期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2021
卷号：118
期号：35
DOI：10.1073/pnas.2100514118
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Significance
Malaria parasites invade and replicate within human red blood cells, which lack nuclei and have minimal metabolic activity. To survive, the parasites create new pathways that alter the permeability of the red blood cell membrane, allowing them to import nutrients and export waste. Here, we present the native structure of the three-membered RhopH protein complex, which plays a key role in this process. We determined the structure of this essential complex from a heterogeneous mixture of proteins enriched directly from parasite cell lysate, using a cryo-electron microscopy–enabled endogenous structural proteomics approach. The native structure of the RhopH complex in a soluble, trafficking state helps elucidate the long-standing question of how parasite transmembrane proteins are trafficked to the erythrocyte membrane.
The RhopH complex is implicated in malaria parasites’ ability to invade and create new permeability pathways in host erythrocytes, but its mechanisms remain poorly understood. Here, we enrich the endogenous RhopH complex in a native soluble form, comprising RhopH2, CLAG3.1, and RhopH3, directly from parasite cell lysates and determine its atomic structure using cryo–electron microscopy (cryo-EM), mass spectrometry, and the cryoID program. CLAG3.1 is positioned between RhopH2 and RhopH3, which both share substantial binding interfaces with CLAG3.1 but make minimal contacts with each other. The forces stabilizing individual subunits include 13 intramolecular disulfide bonds. Notably, CLAG3.1 residues 1210 to 1223, previously predicted to constitute a transmembrane helix, are embedded within a helical bundle formed by residues 979 to 1289 near the C terminus of CLAG3.1. Buried in the core of the RhopH complex and largely shielded from solvent, insertion of this putative transmembrane helix into the erythrocyte membrane would likely require a large conformational rearrangement. Given the unusually high disulfide content of the complex, it is possible that such a rearrangement could be initiated by the breakage of allosteric disulfide bonds, potentially triggered by interactions at the erythrocyte membrane. This first direct observation of an exported
Plasmodium falciparum transmembrane protein—in a soluble, trafficking state and with atomic details of buried putative membrane-insertion helices—offers insights into the assembly and trafficking of RhopH and other parasite-derived complexes to the erythrocyte membrane. Our study demonstrates the potential the endogenous structural proteomics approach holds for elucidating the molecular mechanisms of hard-to-isolate complexes in their native, functional forms.
