Molecular mechanism of complement inhibition by the trypanosome receptor ISG65

Reviewer #1 (Public Review):

The authors set out to use structural biology (cryo-EM), surface plasmon resonance, and complement convertase assays to understand the mechanism(s) by which ISG65 dampens the cytoxicity/cellular clearance to/of trypanosmes opsonised with C3b by the innate immune system.

The cryo-EM structure adds significantly to the author's previous crystallographic data because the latter was limited to the C3d sub-domain of C3b. Further, the in vitro convertase assay adds an additional functional dimension to this study.

The authors have achieved their aims and the results support their conclusions.

The role of complement in immunity to T. brucei (or lack thereof) has been a significant question in molecular parasitology for over 30 years. The identification of ISG65 as the C3 receptor and now this study providing mechanistic insights represents a major advance in the field.

Reviewer #2 (Public Review):

This is an excellent paper that uses structural work to determine the precise role of one of the few invariant proteins on the surface of the African trypanosome. This protein, ISG65, was recently determined to be a complement receptor and specifically a receptor of C3, whose binding to ISG65 led to resistance to complement-mediated lysis. But the molecular mechanism that underlies resistance was unknown.

Here, through cryoEM studies, the authors reveal the interaction interface (two actually) between ISG65 and C3, and based on this, make inferences regarding downstream events in the complement cascade. Specifically, they suggest that ISG65 preferably binds the converted C3b (rather than the soluble C3). Moreover, while conversion to a C3bB complex is not blocked, the ability to bind complement receptors 1 and 3 is likely blocked.

Of course, all this is work on proteins in isolation and the remaining question is - can this in fact happen on the membrane? The VSG-coated membrane is supposed to be incredibly dense (packed at the limits of physical density) and so it is unclear whether the interactions that are implied by the structural work can actually happen on the membrane of a live trypanosome. This is not necessarily a ding but it should be addressed in the manuscript perhaps as a caveat.

Reviewer #3 (Public Review):

The authors investigate the mechanisms by which ISG65 and C3 recognize and interact with each other. The major strength is the identification of eco-site by determining the cryoEM structure of the complex, which suggests new intervention strategies. This is a solid body of work that has an important impact on parasitology, immunology, and structural biology.