Biology subjects

Cook, A. D.

Publications and source records attributed to Cook, A. D..

8 recordsLinked to original sources

bioRxiv2026

An African trypanosome surface protein inhibits amplification of the complement system

African trypanosomes replicate within the blood and tissue spaces of their mammalian hosts. As extracellular pathogens, they are constantly exposed to immune cells and molecules and are susceptible to killing by components of the complement system. In this study, we conducted a large-scale screen of a panel of human complement components against a set of putative T. brucei surface proteins and discovered a novel T. brucei receptor that binds to complement Factor B. Biochemical and structural characterisation of this protein revealed it to be a potent inhibitor of the C3bBb convertase, a central enzyme in amplification of the complement cascade. Structural studies show that this C3bBb receptor bridges C3b and Bb in a conformation which is incompatible with its catalytic activity, directly blocking C3bBb convertase function. This reveals a novel mechanism of C3bBb convertase regulation and deepens insight into how the African trypanosome cell surface has evolved to evade complement-mediated killing.

immunology↗

bioRxiv2026

A Trypanosoma brucei gambiense gene variant confers human serum resistance

Most species of African trypanosomes cannot infect humans due to the presence in our blood of trypanolytic factors (TLFs). These lipoprotein particles contain the apolipoprotein L1 (ApoLI) toxin which, when internalised by trypanosomes, forms pores and causes cell death. However, two subspecies of Trypanosoma brucei have evolved resistance to TLFs and cause Human African Trypanosomiasis (HAT). The mechanism of resistance of T. b. rhodesiense requires a single additional molecule, the serum resistance associated protein SRA. However, the mechanism of resistance of T. b. gambiense, which causes HAT in West Africa, has not been fully understood. Here we identify a single polymorphic variant of a PLAC8-domain containing protein which is required for human serum resistance and call this T. b. gambiense-specific resistance variant, TgsRV. African trypanosomes are coated with a dense layer of many copies of one member of the variant surface glycoprotein protein family (VSGs). For cells expressing some VSGs, TgsRV is sufficient for human serum resistance, while cells which express other VSGs require a second protein, TgsGP in addition to TgsRV. We therefore complete the identification of the molecular players required for human serum resistance by the African trypanosomes.

microbiology↗

bioRxiv2025

Cell surface localisation of GPI-anchored receptors in Trypanosoma brucei

Trypanosoma brucei, the causal agent of Human and Animal African trypanosomiasis proliferates in the extracellular milieu of mammals. It acquires host macromolecular nutrients, by receptor mediated endocytosis. The best characterised cell surface receptor is for transferrin (TfR) and it has been reported to be preferentially localised in the flagellar pocket domain of the plasma membrane, the sole site of endocytosis. In this location the TfR may be inaccessible to adaptive immune system effectors. The T. brucei genome encodes [~]15 TfR variants, and here we compared two, the first attached to the plasma membrane by a single glycosylphosphatidylinositol (GPI)-anchor and the other by two. Transferrin uptake kinetics were similar and rapid for both. Unexpectedly, initial binding of transferrin occurred over the whole cell surface suggesting the TfR was not localised solely in the flagellar pocket. This localisation was confirmed by immunofluorescence assays and was independent of the number of GPI-anchors. Two other GPI-anchored receptors were investigated to determine whether localisation to the whole cell surface was a general property of GPI-anchored receptors. Haptoglobin-haemoglobin uptake assays and immunofluorescence localisation of complement factor H receptor showed both were also whole cell surface localised. The mechanisms by which trypanosome receptors are protected from antibody-mediated attack are more complex than hiding in a pocket.

microbiology↗

bioRxiv2023

Molecular mechanism of complement inhibition by the trypanosome receptor ISG65

African trypanosomes replicate within infected mammals where they are exposed to the complement system. This system centres around complement C3, which is present in a soluble form in serum but becomes covalently deposited onto the surfaces of pathogens after proteolytic cleavage to C3b. Membrane-associated C3b triggers different complement-mediated effectors which promote pathogen clearance. To counter complement-mediated clearance, African trypanosomes have a cell surface receptor, ISG65, which binds to C3b and which decreases the rate of trypanosome clearance in an infection model. However, the mechanism by which ISG65 reduces C3b function has not been determined. We reveal through cryogenic electron microscopy that ISG65 has two distinct binding sites for C3b, only one of which is available in C3 and C3d. We show that ISG65 does not block the formation of C3b or the function of the C3 convertase which catalyses the surface deposition of C3b. However, we show that ISG65 forms a specific conjugate with C3b, perhaps acting as a decoy. ISG65 also occludes the binding sites for complement receptors 2 and 3, which may disrupt recruitment of immune cells, including B cells, phagocytes and granulocytes. This suggests that ISG65 protects trypanosomes by combining multiple approaches to dampen the complement cascade. Impact StatementA structure derived from cryogenic electron microscopy shows how ISG65, an African trypanosome receptor that aids virulence, binds C3b and suggests mechanisms through which ISG65 might aid complement resistance.

microbiology↗

bioRxiv2022

Mechanochemical tuning of a kinesin motor essential for malaria parasite transmission

