Structural and regulatory insights into the glideosome-associated connector from Toxoplasma gondii
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The authors describe the first full-length crystal structure and solution conformation of the GAC protein from Toxoplasma gondii. The data are convincing and support a model in which GAC uses multiple conformations and lipid-binding surfaces. This paper presents an important contribution to our understanding of the molecular machinery involved in host cell invasion, but questions remain about how this protein links to the cytoskeleton and functions during the invasion process.
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Abstract
The phylum of Apicomplexa groups intracellular parasites that employ substrate-dependent gliding motility to invade host cells, egress from the infected cells, and cross biological barriers. The glideosome-associated connector (GAC) is a conserved protein essential to this process. GAC facilitates the association of actin filaments with surface transmembrane adhesins and the efficient transmission of the force generated by myosin translocation of actin to the cell surface substrate. Here, we present the crystal structure of Toxoplasma gondii GAC and reveal a unique, supercoiled armadillo repeat region that adopts a closed ring conformation. Characterisation of the solution properties together with membrane and F-actin binding interfaces suggests that GAC adopts several conformations from closed to open and extended. A multi-conformational model for assembly and regulation of GAC within the glideosome is proposed.
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eLife assessment
The authors describe the first full-length crystal structure and solution conformation of the GAC protein from Toxoplasma gondii. The data are convincing and support a model in which GAC uses multiple conformations and lipid-binding surfaces. This paper presents an important contribution to our understanding of the molecular machinery involved in host cell invasion, but questions remain about how this protein links to the cytoskeleton and functions during the invasion process.
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Reviewer #1 (Public Review):
The glideosome-associated connector is an essential piece of the machinery used by the apicomplexa parasites as they invade host cells. This GAC makes important interactions with the membrane and with actin during this process. Here, Kumar et al present the first structure of the GAC from T. gondii, showing a complex fold in a closed form. This structure was determined at pH 5, and they show that at more physiological pH values the structure is far more open. However, this is not in the context of actin, membrane, or other binding partners, and so the question remains about how open the structure is in its physiological context. The authors next use molecular dynamics, NMR, and mutagenesis to identify the residues involved in membrane binding and also assess actin binding through modelling which is not …
Reviewer #1 (Public Review):
The glideosome-associated connector is an essential piece of the machinery used by the apicomplexa parasites as they invade host cells. This GAC makes important interactions with the membrane and with actin during this process. Here, Kumar et al present the first structure of the GAC from T. gondii, showing a complex fold in a closed form. This structure was determined at pH 5, and they show that at more physiological pH values the structure is far more open. However, this is not in the context of actin, membrane, or other binding partners, and so the question remains about how open the structure is in its physiological context. The authors next use molecular dynamics, NMR, and mutagenesis to identify the residues involved in membrane binding and also assess actin binding through modelling which is not validated by experiment. This paper presents an important contribution to our understanding of the molecular machinery involved in host cell invasion but leaves many questions remaining about how this protein links to the cytoskeleton and functions during the invasion process.
• The structure of TgGAC provides the first such structure of this complex and is an important contribution to our understanding. The structure presented in Figure 1A is a composite, containing the crystal structure of the majority of the protein, determined at pH 5, to which has been docked the PH domain structure, determined by NMR. It would be good to see more clarity in the figure about what is experimentally determined and what is modelled.
• SAXS data shows that, at pH 8, a substantial fraction of the protein is in a very extended conformation, which differs significantly from the compact structure seen in crystals at pH 5. I would prefer to see the models in Figure 2d represented as spheres or surfaces, to prevent over-interpretation associated with showing models with low-resolution data. However, the SAXS findings are robust and this is clearly a dynamic molecule in solution. It will be interesting to see what the situation is in the context of binding partners.
• Molecular dynamic simulations next indicate the region which binds to a lipid bilayer, with contact residues forming a consistent interaction surface in three independent simulations. This identified the PH domain and neighbouring residues as the membrane interaction surface.
• Switching to Plasmodium falciparum protein, the authors next use NMR to investigate the binding of the PH domain to membrane nanodiscs, and show that the same protein region identified in the MD simulations was found to bind in the NMR experiments.
• These membrane binding assays were then followed up through liposome pelleting assays, using TgGAP, which showed that the protein only pellets in the presence of PA lipid and that mutation of residues identified through NMR abolished liposome binding. The mutations didn't have the same effect on full-length and PH domains (noting KER for example) suggesting that lipid binding is not entirely mediated by the PH domain in the full-length protein.
• The authors next put the mutants into toxoplasma and assay the effect on apical localisation and on invasion percentage. Interestingly the mutants had little effect, perhaps due to the role of other regions of the GAC on lipid binding, suggesting that abolishing PH domain lipid binding is not sufficient. Unfortunately, as the mutations only partly reduced lipid binding in the context of full-length GAC, as shown in liposome experiments, it is hard to come to a firm conclusion about the importance of lipid binding from this data as the protein used in this experiment will still have partial lipid binding properties.
