Structural characterization of CD47 and STING in complex with the activating nanobody

Skrobek, Blazej (2023): Structural characterization of CD47 and STING in complex with the activating nanobody. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

cGAS-STING pathway is one of the most important first line defense mechanisms in a human body. Its action begins when a dsDNA from a pathogen enters the cell. Upon its detection by cGAS, the small molecule ligand of STING, called 2’,3’-cGAMP, is synthesized. It binds to STING which resides in form of a dimer in endoplasmic reticulum. This butterfly-shaped molecule then closes its wings and rotates. Afterwards, the partner protein, TBK1 binds at its top and the oligomerization process begins. Such complex migrates to Golgi apparatus and due to several phosphorylation events it activates IRF3 which migrates to the nucleus and initiates the expression of type I interferons. These molecules trigger the further cascade of events that leads to inflammation and defense against the pathogen. This important physiological process can also lead to various disease states and thus is tightly regulated. Despite long-term research efforts, this complex pathway is still not fully understood. Here, I present the STING-activating nanobody which can be a tool to explore this protein’s mysteries. The nanobody was generated using alpaca immunization and found to significantly increase expression of CXCL10 - one of the interferon-stimulated genes, and to cause IRF3 migration to the nucleus. The first insight on how it happens was provided by the crystal structure of the nanobody with a soluble domain of STING. The wings of STING do not close during this interaction, which resembles the ways in which bacterial cyclic dinucleotides activate this protein. For now we may only speculate on how exactly the nanobody activates STING and further structural work is required to explain it. Examples of potential ways would be: release of STING C-terminal tail, positive or negative influence on interaction with partner proteins and triggering an oligomerization. CD47, the marker of self, is a membrane protein present on the surface of every human cell, making it a “friend” of the host immune system. “Foes”, e.g. cancer cells, often use its power to evade detection and thrive. Such observations lead to development of more and more successful therapies based on antibodies that bind to CD47 and prevent its interaction with SIRPα on macrophages, priming cells for destruction. However, the problem is that no one fully determined how actually CD47 works. Here, I present my multiple structural attempts to do that, using cryo-EM. I used multiple powerful tools, like nanodiscs, detergents, affinity binders to facilitate structure determination. Despite that, a high-enough resolution was not obtained, most probably because of too high inherent flexibility of this challenging target. Even recently published, crystal apo structure did not provide enough information to explain how this molecule perform its function. To decipher its mysteries multiple more structures of CD47 with its known and to-be-known partner proteins are needed.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: STING, CD47, cryo-EM, innate immunity, membrane proteins, X-ray crystallography
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 14. Juli 2023
• 1. Bericht­erstatter:in: Hopfner, Karl-Peter