Title The molecular mechanism of cytoadherence to placenta or tumor cells through VAR 2 CSA from Plasmodium falciparum Running Title Structural Insight into VAR 2 CSA Recognizing Both Placental or Tumor Cells

Pregnancy Associated Malaria (PAM) threatens more than one million pregnant women and their infants in endemic regions due to poor outcomes, such as maternal anemia, stillbirth, infant death, etc. VAR2CSA, a 350 kDa transmembrane protein encoded by Plasmodium falciparum (P.falciparum), plays a vital role in the cytoadherence of infected erythrocytes (IEs) to placenta. Chondroitin sulfate A (CSA), displayed mostly on the surface of placental or tumor cells, has been recognized as the specific ligand for VAR2CSA. However, the architecture and CSA binding mechanism of VAR2CSA remain elusive. In this study, we determined the cryo-EM structures of P. falciparum VAR2CSA ectodomain and its complex with CSA at a resolution of 3.6 Å and 3.4 Å, respectively. Most importantly, it was revealed that CSA binding induces significant conformational change to close the binding pocket by turning DBL2X and DBL1X closer to DBL4ɛ, and meanwhile enlarge the inner binding pocket via slightly moving a CSA-binding helix of DBL2X outward. Impressively, the structural analysis indicated that 9 key residues with positive charge in DBL2X might be mainly responsible for CSA binding, which is further validated by multidisciplinary methods, i.e., sequence alignment, site mutagenesis, CSA binding tests, and cytoadherence assays using confocal fluorescence microscopy. In summary, we elucidated the detailed molecular mechanism of cytoadherence to placenta or tumor cells through VAR2CSA, which may facilitate antigen design for PAM vaccine, and improve the drug delivery systems targeting both placenta and tumors.