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?