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.

Trypanosomes and complement: more than one way to die?

African trypanosomes show a remarkable ability to survive as extracellular parasites in the blood and tissue spaces of an infected mammal. Throughout the infection they are exposed to the molecules and cells of the immune system, including complement. In this opinion piece, we review decades-worth of evidence about how complement affects African trypanosomes. We highlight the discovery of a trypanosome receptor for complement C3 and we critically assess three recent studies which attempt to provide a structural and mechanistic view of how this receptor helps trypanosomes to survive in the presence of complement.

Mechanochemical tuning of a kinesin motor essential for malaria parasite transmission.

Plasmodium species cause malaria and kill hundreds of thousands annually. The microtubule-based motor kinesin-8B is required for development of the flagellated Plasmodium male gamete, and its absence completely blocks parasite transmission. To understand the molecular basis of kinesin-8B's essential role, we characterised the in vitro properties of kinesin-8B motor domains from P. berghei and P. falciparum. Both motors drive ATP-dependent microtubule gliding, but also catalyse ATP-dependent microtubule depolymerisation. We determined these motors' microtubule-bound structures using cryo-electron microscopy, which showed very similar modes 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 required for motility and depolymerisation activities of these motors. These data suggest that the mechanochemistry of Plasmodium kinesin-8Bs is functionally tuned to support flagella formation.

Invariant surface glycoprotein 65 of Trypanosoma brucei is a complement C3 receptor.

African trypanosomes are extracellular pathogens of mammals and are exposed to the adaptive and innate immune systems. Trypanosomes evade the adaptive immune response through antigenic variation, but little is known about how they interact with components of the innate immune response, including complement. Here we demonstrate that an invariant surface glycoprotein, ISG65, is a receptor for complement component 3 (C3). We show how ISG65 binds to the thioester domain of C3b. We also show that C3 contributes to control of trypanosomes during early infection in a mouse model and provide evidence that ISG65 is involved in reducing trypanosome susceptibility to C3-mediated clearance. Deposition of C3b on pathogen surfaces, such as trypanosomes, is a central point in activation of the complement system. In ISG65, trypanosomes have evolved a C3 receptor which diminishes the downstream effects of C3 deposition on the control of infection.

Inhibiting parasite proliferation using a rationally designed anti-tubulin agent.

Infectious diseases caused by apicomplexan parasites remain a global public health threat. The presence of multiple ligand-binding sites in tubulin makes this protein an attractive target for anti-parasite drug discovery. However, despite remarkable successes as anti-cancer agents, the rational development of protozoan parasite-specific tubulin drugs has been hindered by a lack of structural and biochemical information on protozoan tubulins. Here, we present atomic structures for a protozoan tubulin and microtubule and delineate the architectures of apicomplexan tubulin drug-binding sites. Based on this information, we rationally designed the parasite-specific tubulin inhibitor parabulin and show that it inhibits growth of parasites while displaying no effects on human cells. Our work presents for the first time the rational design of a species-specific tubulin drug providing a framework to exploit structural differences between human and protozoa tubulin variants enabling the development of much-needed, novel parasite inhibitors.

Cryo-EM structure of a microtubule-bound parasite kinesin motor and implications for its mechanism and inhibition.

Plasmodium parasites cause malaria and are responsible annually for hundreds of thousands of deaths. Kinesins are a superfamily of microtubule-dependent ATPases that play important roles in the parasite replicative machinery, which is a potential target for antiparasite drugs. Kinesin-5, a molecular motor that cross-links microtubules, is an established antimitotic target in other disease contexts, but its mechanism in Plasmodium falciparum is unclear. Here, we characterized P. falciparum kinesin-5 (PfK5) using cryo-EM to determine the motor's nucleotide-dependent microtubule-bound structure and introduced 3D classification of individual motors into our microtubule image processing pipeline to maximize our structural insights. Despite sequence divergence in PfK5, the motor exhibits classical kinesin mechanochemistry, including ATP-induced subdomain rearrangement and cover neck bundle formation, consistent with its plus-ended directed motility. We also observed that an insertion in loop5 of the PfK5 motor domain creates a different environment in the well-characterized human kinesin-5 drug-binding site. Our data reveal the possibility for selective inhibition of PfK5 and can be used to inform future exploration of Plasmodium kinesins as antiparasite targets.

