Structure of Rhoptry Neck Protein 2 is essential for the interaction in vitro with Apical Membrane Antigen 1 in Plasmodium vivax

Abstract

Background

In several Apicomplexa, the formation of moving junctions (MJs) at the interface between the external membranes of the invading parasite and the host cell is essential for the process of parasite invasion. In Plasmodium falciparum and Toxoplasma gondii, the MJ is composed of the Apical Membrane Antigen 1 (AMA1) and Rhoptry Neck Proteins (RONs) complex; specifically, AMA1 interacts with RON2 during host cell invasion.

Methods

Recombinant proteins based on Plasmodium vivax RON2 (A2033-P2100) and its synthetic peptide fragments, one cyclic and one linear, based on PvRON2 (D2035-T2074) were generated and used to evaluate the interaction with P. vivax AMA1 (PvAMA1) by the far western blot, surface plasmon resonance (SPR), and isothermal titration microcalorimetry (ITC) methods. The structural studies of peptides were performed by circular dichroism, and the structural analysis of the complex of PvAMA1 with peptides based on PvRON2 (D2035-T2074) was conducted with small-angle X-ray scattering (SAXS).

Results

Surface plasmon resonance (KD = 23.91 ± 2.078 μmol/L) and ITC (K = 3 × 10⁵ mol/L) studies conclusively showed an interaction between the cyclic peptide based on PvRON2 and PvAMA1-His₆. In contrast, the linear peptide and recombinant PvRON2 (GST fusion protein) did not show an interaction with PvAMA1. However, the interaction among recombinant proteins PvRON2.2 and PvAMA1-His₆ was possible to show by far western blot.

Conclusions

The results show that the PvRON2 structure, particularly the S–S bond between C2051 and C2063, is determinant for the existence of the interaction between PvAMA1 and PvRON2.