4. In solution, P2 forms a stable tetramer which is a molten globule in nature having hydrophobic pockets on the surface
NMR experiments in solution distinctly revealed that recombinant P2 protein has a high propensity to oligomerize and tend to form aggregates by self-association at a millimolar concentration (Mishra et al., 2012; Mishra et al., 2014a). Further exploration using circular dichroism (CD) and solution NMR has also revealed that the recombinant monomeric species of P2 is predominantly α helical but molten globule in nature and the ‘C’ terminal region is intrinsically disordered in structure (Mishra et al., 2014b, Mishra et al., 2015). At physiological pH 7.4, the thermodynamic stability of the monomer shifts towards tetramerization maintaining the molten globule nature of each monomer in the tetramer (Mishra et al., 2015), thereby keeping the flexibility of the entire tetrameric complex. Urea denaturation of recombinant P2 followed by residue level interrogation using NMR further revealed that two monomeric P2 molecules associate to form a dimer and two such dimer molecules packaged closely at their N terminus having α helices to form the tetramer (Mishra et al., 2015). 2D 1H –15N HSQC spectra of the native deuterated tetrameric form of P2 exhibited hydrophobic surface/pocket mostly contributed by the N terminal α helices as shown using 8-anilinonaphthalene-1-sulfonic acid (ANS) (Mishra et al., 2015) indicating that P2 tetramer could provide sites for intermolecular association in an aqueous environment and may have the possibility to bind non-polar molecules in its natively localized environment, i.e., iRBCs surface. Between P. falciparumP2 and Human P2, there is 69% amino acid sequence homology but they differ in their oligomerization pattern and behavior as Human P2 at physiological pH forms a stable dimer (ref) but in the same conditionP. falciparum P2 forms a molten globule tetramer indicating some functional implications of tetrameric P2 on the surface of iRBCs at late trophozoite stage. In a recent discovery, it has been demonstrated that P2 tetramers stabilize themselves on the iRBC surface by interacting with RBC Band3 protein where N terminal 70 amino acids of P2 interact to form the oligomers and associate with Band 3 protein (Mishra et al., 2020).
5. What could be the possible function of oligomeric/tetrameric P2 on the iRBC surface?
The selectivity of the infected red blood cell membrane is compromised due to the translocation of several effector proteins into the host cells. Virulence factors and channel proteins both are predominant components of the exportome which plays important role in disease biology and solute uptake respectively. After the erythrocyte invasion by malaria parasites, induction of a broad specificity channel known as the new permeability pathway (NPP) into the host cells renders the red blood cell membrane non-selective and promiscuous for a range of small molecule solutes including ions (Kirk et al., 2015). Blockage of NPP by furosemide resulted in the pronounced inhibition of parasite growth in culture suggesting the indispensability of NPP in iRBCs (Staines et al., 2004). One of the channel molecules of NPP has recently been extensively characterized as a plasmodial surface anion channel (PSAC), a widely accepted iRBC channel protein shown to enhance nutrient permeability of iRBCs (Kirk et al., 2015; Alkhalil et al., 2009, Desai et al., 2012). While the enhanced permeability of iRBCs has been experimented out for decades, the molecular identity of PSAC was largely unknown until recently a cytoadherence-linked antigen 3 (Clag3) is the key player of the channel for nutrient uptake (Alkhalil et al., 2009, Desai et al., 2012; Desai et al., 2014; Nguitragool et al., 2014; Gupta et al., 2018). In the formation of PSAC, Clag3 protein forms a homodimer, and RhopH2 and RhopH3 do associate with the dimer for the construction of functional channel (Nguitragool et al., 2014; Gupta et al., 2018; Kaneko et al., 2005; Ito et al., 2017; Sherling et al., 2017; Counihan et al., 2017; Schureck et al., 2021). Under PTEX suppressed condition, Clag3 still translocate into the host cell suggesting an alternative mechanism of Clag3 export but in the PTEX suppressed parasite lines, the transport of solutes by PSAC was diminished (Beck et al., 2014; Comeaux et al., 2011) indicating that other exported proteins are required for channel formation either in association with or independent of Clag3.
Based on experimental evidence, the non-ribosomal role (s) of oligomeric/tetrameric P2 on the iRBC surface appears to be important but currently is at the stage of speculation. The tight regulation of oligomerization and subsequent localization of oligomer/tetramer on the iRBC surface at the trophozoite stage drives the attention towards its direct possible role in the formation of some channel either in association with Clag3 or independently for small molecule transport. P2 has one putative transmembrane (TM) domain from amino acid N’64-84C’ but in the oligomeric state how this TM domain is important for host membrane insertion is currently elusive. Immunofluorescence assay (IFA) at mid to late trophozoite stage showed the presence of Clag3 on iRBC surface co-localized with RhopH3 (Sherling et al., 2017). But at this stage of mid to late trophozoite, P2 oligomers were diminished as IFA using E2G12 did not stain the iRBC surface suggesting that oligomeric P2 may not be a component of PSAC but there is a possibility of channel formation by P2 oligomers either in association with other export proteins or independently. If oligomeric P2 is forming a channel with/without other parasitic proteins then this channel does not seem to complement the function of PSAC under the null state of Clag3.1 and Clag3.2 as these null parasites showed significant growth inhibition (Nguitragool et al., 2014; Gupta et al., 2018; Kaneko et al., 2005), hence there is a possibility that PSAC and putative channel of oligomeric P2 both are working independently.
Oligomeric/tetrameric P2 being molten globule in nature and having hydrophobic pockets on the surface also drives the speculation towards its possible direct interaction with hydrophobic molecules such as lipids. Parasite culture medium devoid of oleic acid and palmitic acid resulted in parasite cell cycle arrest (Mitamura et al., 2000) similar to P2 antibody-mediated arrest (Das et al., 2012a). Hence it could be logical to think and rationale to design experiments to validate the interaction of P2 oligomers with lipids which are already reported to be crucial for parasite progression. In addition to possible channel formation or P2-lipid interaction, oligomeric P2 might have other non-ribosomal function (s) depending on the interaction of P2 with other partner proteins on the iRBC surface and the structure of the entire oligomeric complex.
6. Frontier questions
Finding oligomeric parasite P2 protein on the iRBC surface was puzzling but at the same time raises the possibility to target it. Unraveling the function of P2 on the iRBCs surface could open several avenues towards our effort to develop novel anti-malarial small molecules and might also come under consideration as a possible vaccine candidate as recently reported (Szuster-Ciesielska et al., 2019). Understanding a fundamental non-ribosomal role (s) of a ribosomal protein in the propagation of Plasmodium parasite in red blood cells can be harnessed to develop new strategies to target this highly resilient parasite and will unmask novel biochemical pathways operational at the trophozoite stage of parasite propagation. Based on current understanding, below there are three key unresolved questions depicted in Figure 1 that need to be answered to understand the eccentric P2 cell biology in Plasmodium parasites, and towards that, my laboratory is fully engaged.
What is the function (s) of oligomeric/tetrameric P2 on the iRBC surface at the late trophozoite/early schizogonic stage?
What is the mechanism of P2 translocation from the parasite cytoplasm to the iRBC surface?
What is the nature of P2 oligomers on the iRBC surface? Does it form a channel complex?