An inhibitor of complement C5 provides structural insights into activation

Abstract

The complement system is a crucial part of innate immune defenses against invading pathogens. The blood-meal of the tickRhipicephalus pulchelluslasts for days, and the tick must therefore rely on inhibitors to counter complement activation. We have identified a class of inhibitors from tick saliva, the CirpT family, and generated detailed structural data revealing their mechanism of action. We show direct binding of a CirpT to complement C5 and have determined the structure of the C5–CirpT complex by cryoelectron microscopy. This reveals an interaction with the peripheral macro globulin domain 4 (C5_MG4) of C5. To achieve higher resolution detail, the structure of the C5_MG4–CirpT complex was solved by X-ray crystallography (at 2.7 Å). We thus present the fold of the CirpT protein family, and provide detailed mechanistic insights into its inhibitory function. Analysis of the binding interface reveals a mechanism of C5 inhibition, and provides information to expand our biological understanding of the activation of C5, and thus the terminal complement pathway.

The allosteric modulation of complement C5 by knob domain peptides

“…Aspects of the structural biology of C5 are well understood due to a crystal structure of the apo form ( Fredslund et al, 2008 ) and a number of co-crystal structures of C5 with various modulators. By virtue of its constitutive role in the terminal pathway, C5 is a recurrent target for immune evasion molecules and structures have been determined of C5 in complex with an inhibitory molecule derived from Staphylococcus aureus , SSL-7 ( Laursen et al, 2010 ), as well as several structurally distinct examples from ticks: OmCI ( Jore et al, 2016 ), RaCI ( Jore et al, 2016 ), and Cirp-T ( Reichhardt et al, 2020 ). Additionally, the structures of C5 with the inhibitory monoclonal antibody (mAb) eculizumab ( Schatz-Jakobsen et al, 2016 ), of C5 with a small molecule inhibitor ( Jendza et al, 2019 ), and of C5 with the complement-depleting agent cobra venom factor (CVF) ( Laursen et al, 2011 ) have all been determined.…”

The allosteric modulation of Complement C5 by knob domain peptides

“…Additionally, another tick-derived inhibitor, Cirp-T, was also recently reported as predominantly binding to the MG4 domain, with an orthosteric mechanism of action attributed. However, we note that published data only showed an Emax of < 90 % in AP driven assays (Reichhardt et al, 2020), indicating it is an allosteric C5 inhibitor for the AP, and potentially also the CP, C5 convertase, which may merit further investigation. This study highlights the importance of orthogonal biophysical techniques for the accurate interpretation of crystal structures.…”

“…Aspects of the structural biology of C5 are well understood, due to a crystal structure of the apo form (Fredslund et al, 2008) and a number of co-crystal structures of C5 with various modulators. By virtue of its constitutive role in the terminal pathway, C5 is a recurrent target for immune evasion molecules and structures have been solved of C5 in complex with an inhibitory molecule derived from Staphylococcus aureus, SSL-7 (Laursen et al, 2010), as well as several structurally distinct examples from ticks: OmCI (Jore et al, 2016), RaCI (Jore et al, 2016) and Cirp-T (Reichhardt et al, 2020). Additionally, the structures of C5 with the inhibitory monoclonal antibody (mAb) eculizumab (Schatz-Jakobsen et al, 2016), of C5 with a small molecule inhibitor (Jendza et al, 2019), and of C5 with the complement depleting agent Cobra Venom Factor (CVF) (Laursen et al, 2011), have all been determined.…”

De novo assembled salivary gland transcriptome and expression pattern analyses for Rhipicephalus evertsi evertsi Neuman, 1897 male and female ticks

“…Recently, complement inhibitors have been found for R. appendiculatus that specifically targets complement C5 to control complement-mediated inflammation 52 , 53 . Complement inhibitor (CirPT1) presents the knottin fold (inhibitor cysteine knot (aka ICK or Knottin)), while R. appendiculatus complement inhibitor (RaCI) presents a unique fold that resembles the disulphide-rich structure of small toxins from snake venom 53 .…”