Fig. 2. Crystal structure of the N-terminal domain of PvRBP2b and its functional requirement.

(A) Structure of the N-terminal domain of PvRBP2b from amino acid 169 to 470 shown in two orthogonal views. (B) Electrostatic surface potential on the PvRBP2b structure. (C) Superimposition of the PvRBP2b structure (green) with PvRBP2a (purple) and PfRh5 (orange). The PDB ID codes for PfRh5 and PvRBP2a are 4WAT and 4Z8N, respectively. (D) Crystal structure of the N-terminal domain superimposed with SAXS ab initio bead model of PvRBP2b_169-470 (left) and PvRBP2b_169-652 (right). (E) Sliding window analysis showing nucleotide diversity (π) values and Tajima’s D statistic in PvRBP2b. The grey box refers to a highly polymorphic region at amino acid positions 169 to 470 that appears to be under balancing selection. (F) Schematic representation of full-length PvRBP2b and recombinant protein fragments (left). Signal peptide (SP), transmembrane domain (TM) and N-terminal domain (yellow) are indicated. (G) PvRBP2b binding results by flow cytometry where PvRBP2b_161-1454 binding was arbitrarily assigned to be 100%. (H) Unlabeled recombinant PvRBP2b fragments or PfRh4 were mixed at 10-fold molar excess relative to the labeled PvRBP2b_161-1454-TfR1 FRET pair. The FRET intensity was relative to buffer control. For (G to H), Mean ± S.E.M, n = 4, open dots represent biological replicates. The Mann-Whitley test was used to calculate the P value using the binding of 2b_474-1454 that was considered no binding, * P≤ 0.05, ** P ≤ 0.001.