Dietrich, Melanie H.Chan, Li JinAdair, AmyBoulet, CoralieO'Neill, Matthew T.Tan, Li LynnKeremane, SravyaMok, Yee FoongLo, Alvin W.Gilson, PaulTham, Wai Hong2025-05-262025-05-26ORCID:/0000-0001-7950-8699/work/218987879http://www.scopus.com/inward/record.url?scp=85134698546&partnerID=8YFLogxKhttps://hdl.handle.net/1885/733753761During 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.We acknowledge the traditional custodians of the lands on which this project was conducted, the Wurundjeri and the Bunurong people of the Kulin Nation. We thank Janet Newman and Bevan Marshall from the CSIRO Collaborative Crystallization Centre ( www.csiro.au/C3 ) (CSIRO; Parkville, Australia) for assistance with setting up the crystallization screens. This research was undertaken using the MX2 beamline at the Australian Synchrotron, part of ANSTO, and made use of the Australian Cancer Research Foundation (ACRF) detector. We also thank the MX2 beamline staff at the Australian Synchrotron for their assistance during data collection. We thank Michael Lawrence for his invaluable advice on the structure refinement of the heterodimeric complex of Pf12 and Pf41. W.-H.T. is a Howard Hughes Medical Institute-Wellcome Trust International Research Scholar (208693/Z/17/Z) and supported by National Health and Medical Research Council of Australia (APP2001385, GNT1143187, GNT1160042, GNT1160042, GNT1154937). We thank The Australian Red Cross Blood Bank for the supply of human erythrocytes.enPublisher Copyright: © 2022 The Author(s). Published by Oxford University Press on behalf of FEMS.6-cysteine proteinsblood stagesmalariaNanobodiesPlasmodium falciparumX-ray crystallography202210.1093/femsmc/xtac00585134698546