High-throughput hit-squad tackles trypanosomes.

African trypanosomes cause diseases of humans and their livestock. To date, a much-desired vaccine has been elusive, due in part to the immune evasion mechanisms of these cunning parasites. However, Autheman et al. have used a bold, high-throughput screen to provide hope that vaccines may be on the way.

Structure of Microtubule-Trapped Human Kinesin-5 and Its Mechanism of Inhibition Revealed Using Cryoelectron Microscopy.

Kinesin-5 motors are vital mitotic spindle components, and disruption of their function perturbs cell division. We investigated the molecular mechanism of the human kinesin-5 inhibitor GSK-1, which allosterically promotes tight microtubule binding. GSK-1 inhibits monomeric human kinesin-5 ATPase and microtubule gliding activities, and promotes the motor's microtubule stabilization activity. Using cryoelectron microscopy, we determined the 3D structure of the microtubule-bound motor-GSK-1 at 3.8 Å overall resolution. The structure reveals that GSK-1 stabilizes the microtubule binding surface of the motor in an ATP-like conformation, while destabilizing regions of the motor around the empty nucleotide binding pocket. Density corresponding to GSK-1 is located between helix-α4 and helix-α6 in the motor domain at its interface with the microtubule. Using a combination of difference mapping and protein-ligand docking, we characterized the kinesin-5-GSK-1 interaction and further validated this binding site using mutagenesis. This work opens up new avenues of investigation of kinesin inhibition and spindle perturbation.

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

Microtubules are polar filaments built from αß-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 α- and ß-tubulin. The determination of the αß-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.

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 lacks minus-end specificity. Here we report near-atomic cryo-electron microscopy structures of HsCKK- and NgCKK-microtubule complexes, which show that these CKK domains share the same protein fold, bind at the intradimer interprotofilament tubulin junction, but exhibit different footprints on microtubules. NMR experiments show that both HsCKK and NgCKK are remarkably rigid. However, 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. Thus, in contrast to many MAPs, the HsCKK domain can differentiate subtly specific tubulin conformations to enable microtubule minus-end recognition.

Enhancement of T cell responses as a result of synergy between lower doses of radiation and T cell stimulation.

As a side effect of cancer radiotherapy, immune cells receive varying doses of radiation. Whereas high doses of radiation (>10 Gy) can lead to lymphopenia, lower radiation doses (2-4 Gy) represent a valid treatment option in some hematological cancers, triggering clinically relevant immunological changes. Based on our earlier observations, we hypothesized that lower radiation doses have a direct positive effect on T cells. In this study, we show that 0.6-2.4 Gy radiation enhances proliferation and IFN-γ production of PBMC or purified T cells induced by stimulation via the TCR. Radiation with 1.2 Gy also lowered T cell activation threshold and broadened the Th1 cytokine profile. Although radiation alone did not activate T cells, when followed by TCR stimulation, ERK1/2 and Akt phosphorylation increased above that induced by stimulation alone. These changes were followed by an early increase in glucose uptake. Naive (CD45RA(+)) or memory (CD45RA(-)) T cell responses to stimulation were boosted at similar rates by radiation. Whereas increased Ag-specific cytotoxic activity of a CD8(+) T cell line manifested in a 4-h assay (10-20% increase), highly significant (5- to 10-fold) differences in cytokine production were detected in 6-d Ag-stimulation assays of PBMC, probably as a net outcome of death of nonstimulated and enhanced response of Ag-stimulated T cells. T cells from patients receiving pelvic radiation (2.2-2.75 Gy) also displayed increased cytokine production when stimulated in vitro. We report in this study enhanced T cell function induced by synergistic radiation treatment, with potential physiological significance in a wide range of T cell responses.