Properdin enhances complement-mediated opsonization of targeted cells and particles for immune clearance. Properdin occurs as dimers, trimers and tetramers in human plasma, which recognize C3b-deposited surfaces, promote formation, and prolong the lifetime of C3bBb-enzyme complexes that convert C3 into C3b, thereby enhancing the complement-amplification loop. Here, we report crystal structures of monomerized properdin, which was produced by co-expression of separate N- and C-terminal constructs that yielded monomer-sized properdin complexes that stabilized C3bBb. Consistent with previous low-resolution X-ray and EM data, the crystal structures revealed ring-shaped arrangements that are formed by interactions between thrombospondin type-I repeat (TSR) domains 4 and 6 of one protomer interacting with the N-terminal domain (which adopts a short transforming-growth factor B binding protein-like fold) and domain TSR1 of a second protomer, respectively. Next, a structure of monomerized properdin in complex with the C-terminal domain of C3b showed that properdin-domain TSR5 binds along the C-terminal α-helix of C3b, while two loops, one from domain TSR5 and one from TSR6, extend and fold around the C3b C-terminus like stirrups. This suggests a mechanistic model in which these TSR5 and TSR6 “stirrups” bridge interactions between C3b and factor B or its fragment Bb, and thereby enhance formation of C3bB pro-convertases and stabilize C3bBb convertases. In addition, properdin TSR6 would sterically block binding of the protease factor I to C3b, thus limiting C3b proteolytic degradation. The presence of a valine instead of a third tryptophan in the canonical Trp-ladder of TSR domains in TSR4 allows a remarkable ca. 60°-domain bending motion of TSR4. Together with variable positioning of TSR2 and, putatively, TSR3, this explains the conformational flexibility required for properdin to form dimers, trimers, and tetramers. In conclusion, the results indicate that binding avidity of oligomeric properdin is needed to distinguish surface-deposited C3b molecules from soluble C3b or C3 and suggest that properdin-mediated interactions bridging C3b-B and C3b-Bb enhance affinity, thus promoting convertase formation and stabilization. These mechanisms explain the enhancement of complement-mediated opsonization of targeted cells and particle for immune clearance.

C3 glomerulopathy (C3G) is an umbrella classification for severe renal diseases characterized by predominant staining for complement component C3 in the glomeruli. The disease is caused by a dysregulation of the alternative pathway (AP) of the complement system. In more than half of C3G patients C3 nephritic factors (C3NeFs) are found. These autoantibodies bind to the AP C3 convertase, prolonging its activity. C3NeFs can be dependent or independent of the complement regulator properdin for their convertase-stabilizing function. However, studies to determine the properdin-dependency of C3NeFs are rare and not part of routine patient workup. Until recently, only supportive treatments for C3G were available. Complement-directed therapies are now being investigated. We hypothesized that patients with properdin-dependent C3NeFs may benefit from properdin-inhibiting therapy to normalize convertase activity. Therefore, in this study we validated two methods to distinguish between properdin-dependent and properdin-independent C3NeFs. These methods are hemolytic assays for measuring convertase activity and stability in absence of properdin. The first assay assesses convertase stabilization by patient immunoglobulins in properdin-depleted serum. The second assay measures convertase stabilization directly in patient serum supplemented with the properdin-blocking agent Salp20. Blood samples from 13 C3NeF-positive C3G patients were tested. Three patients were found to have properdin-dependent C3NeFs, whereas the C3NeF activity of the other ten patients was independent of properdin. The convertase-stabilizing activity in the samples of the patients with properdin-dependent C3NeFs disappeared in absence of properdin. These data indicate that inhibition of properdin in patients with properdin-dependent C3NeFs can normalize convertase activity and could represent a novel therapy for normalizing AP hyperactivity. Our assays provide a tool for identifying C3G patients who may benefit from properdin-inhibiting therapy and can be incorporated into standard C3G laboratory investigations.

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Cleavage of the mammalian plasma protein C4 into C4b initiates opsonization, lysis, and clearance of microbes and damaged host cells by the classical and lectin pathways of the complement system. Dysregulated activation of C4 and other initial components of the classical pathway may cause or aggravate pathologies, such as systemic lupus erythematosus, Alzheimer disease, and schizophrenia. Modulating the activity of C4b by small-molecule or protein-based inhibitors may represent a promising therapeutic approach for preventing excessive inflammation and damage to host cells and tissue. Here, we present seven nanobodies, derived from llama ( Lama glama ) immunization, that bind to human C4b ( Homo sapiens ) with high affinities ranging from 3.2 nM to 14 pM. The activity of the nanobodies varies from no to complete inhibition of the classical pathway. The inhibiting nanobodies affect different steps in complement activation, in line with blocking sites for proconvertase formation, C3 substrate binding to the convertase, and regulator-mediated inactivation of C4b. For four nanobodies, we determined single-particle cryo-electron microscopy structures in complex with C4b at 3.4–4 Å resolution. The structures rationalize the observed functional effects of the nanobodies and define their mode of action during complement activation. Thus, we characterized seven anti-C4b nanobodies with diverse effects on the classical pathway of complement activation that may be explored for imaging, diagnostic, or therapeutic applications.

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