partner of Pf12. We generated 10 distinct Pf12p-specific nanobodies which map into two separate epitope groups; one group which binds within the D2 domain, while several members of the second group bind at the interface of the D1 and D2 domain of Pf12p. Characterization of the structural features of the 6-cys family and their associated nanobodies provide a framework for generating new tools to study the diverse functions of the 6-cys protein family in the Plasmodium life cycle.
Highlights
Plasmodium falciparum is the most lethal of human malaria species and responsible for the majority of malaria-related deaths [1]
After two rounds of biopanning, we identified 10 distinct nanobody clonal groups based on differences in the amino acid sequence of the complementary determining region 3 (CDR3) (Figure 1A)
Members of the 6-cys family of proteins are conserved across Plasmodium species and play critical roles in parasite invasion, fertilization, transmission, and host immune evasion
Summary
Plasmodium falciparum is the most lethal of human malaria species and responsible for the majority of malaria-related deaths [1]. One of the key protein families in P. falciparum is the 6-cysteine (6-cys) protein family with members representing some of the most abundant surface-expressed proteins across all stages of the malaria parasite life cycle [2]. The 6-cys proteins interact with specific human or mosquito proteins for entry into host tissues or to evade the host immune response to promote survival of the malaria parasites [3,4,5,6]. Several members of the 6-cys proteins are involved in parasite sexual development and fertilization of gametes [7,8]. Pf92 plays a role in immune evasion by recruiting human complement regulator Factor H, which is the major complement regulator of the alternative pathway of complement This recruitment serves to downregulate complement activation on the merozoite surface and protect P. falciparum merozoites from complement-mediated lysis [5]
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