• The authors next investigate actin binding by TgGAC and show that most of the N-terminal half of the protein is required for this function. The authors propose, using AlphaFold2 and similarities to catenins, how GAC might bind to actin. In the absence of any validation from experimental data, caution is needed here, and I would personally not rely on the accuracy of these models. -
Reviewer #2 (Public Review):
Toxoplasma gondii (Tg) and Plasmodium falciparum (Pf) are two protozoan parasites that both present threats to global human health as the causal agents of toxoplasmosis and malaria, respectively. In absence of effective vaccines, disease control relies heavily on the use of drugs aimed at treating infected patients to inhibit parasitic growth and eventually kill parasites to interrupt the parasitic lifecycle. These obligate intracellular vacuole-dwelling parasites quickly attach to their host cells before actively pinching through their plasma membranes and completing their complex respective lifecycles.
Kumar et al. seek to understand the complex process of host cell recognition, attachment, and invasion in order to devise possible strategies to possibly interfere and/or block to prevent invasion of the …
Reviewer #2 (Public Review):
Toxoplasma gondii (Tg) and Plasmodium falciparum (Pf) are two protozoan parasites that both present threats to global human health as the causal agents of toxoplasmosis and malaria, respectively. In absence of effective vaccines, disease control relies heavily on the use of drugs aimed at treating infected patients to inhibit parasitic growth and eventually kill parasites to interrupt the parasitic lifecycle. These obligate intracellular vacuole-dwelling parasites quickly attach to their host cells before actively pinching through their plasma membranes and completing their complex respective lifecycles.
Kumar et al. seek to understand the complex process of host cell recognition, attachment, and invasion in order to devise possible strategies to possibly interfere and/or block to prevent invasion of the host cell or compromise egress from the infected cell. Characterizing the 3D structure at atomic resolution and dynamics of the glideosome molecular machinery involved in parasite attachment and invasion/egress provides grounds for the future rational design of novel anti-parasitic therapies targeting novel molecular targets and phylum-specific biological processes. Toxoplasma belongs to the same large family of obligate intracellular parasites such as the malaria parasite Plasmodium. These protozoa actively attach and glide at the surface of their target host cell before invading it. Such motility and propulsion at the surface of the host cell are powered by a large protein complex, the glideosome.
The article elegantly combines structural, biophysical, biochemical, computational, and cell biology approaches to dissect the structure and mechanism of action of TgGAC (and PfGAC).
The crystal structure of TgGAC was solved at an apparent 2.7A resolution by se-mad and although it is overall well described it requires further polishing in terms of model quality and accuracy. This is a very large protein, so it represents a considerable amount of work to build and refine. We note deficiencies in the way refinement (atomic displacement parameters and model building in general) and phasing statistics description were carried out or presented. This warrants further inspection and requires significant improvement and corrections to meet the usual standards expected from this field of research.
Solution scattering data while supporting the model of a conformational change between a compact (closed) conformation observed in the crystal obtained at pH 5 and an extended monomeric conformation observed at pH 8 more amenable to interactions with other cellular partners in the context of a functional glideosome needs some clarification. Because of the way proteins seem to be prepared for the SAXS analysis, I have some objections to the interpretation of some of the data.
The biochemical analysis of lipid binding specificity of the small c-terminal pleckstrin-like domain of TgGAC and PfGAC (full-length or c-terminal domain) using liposome binding assays, elegant NMR relaxation methods but also molecular dynamics on full-length GAC models are extremely convincing and support all authors claim.
The fact that however the CTD lipid binding activity is not required in vivo is a bit surprising although CTD seems required to stabilize the protein in vitro.
The section describing the hydrogen-deuterium exchange analysis of TgGAC conformation is confusing as it stands and requires clarification. It fails to be compelling in my personal opinion.
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Reviewer #3 (Public Review):
The authors present a multi-disciplinary structural analysis of the glideosome-associated connector (GAC), which is important for the motility of parasites within the Apicomplexa phylum. Strengths of the study include the first crystal of the GAC, revealing an elaborate pyramid structure with a protruding arch bearing a PH domain. The lipid binding analyses, featuring NMR experiments and simulations to identify key residues, provide a nice complement to the crystal structure. There are interesting differences between the structure obtained and the small-angle X-ray scattering data, which are plausibly (but not conclusively) explained by a model in which GAC uses multiple conformations. It is also puzzling that the lipid binding residues in the PH domain do not seem vital for parasite invasion, although this …
Reviewer #3 (Public Review):
The authors present a multi-disciplinary structural analysis of the glideosome-associated connector (GAC), which is important for the motility of parasites within the Apicomplexa phylum. Strengths of the study include the first crystal of the GAC, revealing an elaborate pyramid structure with a protruding arch bearing a PH domain. The lipid binding analyses, featuring NMR experiments and simulations to identify key residues, provide a nice complement to the crystal structure. There are interesting differences between the structure obtained and the small-angle X-ray scattering data, which are plausibly (but not conclusively) explained by a model in which GAC uses multiple conformations. It is also puzzling that the lipid binding residues in the PH domain do not seem vital for parasite invasion, although this may be explained by the second lipid binding site in the GAC arch. The AlphaFold prediction of the interface between the GAC and a peptide from MIC2 is interesting, in that it is reminiscent of the B-catenin/E-cadherin interaction, but requires validation. The study will be useful for researchers investigating the structural mechanism of parasite motility.
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