Periodic Reporting for period 4 - C-CLEAR (Complement: to clear or not to clear)
Periodo di rendicontazione: 2023-01-01 al 2023-12-31
Mammalian complement recognizes a variety of cell-surface danger and damage signals to clear invading microbes and injured host cells, while protecting healthy host cells. Improper complement responses contribute to diverse pathologies, ranging from bacterial infections up to paralyzing Guillain-Barré syndrome and schizophrenia. What determines the balance between complement attack reactions and host-cell defense measures and, thus, what drives cell fate is unclear. The project tries to elucidate molecular mechanisms that underlie the balance between complement attack and host-cell defense, which determine cell fate. Better understanding of the underlying molecular mechanisms of complement activation will be instrumental in developing new therapeutic approaches to fight infections and complement-dependent disorders.
Due to the COVID-19 outbreak, initial progress was hampered due to a complete shut-down of all experimental activities not directly related to SARS-2 research for 2.5 months followed by restricted access to lab spaces for 9 months. During these times, we developed alternative computational projects focusing on protein dynamics in crystallographic models (DOI: 10.1107/S2059798321010044) and on modelling reaction rate kinetics (by differential equations) in complement activation. Moreover, we initiated experimental research into membrane protein organization (by tetraspanins) focusing on SARS-2.
After the restart, we determined several atomic structures of complement components in complex with newly generated nanobodies (Llama derived 12.5 kDa antigen-binding antibody fragments). Nanobodies are popular tools in structural biology, but also have a potential use in diagnostics and therapeutics. A diverse set of mode-of-actions (inhibitory and non-inhibitory) of seven nanobodies for specific C4b binding was determined, highlighting the insights into C4b functioning (DOI: 10.4049/jimmunol.2100647) Furthermore (in collaboration), we resolved structures of nanobody-C5 complexes (DOI: DOI: 10.1016/j.jbc.2023.104956). For example, anti-C5 nanobody UNbC5-1 is of potential clinical interest because it binds and inhibits C5 R885H, a genetic variant of C5 resistant to treatment with eculizumab (widely used for clinical treatment for complement diseases such as atypical hemolytic uremic syndrome and paroxysmal nocturnal hemoglobinuria).
We hypothesized a role for (pre-)organization of antigenic membrane-protein into large multivalent platforms in potent complement activation. Antibodies against antigens, such as CD20, CD37 and HLA, may give rise to a very potent complement activation. Both anti-CD37 and anti-CD20 antibodies are widely used in the treatment of B-cell malignancies and bi-specific antibodies against these proteins are the most potent complement activators found to date. CD37 belongs to a family of tetraspanins emerging as molecular organizers of the membrane. Our interest in tetraspanins was further raised due to the potential interaction of tetraspanins with ACE2, the cellular receptor for the SARS-CoV-2 virus. Previously, the tetraspanin CD9 was found to facilitate cell-entry of the closely related virus that causes MERS. Humans express 33 tetraspanins, some of which are found on almost all cell types and others with more limited expression. An example of tetraspanins that form stable dimers and higher-order structures are the retina-specific peripherin-2 and ROM-1, which are essential for the formation of the outer segments in rods and cones. Many genetic variants of peripherin-2 have been linked to blindness. We solved the molecular structure of peripherin-2/ROM-1 dimers and heterodimers and higher order oligomers. These structures provide a model showing how these complexes may affect membrane curvature and show that most disease-related variant map to the dimer interface and are thus likely to disrupt oligomer formation. This work has been published (DOI: 10.1126/sciadv.add3677). Work on CD20-CD37 and CD37 complexes continued and resulted in a 2.8 Å resolution map of a large CD37 complex. This work is part of a thesis and will be submitted for publication.
A major part of the work in this project focused on cryo-EM structure determination of the large enzyme complexes critical for complement activation. Our work provided insights into these large multi-protein complexes and their dynamics. The analyses are in their final stages and two manuscripts on these enzyme complexes are in preparation.
After the restart, we determined several atomic structures of complement components in complex with newly generated nanobodies (Llama derived 12.5 kDa antigen-binding antibody fragments). Nanobodies are popular tools in structural biology, but also have a potential use in diagnostics and therapeutics. A diverse set of mode-of-actions (inhibitory and non-inhibitory) of seven nanobodies for specific C4b binding was determined, highlighting the insights into C4b functioning (DOI: 10.4049/jimmunol.2100647) Furthermore (in collaboration), we resolved structures of nanobody-C5 complexes (DOI: DOI: 10.1016/j.jbc.2023.104956). For example, anti-C5 nanobody UNbC5-1 is of potential clinical interest because it binds and inhibits C5 R885H, a genetic variant of C5 resistant to treatment with eculizumab (widely used for clinical treatment for complement diseases such as atypical hemolytic uremic syndrome and paroxysmal nocturnal hemoglobinuria).
