Trypanosomes are known to activate the complement system on their surface, but they control the cascade in a manner such that the cascade does not progress into the terminal pathway. It was recently reported that the invariant surface glycoprotein ISG65 from Trypanosoma brucei interacts reversibly with complement C3 and its degradation products, but the molecular mechanism by which ISG65 interferes with complement activation remains unknown. In this study, we show that ISG65 does not interfere directly with the assembly or activity of the two C3 convertases. However, ISG65 acts as a potent inhibitor of C3 deposition through the alternative pathway in human and murine serum. Degradation assays demonstrate that ISG65 stimulates the C3b to iC3b converting activity of complement factor I in the presence of the cofactors factor H or complement receptor 1. A structure-based model suggests that ISG65 promotes a C3b conformation susceptible to degradation or directly bridges factor I and C3b without contact with the cofactor. In addition, ISG65 is observed to form a stable ternary complex with the ligand binding domain of complement receptor 3 and iC3b. Our data suggest that ISG65 supports trypanosome complement evasion by accelerating the conversion of C3b to iC3b through a unique mechanism.

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Structure determination of macromolecular complexes is challenging if subunits can dissociate during crystallization or preparation of electron microscopy grids. We present an approach where a labile complex is stabilized by linking subunits though introduction of a peptide tag in one subunit that is recognized by a nanobody tethered to a second subunit. This allowed crystal structure determination at 3.9 Å resolution of the highly non‐globular 320 kDa proconvertase formed by complement components C3b, factor B, and properdin. Whereas the binding mode of properdin to C3b is preserved, an internal rearrangement occurs in the zymogen factor B von Willebrand domain type A domain compared to the proconvertase not bound to properdin. The structure emphasizes the role of two noncanonical loops in thrombospondin repeats 5 and 6 of properdin in augmenting the activity of the C3 convertase. We suggest that linking of subunits through peptide specific tethered nanobodies represents a simple alternative to approaches like affinity maturation and chemical cross‐linking for the stabilization of large macromolecular complexes. Besides applications for structural biology, nanobody bridging may become a new tool for biochemical analysis of unstable macromolecular complexes and in vitro selection of highly specific binders for such complexes.

The complement cascade is activated when pattern recognition molecules recognize pathogens, dying host cells or immune complexes. The proteolytic cascades in complement can initiate through the classical pathway (CP) or the lectin pathway (LP) which leads to assembly of the CP/LP C3 convertase C4b2a. This proteolytic enzyme turns over complement C3 into the anaphylatoxin C3a and the opsonin C3b that may become covalently linked to the surface of the complement activator (Figure 1A). The downstream alternative pathway (AP) provides an amplification loop for the two other pathways. 1 The C3b initially deposited by the CP C3 convertase can associate with Factor B (FB) and form the AP proconvertase C3bB that upon cleavage by Factor D (FD) becomes the active AP C3 convertase C3bBb. 1 This initiates a positive feedback loop, which markedly amplifies the complement activation through the CP and LP. 2,3 FP is central in the AP amplification loop and binds directly to C3b, the C3bB proconvertase, and the C3bBb convertase (Figure 1A). The alternative pathway may also initiate in the

The integrin receptor αMβ2 mediates phagocytosis of complement-opsonized objects, adhesion to the extracellular matrix and trans-endothelial migration of leukocytes. Here we present the first atomic structure of the human αMβ2 headpiece fragment in complex with the nanobody hCD11bNb1 determined at a resolution of 3.2 Å. The receptor headpiece adopts the closed conformation expected to have low ligand affinity. The crystal structure advocates that in the R77H αM variant associated with systemic lupus erythematosus, the modified allosteric coupling between ligand coupling and integrin outside-inside signalling is due to subtle conformational effects transmitted over 40 Å. The nanobody binds to the αI domain of the αM subunit in an Mg2+ independent manner with low nanomolar affinity. Biochemical and biophysical experiments with purified proteins argue that the nanobody acts as a competitive inhibitor through steric hindrance exerted on the thioester domain of iC3b attempting to bind the αM subunit. Surprisingly, the nanobody stimulates the interaction of cell-bound αMβ2 with iC3b suggesting that it represents a novel high-affinity proteinaceous αMβ2 specific agonist. We propose a model based on the conformational spectrum of the receptor to reconcile these conflicting observations regarding the functional consequences of hCD11bNb1 binding to αMβ2. Furthermore, our data suggest that the iC3b-αMβ2 complex may be more dynamic than predicted from the crystal structure of the core complex.