Exploring the oligomeric state of TLR4 and downstream regulators RIPK1 and RIPK3

Toll-like receptors (TLRs) are a group of pattern recognition receptors involved in innate immunity. Their extracellular domain is made of leucine-rich repeats that recognise ligands, leading to the dimerisation of their intracellular Toll/interleukin 1 receptor (TIR) domain, which builds a platform for signalling via the MyD88- and TRIF-pathway. TLR4, with its co-receptor MD2, unlike the other TLRs, can signal via the MyD88- and TRIF-pathway when activated by LPS as well as non-LPS ligands such as the Zaire ebolavirus glycoprotein. Key regulators in the TLR4 pathway are RIPK1 and RIPK3, which are essential during programmed cell death, and therefore, signalling via TLR4 is key in the decision between pro immunity or cell death during infection. Whilst much is known about which molecules are involved in TLR4 signalling, the molecular details of this pathway are lacking, primarily due to the lack of structures of the signalling complexes involved. This study utilises biophysical and structural analyses to elucidate three hypotheses on the TLR4 signalling pathway. The structure of each domain of TLR4 is known; however, the lack of full-length structures hinders the current understanding of the activation mechanism. TLR4 has been proposed to signal via a two-step mechanism where ligand binding stabilises the dimerisation of the extracellular domains, leading to a conformational change in the whole complex that promotes dimerisation of the TIR domains. A robust method to express and purify the TLR4/MD2 complex was developed to confirm this hypothesis. Despite TLR4/MD2 forming mainly a tetramer in solution, in line with previous experimentation, the full-length structure remains to be elucidated due to difficulties facing aggregation at the air-water interface during cryo-electron microscopy analyses. Key signalling molecules in the TLR signalling pathway have been shown to form large supramolecular signalling complexes, including RIPK1 and RIPK3. However, the oligomerisation and full length structures of RIPK1 and RIPK3 are unknown. To illuminate RIPK1 and RIPK3 structures, they were expressed and purified. Biophysical analyses of RIPK1 and RIPK3 show these proteins can form dimers and higher oligomerisation that increase with concentration. Cryo-electron microscopy analysis of RIPK1 reveals a hexamer where the death domains are likely forming a ring, with the RHIM forming a stalk in the centre. The kinase domain was not resolved; this was potentially due to flexibility connecting the kinase domain to the rest of the domains, or that preferred orientation limited resolving this domain. These results highlight how key regulators of TLR4 signalling can form large oligomers. How LPS binds and promotes dimerisation of TLR4’s extracellular domain is well understood, but TLR4 has also been shown to be activated by many non LPS ligands with diverse structures, including Zaire ebolavirus glycoprotein. It was shown previously that the internal fusion loop of this glycoprotein was required for signalling via TLR4/MD2. As some ligands for TLR4/MD2 have been shown to present LPS to the complex instead of binding directly, to determine if the internal fusion loop can mimic LPS by binding inside MD2’s pocket, MD2 was purified, and its binding to the internal fusion loop was tested. As no direct binding was seen between MD2 and the internal fusion loop, it is possible that other features outside of the internal fusion loop of the Zaire ebolavirus glycoprotein are required to bind MD2 and that the internal fusion loop alone does not bind MD2 with high affinity.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2025

Author dc:creator

• Crabb, Alyssa

Advisor dc:contributor.advisor

• Gay, Nicholas

• RIPK signalling
• RIPK1
• RIPK3
• TLR4
• TLR4 signalling

Licence

• https://www.repository.cam.ac.uk/bitstreams/294ea71e-aa88-4654-b42f-925131301aef/download
• http://purl.org/NET/rdflicense/allrightsreserved

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.123034

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/392278