Kinesins are a superfamily of molecular motors that undertake ATP-dependent microtubule-based movement or regulate microtubule dynamics. Plasmodium species, which cause malaria and kill hundreds of thousands annually, encode 8-9 kinesins in their genomes. Of these, two - kinesin-8B and kinesin-8X -are canonically classified as kinesin-8s, which in other eukaryotes typically modulate microtubule dynamics. Unexpectedly, Plasmodium kinesin-8B is required for development of the flagellated male gamete in the mosquito host, and its absence completely blocks parasite transmission. To understand the molecular basis of kinesin-8Bs essential role, we characterised the in vitro properties of the kinesin-8B motor domains from P. berghei and P. falciparum. Both motors drive plus-end directed ATP-dependent microtubule gliding, but also catalyse ATP-dependent microtubule depolymerisation. We determined the microtubule-bound structures of these motors using cryo-electron microscopy. P. berghei and P. falciparum kinesin-8B exhibit a very similar mode of microtubule interaction, in which Plasmodium-distinct sequences at the microtubule-kinesin interface influence motor function. Intriguingly, however, P. berghei kinesin-8B exhibits a non-canonical structural response to ATP analogue binding such that neck linker docking is not induced. Nevertheless, the neck linker region is absolutely required for motility and depolymerisation activities of these motors. Taken together, these data suggest that the mechanochemistry of Plasmodium kinesin-8Bs has been functionally tuned to efficiently contribute to flagella formation.

biochemistry↗

bioRxiv2021

Molecular mechanism of a parasite kinesin motor and implications for its inhibition

Plasmodium parasites cause malaria and are responsible annually for hundreds of thousands of deaths. They have a complex life cycle in which distinct stages are transmitted between, and reproduce in, human and mosquito hosts. In the light of emerging resistance to current therapies, components of the parasite replicative machinery are potentially important targets for anti-parasite drugs. Members of the superfamily of kinesin motors play important roles in the microtubule-based replicative spindle machinery, and kinesin-5 motors are established anti-mitotic targets in other disease contexts. We therefore studied kinesin-5 from Plasmodium falciparum (PfK5) and characterised the biochemical properties and structure of the PfK5 motor domain. We found that the PfK5 motor domain is an ATPase with microtubule plus-end directed motility. We used cryo-EM to determine the motors microtubule-bound structure in no nucleotide and AMPPNP-bound states. Despite significant sequence divergence in this motor, these structures reveal that this parasite motor exhibits classical kinesin mechanochemistry. This includes ATP-induced neck-linker docking to the motor domain, which is consistent with the motors plus-ended directed motility. Crucially, we also observed that a large insertion in loop5 of the PfK5 motor domain creates a dramatically different chemical environment in the well characterised human kinesin-5 drug-binding site. Our data thereby reveal the possibility for selective inhibition of PfK5 and can be used to inform future exploration of Plasmodium kinesins as anti-parasite targets.

biophysics↗

bioRxiv2019

A microtubule RELION-based pipeline for cryo-EM image processing

Microtubules are polar filaments built from {beta}-tubulin heterodimers that exhibit a range of architectures in vitro and in vivo. Tubulin heterodimers are arranged helically in the microtubule wall but many physiologically relevant architectures exhibit a break in helical symmetry known as the seam. Noisy 2D cryo-electron microscopy projection images of pseudo-helical microtubules therefore depict distinct but highly similar views owing to the high structural similarity of - and {beta}-tubulin. The determination of the {beta}-tubulin register and seam location during image processing is essential for alignment accuracy that enables determination of biologically relevant structures. Here we present a pipeline designed for image processing and high-resolution reconstruction of cryo-electron microscopy microtubule datasets, based in the popular and user-friendly RELION image-processing package, Microtubule RELION-based Pipeline (MiRP). The pipeline uses a combination of supervised classification and prior knowledge about geometric lattice constraints in microtubules to accurately determine microtubule architecture and seam location. The presented method is fast and semi-automated, producing near-atomic resolution reconstructions with test datasets that contain a range of microtubule architectures and binding proteins.\n\nAbbreviationsMiRP, Microtubule RELION-based Pipeline; cryo-EM, cryo-electron microscopy; MT, microtubule; CTF, contrast transfer function; PF, protofilament.

molecular biology↗

bioRxiv2019

Structural determinants of microtubule minus end preference in CAMSAP CKK domains

CAMSAP/Patronins regulate microtubule minus-end dynamics. Their end specificity is mediated by their CKK domains, which we proposed recognise specific tubulin conformations found at minus ends. To critically test this idea, we compared the human CAMSAP1 CKK domain (HsCKK) with a CKK domain from Naegleria gruberi (NgCKK), which has lost minus-end specificity. Near-atomic cryo-electron microscopy structures of HsCKK- and NgCKK-microtubule complexes show that these CKK domains share the same protein fold, bind at the intradimer interprotofilament tubulin junction, but exhibit subtly different footprints on microtubules. Whereas NgCKK binding does not alter the microtubule architecture, HsCKK remodels its microtubule interaction site and changes the underlying polymer structure because the tubulin lattice conformation is not optimal for its binding. NMR experiments show that HsCKK is remarkably rigid, supporting this remodelling ability. Thus, in contrast to many MAPs, CKK domains can differentiate subtly specific tubulin conformations to enable microtubule minus-end recognition.

biophysics↗