Efforts Towards the Cryo-EM Structural Characterization of Plasmodium Falciparum Gametocyte Development 1 Protein
Malaria remains a global public health crisis, resulting in almost 250 million cases and causing over half a million deaths in 2022. The most virulent parasite strain P. falciparum is known to cause the most severe and deadly form of the disease in humans. Resistance to artemisinin-based anti-malarial therapeutics widely used to treat P. falciparum infections has spread across several endemic countries and put an emphasis on the development of new therapeutics to combat this growing resistance. At the forefront of the discovery of anti-malarial therapeutics has been the design of transmission-blocking agents targeting the parasite’s ability to differentiate into its transmissible forms and spread among populations. With the development of transmission-blocking therapeutics and prevention of the spread of P. falciparum, this would not only help with efforts towards malaria eradication but would combat the spread of resistant parasite strains to more severely affected endemic countries.
To stop the parasite from differentiating into its transmissible forms, we must better understand the molecular mechanisms governing the parasite’s decision to commit to gametocytogenesis. Along with many other essential processes within P. falciparum, epigenetic regulation plays a key role in controlling the initiation of sexual commitment within the parasite. Recently, it has been shown that induced expression of master regulator of gametocytogenesis, AP2-G, is governed by eviction of P. falciparum heterochromatin protein 1 (HP1) from this gene locus by upstream activator protein P. falciparum Gametocyte Development 1 (GDV1). The exact function of GDV1 and the mechanism by which it evicts HP1 is still unknown.
Here, we aim to solve a high-resolution structure of GDV1 and the loss-of-function mutant GDV1Δ39aa using cryogenic electron microscopy (cryo-EM) to gain a better understanding of how this protein may function to evict HP1, leading to the initiation of sexual commitment within P. falciparum. We hope to elucidate a possible structure-function relationship of GDV1, a protein with no known homologs, in addition to gaining an understanding of specific interactions between GDV1 and HP1 through structural characterization of these two proteins in complex.
Initial efforts towards the purification of P. falciparum GDV1 from expression in a recombinant E. coli system were seen to yield low levels of protein expression. Due to the lack of sufficient expression and solubility of GDV1, we unexpectedly characterized a 2.79Å structure of endogenous E. coli protein, Maltodextrin Phosphorylase (MalP). Optimization of GDV1 expression was done in collaboration with Dr. Isadora Prata and the Llinás lab at Penn State to yield high levels of induced expression of our target protein as compared to levels observed previously. Further optimization of protein solubility is still in progress.
Once a significant level of soluble protein is obtained, we will proceed towards the purification and eventual cryo-EM structural characterization of P. falciparum GDV1, GDV1Δ39aa, and a GDV1-HP1 complex. Overall, these initial efforts in the attempted purification of GDV1 and the unexpected purification and cryo-EM characterization of MalP yielded results that allowed for more targeted optimization of GDV1 expression and now solubility. Lastly, the unexpected purification and characterization of E. coli MalP also provides us with potential insight into a possible contaminant that may create obstacles in future purification attempts of GDV1.
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| Work Title | Efforts Towards the Cryo-EM Structural Characterization of Plasmodium Falciparum Gametocyte Development 1 Protein |
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| License | CC BY 4.0 (Attribution) |
| Work Type | Research Paper |
| Publication Date | May 2, 2024 |
| Deposited | May 02, 2024 |
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