Structural analyses of Trichomonas vaginalis pyrophosphate-dependent phosphofructokinase (TvPPi-PFK)

Chiu, A.; Liu, L.; Seibold, S.; Battaile, K.; Craig, J.; Harmon, E.; Subramanian, S.; Chakafana, G.; Early, J.; Cron, L.; Staker, B.; Myler, P. J.; Lovell, S. J.; Van Voorhis, W.; Asojo, O.

doi:10.64898/2026.03.28.715000

bioRxiv · 10.64898/2026.03.28.715000

Structural analyses of Trichomonas vaginalis pyrophosphate-dependent phosphofructokinase (TvPPi-PFK)

Chiu, A.·Liu, L.·Seibold, S.·Battaile, K.·Craig, J.·Harmon, E.·Subramanian, S.·Chakafana, G.·Early, J.·Cron, L.·Staker, B.·Myler, P. J.·Lovell, S. J.·Van Voorhis, W.·Asojo, O.

View original source ↗Export metadata ↓

Abstract

Trichomonas vaginalis causes trichomoniasis, the most common non-viral sexually transmitted disease in humans. T. vaginalis pyrophosphate-dependent phosphofructokinase (TvPPi-PFK) is a putative target for rational, structure-based drug discovery, given its absence in mammals and its importance for parasite survival. TvPPi-PFK is a cytosolic enzyme that catalyzes the phosphorylation of fructose-6-phosphate using pyrophosphate (PPi) as the phosphoryl donor. This reversible reaction, catalyzed by TvPPi-PFK, is the first committed step in glycolysis. Its reverse reaction is vital for gluconeogenesis in T. vaginalis. The purification, crystallization, structure determination, and crystal structures of TvPPi-PFK are reported. TvPPi-PFK is the first reported eukaryotic PPi-PFK structure. TvPPi-PFK retains the overall PPi-PFK topology observed in bacterial PPi-PFK including conserved motifs essential for pyrophosphate binding and PPi-PFK catalytic activity. In addition to the catalytic PPi-PFK binding sites, TvPPi-PFK has two additional ligand binding sites. The first binds AMP usurped during protein production and helps stabilize the TvPPi-PFK tetramer. A second ligand binding site was observed in proximity to the AMP-binding site and accommodates sugar phosphates soaked into preformed crystals. This sugar phosphates binding site is distinct from the TvPPi-PFK active site that binds fructose-6-phosphate. Future mutagenesis and activity studies are planned to determine the relevance of both sites. SynopsisThe production, crystallization, and crystal structures of a pyrophosphate-dependent phosphofructokinase from Trichomonas vaginalis (TvPPi-PFK) are reported. TvPPi-PFK has a prototypical PPi-PFK active site as well as unexpected AMP and sugar-phosphate binding sites at the dimer interface.

Explore related subjects

biochemistry Preprint

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Chiu, A., Liu, L., Seibold, S., Battaile, K., Craig, J., Harmon, E., Subramanian, S., Chakafana, G., Early, J., Cron, L., Staker, B., Myler, P. J., Lovell, S. J., Van Voorhis, W., Asojo, O.. 2026-03-28. Structural analyses of Trichomonas vaginalis pyrophosphate-dependent phosphofructokinase (TvPPi-PFK). https://doi.org/10.64898/2026.03.28.715000

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

aaRSID, an engineered pyrrolysyl-tRNA synthetase platform for multi-probe proximity proteomics

Proximity labeling (PL) methods utilize spatially targeted chemical or enzymatic generation of a diffusible, reactive intermediate to covalently tag neighboring proteins in living systems. Unlike other tools for studying molecular interactions, PL can detect transient protein relationships with high spatial and temporal sensitivity, allowing for insight into their roles in biological processes. However, current enzymatic PL tools, such as TurboID and APEX2, are limited by their substrate structure and chemistry, which can generate significant background and/or perturb cellular physiology. To address these limitations, we have developed aminoacyl-tRNA synthetase ID (aaRSID), a PL tool that leverages an engineered pyrrolysyl tRNA synthetase (PylRS) for proximity labeling of proteins. We chose PylRS because it can catalyze promiscuous lysine labeling in the absence of its cognate tRNA and utilize a variety of non-canonical amino acids (ncAAs) as substrates. Here, we demonstrate aaRSID's intrinsic proximity labeling activity, use directed evolution to improve this activity, and apply the improved mutant (aaRSID-Ma1.3) for subcellular proteomics and multiplexed imaging. Our work establishes aminoacyl-tRNA synthetases as a new PL enzyme class and introduces a versatile chemical platform for developing ncAA-derived probes to map cellular microenvironments, greatly expanding the applications possible of PL technology.

biochemistry↗

bioRxiv2026

Cellular uptake of folate-olaparib conjugates via folate receptor-mediated endocytosis: Potential for selective delivery of DNA damage response inhibitors into tumour cells

The folate receptor (FR) is overexpressed in a range of human tumours including ovarian cancer cells. We propose that the overexpression of the FR on the surface of ovarian tumour cells could be exploited for the selective delivery of a DNA damage response inhibitor (DDRi) in the form of an intact folate drug conjugate (FDC). This approach would improve the therapeutic index of the parent DDRi facilitating combination studies of the DDRi-based FDC with DNA damaging chemotherapy. FR-mediated cellular uptake of the proposed folate drug conjugates is requisite for FDC selective delivery into tumours. In this study, we synthesised a series of olaparib-based folate conjugates that maintained the biochemical PARP1 inhibition associated with olaparib and showed binding affinity for the folate receptor. Significantly, we identified compounds 10b and 11 that selectively enter FR overexpressing tumour cells via folate receptor-mediated endocytosis in their intact form and engage with their target as demonstrated by the potent inhibition of PARylation (KB cells, PARylation IC50 = 5.7 and 3.9 nM; respectively).

biochemistry↗

bioRxiv2026

Architecture and Energy Transfer of the Bacterial Photosynthetic Unit

In phototrophic organisms, pigment-protein membrane complexes are densely packed to form photosynthetic units (PSUs) that capture solar energy and convert it into chemical energy. Although the structures of many individual photosynthetic complexes have been resolved, how they are arranged and interact with others within photosynthetic membranes to enable efficient excitation energy transfer (EET) remains poorly understood. Here, we report cryo-electron microscopy structures of PSU supercomplex assemblies from the phototrophic a-proteobacterium Rhodovulum viride, including an RC-LH1 core associated with one or two peripheral LH2 complexes and a curved LH2 tetramer. These membrane-derived assemblies define the relative positions and orientations of neighboring photosynthetic complexes and place their pigment arrays in proximity across antenna-antenna and antenna-core interfaces. Structure-based simulations identify potential EET pathways within the PSU assemblies and reveal rapid energy transfer across both LH2-LH2 and LH2-LH1 interfaces. Collectively, these findings provide insights into the assembly and structural modularity of bacterial PSUs and elucidate how the lateral organization of membrane protein complexes facilitates efficient energy transfer. This work extends structural studies of bacterial photosynthesis from individual complexes to their native higher-order assembly, providing a framework for understanding how photosynthetic supercomplex organization shapes energy migration and for guiding the design of artificial photosynthesis.

biochemistry↗