Final Report Summary - COCO (The molecular complexity of the complement system)
The project "The molecular complexity of the complement system" focussed on deciphering the molecular steps and mechanisms that take place when the immune complement system is activated upon invasion by microbes. Besides an important role in innate and adaptive immunity, complement is also important in clearance of dying host cells or tissue debris. Given these key roles in maintenance of the host, dysregulation of complement activation and host-cell protection have been linked to various disease and pathological conditions. Our general research goal is to provide a detailed understanding of the molecular processes and mechanisms of complement activation. A proper understanding of the underlying mechanisms will facilitate develop of new therapeutics to treat specific complement diseases, such atypical hemolytic uremic syndrome, dense-deposit disease and age-related macular degeneration, up to acute and systemic inflammations, such as in rheumatoid arthritis and heart attacks or strokes. Moreover, the results from our research indicated how complement activation may be employed to target tumor cells, which potentially may enhance the use of humanized antibody therapeutics against a wider range of tumors.
In a major part of the work in this project, we aimed at determining the complement initiating complex that is formed when antibody recognizes antigens on a target. Antibody-mediated complement activation (also referred to as the classical pathway of complement activation) requires multiple antibodies to bind antigens, forming immune complexes, on the target surfaces before the first complement complex, called C1, can bind and becomes activated. However, the stoichiometry and the molecular arrangement needed for C1 binding and activation were unknown despite decades of research. In this project, we used cryo-electron microscopy to visualize the antibody complexes formed on a surface that bind and activate C1. This work was facilitated by the use of the most advanced microscope (Titan Krios) that allowed use to observe a few hundred of these complexes on a vesicle surface in tilt series (i.e. under a range of different orientation angles by systematically tilting the specimens). Subsequent data processing and averaging yielded an in situ image of complement activation at a resolution of 6 nm. Most strikingly, the arrangement consisted of a hexameric platform of antibodies with C1 bound. We demonstrated that this IgG-antibody hexamer is formed by a six-fold ring of Fc segments, with one Fab per IgG binding to the antigen on the surface and one Fab arm "free". The hexameric C1 recognition part (ie. C1q) binds with its globular headpieces to the Fc ring. In collaboration with colleagues from Genmab (Utrecht), it was shown that mutants could disrupt and even enhance the Fc-Fc interactions. Enhanced complement-activating capacity of these antibodies suggests a potential therapeutic use to enhance complement-dependent cytolysis of antibodies directed against tumor cells. This work culminated in a recent publication by Diebolder et al. (Science 2014).
Following one of the three initiation events of complement, the complement cascade is amplified and regulated, where regulation is critical to avoid damage to healthy host cells and tissue. We revealed the key steps taking place in the amplification of complement activation (ie. the so-called alternative pathway of complement activation). In Forneris et al. (Science 2010), followed up by Forneris et al. (Acta Cryst D 2014) using methods developed by Burnley et al. (eLife 2012), we captured the dynamical steps needed to form the central C3 convertase, which is central to the amplification. Following the initial insights on how host cells protect themselves from inappropriate attack by the complement system (Wu et al. Nature 2009), we established in this project how other regulators bind the central C3b molecular and exert their activities. These data provide a common evolutionary basis and general models for the two main mechanisms of critical host protection by regulators (Forneris, Wu, Xue in preparation).
Furthermore, the project provided a structural basis for the initiation of the so-called terminal pathway of complement activation, ie. the formation of the membrane-attack complex (MAC). We showed that initiation of MAC formation involve large conformational changes from C5 to C5b, which are similar to the changes we observed for the conversion of C3 to C3b, except that the conformational change is captured half-way by binding of C6 (Hadders et al. Cell Reports 2012). In addition, cryo-EM data obtained in collaboration with S. Lea (Oxford) showed how the formation of C5b6 (as clarified by crystallography) provide a molecular framework for associating C7, C8 and C9 by sideways alignment of the MACPF domains of C6-9. These structural data support the large beta-barrel MAC pore that perforates the membrane leading lysis of the invading microbe.
In a major part of the work in this project, we aimed at determining the complement initiating complex that is formed when antibody recognizes antigens on a target. Antibody-mediated complement activation (also referred to as the classical pathway of complement activation) requires multiple antibodies to bind antigens, forming immune complexes, on the target surfaces before the first complement complex, called C1, can bind and becomes activated. However, the stoichiometry and the molecular arrangement needed for C1 binding and activation were unknown despite decades of research. In this project, we used cryo-electron microscopy to visualize the antibody complexes formed on a surface that bind and activate C1. This work was facilitated by the use of the most advanced microscope (Titan Krios) that allowed use to observe a few hundred of these complexes on a vesicle surface in tilt series (i.e. under a range of different orientation angles by systematically tilting the specimens). Subsequent data processing and averaging yielded an in situ image of complement activation at a resolution of 6 nm. Most strikingly, the arrangement consisted of a hexameric platform of antibodies with C1 bound. We demonstrated that this IgG-antibody hexamer is formed by a six-fold ring of Fc segments, with one Fab per IgG binding to the antigen on the surface and one Fab arm "free". The hexameric C1 recognition part (ie. C1q) binds with its globular headpieces to the Fc ring. In collaboration with colleagues from Genmab (Utrecht), it was shown that mutants could disrupt and even enhance the Fc-Fc interactions. Enhanced complement-activating capacity of these antibodies suggests a potential therapeutic use to enhance complement-dependent cytolysis of antibodies directed against tumor cells. This work culminated in a recent publication by Diebolder et al. (Science 2014).
Following one of the three initiation events of complement, the complement cascade is amplified and regulated, where regulation is critical to avoid damage to healthy host cells and tissue. We revealed the key steps taking place in the amplification of complement activation (ie. the so-called alternative pathway of complement activation). In Forneris et al. (Science 2010), followed up by Forneris et al. (Acta Cryst D 2014) using methods developed by Burnley et al. (eLife 2012), we captured the dynamical steps needed to form the central C3 convertase, which is central to the amplification. Following the initial insights on how host cells protect themselves from inappropriate attack by the complement system (Wu et al. Nature 2009), we established in this project how other regulators bind the central C3b molecular and exert their activities. These data provide a common evolutionary basis and general models for the two main mechanisms of critical host protection by regulators (Forneris, Wu, Xue in preparation).
Furthermore, the project provided a structural basis for the initiation of the so-called terminal pathway of complement activation, ie. the formation of the membrane-attack complex (MAC). We showed that initiation of MAC formation involve large conformational changes from C5 to C5b, which are similar to the changes we observed for the conversion of C3 to C3b, except that the conformational change is captured half-way by binding of C6 (Hadders et al. Cell Reports 2012). In addition, cryo-EM data obtained in collaboration with S. Lea (Oxford) showed how the formation of C5b6 (as clarified by crystallography) provide a molecular framework for associating C7, C8 and C9 by sideways alignment of the MACPF domains of C6-9. These structural data support the large beta-barrel MAC pore that perforates the membrane leading lysis of the invading microbe.