Structure–function analysis of nucleotide housekeeping protein HAM1 from human malaria parasite Plasmodium falciparum

Saha, Debanjan; Pramanik, Atanu; Freville, Aline; Siddiqui, Asim Azhar; Pal, Uttam; Banerjee, Chinmoy; Nag, Shiladitya; Debsharma, Subhashis; Pramanik, Saikat; Mazumder, Somnath; Maiti, Nakul C.; Datta, Saumen; van Ooij, Christiaan; Bandyopadhyay, Uday

Abstract

Non-canonical nucleotides, generated as oxidative metabolic by-products, significantly threaten the genome integrity of Plasmodium falciparum and thereby, their survival, owing to their mutagenic effects. PfHAM1, an evolutionarily conserved inosine/xanthosine triphosphate pyrophosphohydrolase, maintains nucleotide homeostasis in the malaria parasite by removing non-canonical nucleotides, although structure–function intricacies are hitherto poorly reported. Here, we report the X-ray crystal structure of PfHAM1, which revealed a homodimeric structure, additionally validated by size-exclusion chromatography–multi-angle light scattering analysis. The two monomeric units in the dimer were aligned in a parallel fashion, and critical residues associated with substrate and metal binding were identified, wherein a notable structural difference was observed in the β-sheet main frame compared to human inosine triphosphate pyrophosphatase. PfHAM1 exhibited Mg++-dependent pyrophosphohydrolase activity and the highest binding affinity to dITP compared to other non-canonical nucleotides as measured by isothermal titration calorimetry. Modifying the pfham1 genomic locus followed by live-cell imaging of expressed mNeonGreen-tagged PfHAM1 demonstrated its ubiquitous presence in the cytoplasm across erythrocytic stages with greater expression in trophozoites and schizonts. Interestingly, CRISPR-Cas9/DiCre recombinase-guided pfham1-null P. falciparum survived in culture under standard growth conditions, indicating its assistive role in non-canonical nucleotide clearance during intra-erythrocytic stages. This is the first comprehensive structural and functional report of PfHAM1, an atypical nucleotide-cleansing enzyme in P. falciparum.

Citation

Saha, D., Pramanik, A., Freville, A., Siddiqui, A. A., Pal, U., Banerjee, C., Nag, S., Debsharma, S., Pramanik, S., Mazumder, S., Maiti, N. C., Datta, S., van Ooij, C., & Bandyopadhyay, U. (2024). Structure–function analysis of nucleotide housekeeping protein HAM1 from human malaria parasite Plasmodium falciparum. The FEBS Journal, 291(19), 4349-4371. https://doi.org/10.1111/febs.17216

• Journal Article Type: Article
• Acceptance Date: Jun 20, 2024
• Online Publication Date: Jul 14, 2024
• Publication Date: 2024-10
• Deposit Date: Feb 25, 2025
• Journal: The FEBS Journal
• Print ISSN: 1742-464X
• Electronic ISSN: 1742-4658
• Publisher: Wiley
• Peer Reviewed: Peer Reviewed
• Volume: 291
• Issue: 19
• Pages: 4349-4371
• DOI: https://doi.org/10.1111/febs.17216
• Keywords: CRISPR-Cas9/DiCre; molecular dynamics; non-canonical bases; Plasmodium falciparum; X-ray crystallography
• Public URL: https://keele-repository.worktribe.com/output/1079173
• Publisher URL: https://febs.onlinelibrary.wiley.com/doi/10.1111/febs.17216
• Additional Information: Received: 2024-01-20; Accepted: 2024-06-20; Published: 2024-07-14