We hypothesized a role for (pre-)organization of antigenic membrane-protein into large multivalent platforms in potent complement activation. Antibodies against antigens, such as CD20, CD37 and HLA, may give rise to a very potent complement activation. Both anti-CD37 and anti-CD20 antibodies are widely used in the treatment of B-cell malignancies and bi-specific antibodies against these proteins are the most potent complement activators found to date. CD37 belongs to a family of tetraspanins emerging as molecular organizers of the membrane. Our interest in tetraspanins was further raised due to the potential interaction of tetraspanins with ACE2, the cellular receptor for the SARS-CoV-2 virus. Previously, the tetraspanin CD9 was found to facilitate cell-entry of the closely related virus that causes MERS. Humans express 33 tetraspanins, some of which are found on almost all cell types and others with more limited expression. An example of tetraspanins that form stable dimers and higher-order structures are the retina-specific peripherin-2 and ROM-1, which are essential for the formation of the outer segments in rods and cones. Many genetic variants of peripherin-2 have been linked to blindness. We solved the molecular structure of peripherin-2/ROM-1 dimers and heterodimers and higher order oligomers. These structures provide a model showing how these complexes may affect membrane curvature and show that most disease-related variant map to the dimer interface and are thus likely to disrupt oligomer formation. This work has been published (DOI: 10.1126/sciadv.add3677). Work on CD20-CD37 and CD37 complexes continued and resulted in a 2.8 Å resolution map of a large CD37 complex. This work is part of a thesis and will be submitted for publication.
A major part of the work in this project focused on cryo-EM structure determination of the large enzyme complexes critical for complement activation. Our work provided insights into these large multi-protein complexes and their dynamics. The analyses are in their final stages and two manuscripts on these enzyme complexes are in preparation.
We used biochemical and structural techniques, mammalian expression, purification and reconstitution of large (membrane bound) protein complexes and resolved them by cryo-EM single-particle analysis and cryo-EM tomography. These approaches yielded mechanistic insights into multivalent recognition of cell-surface danger signals through large complex formations and subsequent reactions on membranes leading to cell lysis and clearance.
Massive cryo-EM data analysis addressed the inherent flexibility of complexes yielding insights into: 1) complement activation focusing on the pivotal enzyme complexes, i.e. the multivalent initiating (antibody-mediated) C1 complex and the amplifying C3 convertase (part I); 2) the emergence of a new concept of membrane organization, where we advanced in understanding of tetraspanin membrane-protein complex formation; however, the perceived large complex formation that give rise to multivalent signaling and potent complement activation needs further substantiation (part II); and, 3) initiation of the terminal pathway leading to lysis through MAC pore formation, where we used cryo-EM tomography for in situ visualization and, most recently, initiated a new approach to obtain structural insights into the C5 convertases (part III).
The results of the project have been presented at international meetings on antibodies (Antibodies and Complement 2019), complement (Keystone Symposium Antibodies as Drugs: From B-cell Biology to new treatments, 2020; 13th International conference on Complement Therapeutics, 2021; GRC Proteolytic enzymes & Their Inhibitors 2022; 14th International conference on Complement Therapeutics, 2022) and electron microscopy (Gordon Research Conference: Three Dimensional Electron Microscopy 2022). Currently, we have published four papers. Two theses resulted from this project (one submitted and one near completion). Two manuscripts on the complement enzymatic complexes and two on membrane protein organization are in preparation
Massive cryo-EM data analysis addressed the inherent flexibility of complexes yielding insights into: 1) complement activation focusing on the pivotal enzyme complexes, i.e. the multivalent initiating (antibody-mediated) C1 complex and the amplifying C3 convertase (part I); 2) the emergence of a new concept of membrane organization, where we advanced in understanding of tetraspanin membrane-protein complex formation; however, the perceived large complex formation that give rise to multivalent signaling and potent complement activation needs further substantiation (part II); and, 3) initiation of the terminal pathway leading to lysis through MAC pore formation, where we used cryo-EM tomography for in situ visualization and, most recently, initiated a new approach to obtain structural insights into the C5 convertases (part III).
The results of the project have been presented at international meetings on antibodies (Antibodies and Complement 2019), complement (Keystone Symposium Antibodies as Drugs: From B-cell Biology to new treatments, 2020; 13th International conference on Complement Therapeutics, 2021; GRC Proteolytic enzymes & Their Inhibitors 2022; 14th International conference on Complement Therapeutics, 2022) and electron microscopy (Gordon Research Conference: Three Dimensional Electron Microscopy 2022). Currently, we have published four papers. Two theses resulted from this project (one submitted and one near completion). Two manuscripts on the complement enzymatic complexes and two on membrane protein organization are in preparation