Signals arising from bacterial infections are detected by pathogen recognition receptors (PRRs) and are transduced by specialized adapter proteins in mammalian cells. The Receptor-interacting-serine/threonine-protein kinase 2 (RIPK2 or RIP2) is such an adapter protein that is critical for signal propagation of the Nucleotide-binding-oligomerization-domain-containing proteins 1/2 (NOD1 and NOD2). Dysregulation of this signaling pathway leads to defects in bacterial detection and in some cases autoimmune diseases. Here, we show that the Caspase-activation-and-recruitment-domain (CARD) of RIP2 (RIP2-CARD) forms oligomeric structures upon stimulation by either NOD1-CARD or NOD2-2CARD. We reconstitute this complex, termed the RIPosome in vitro and solve the cryo-EM filament structure of the active RIP2-CARD complex at 4.1 Å resolution. The structure suggests potential mechanisms by which CARD domains from NOD1 and NOD2 initiate the oligomerization process of RIP2-CARD. Together with structure guided mutagenesis experiments at the CARD-CARD interfaces, we demonstrate molecular mechanisms how RIP2 is activated and self-propagating such signal.

Interleukin 27 (IL-27) is a heterodimeric cytokine that elicits potent immunosuppressive responses. Comprised of EBI3 and p28 subunits, IL-27 binds GP130 and IL-27Ra receptor chains to activate the JAK/STAT signaling cascade. However, how these receptors recognize IL-27 and form a complex capable of phosphorylating JAK proteins remains unclear. Here, we used cryo electron microscopy (cryoEM) and AlphaFold modeling to solve the structure of the IL-27 receptor recognition complex. Our data show how IL-27 serves as a bridge connecting IL-27Ra (domains 1-2) with GP130 (domains 1-3) to initiate signaling. While both receptors contact the p28 component of the heterodimeric cytokine, EBI3 stabilizes the complex by binding a positively charged surface of IL-27Ra and Domain 1 of GP130. We find that assembly of the IL-27 receptor recognition complex is distinct from both IL-12 and IL-6 cytokine families and provides a mechanistic blueprint for tuning IL-27 pleiotropic actions.

Interleukin 27 (IL-27) is a heterodimeric cytokine that elicits potent immuno-suppressive responses. Comprised of EBI3 and p28 subunits, IL-27 binds GP130 and IL-27Rα receptor chains to activate the JAK/STAT signalling cascade. However, how these receptors recognize IL-27 and form a complex capable of phosphorylating JAK proteins remains unclear. Here, we used cryo electron microscopy (cryoEM) to solve the structure of the IL-27 receptor recognition complex. Our data show how IL-27 serves as a bridge connecting IL-27Rα with GP130 to initiate signalling. While both receptors weakly bind the p28 component of the heterodimeric cytokine, EBI3 stabilizes the complex by binding a positively charged surface of IL-27Rα. We observe a large degree of flexibility in the rotation of D1 and the two CHR domains of GP130 contacting p28, which could contribute to GP130 binding degeneracy. We find that assembly of the IL-27 receptor recognition complex is distinct from both IL-12 and IL-6 cytokine families and provides a mechanistic blueprint for tuning IL-27 pleiotropic actions.

Chromosomal DNA ends shorten after each round of replication due to the end-replication problem and post replication processing. The ends of linear eukaryotic chromosomes are capped and protected by protein-DNA complexes forming a structure called the telomere.The telomere length is crucial as critically short telomeres activate the DNA damage response pathway leading to cell senescence. The telomere is extended by a protein-RNA enzyme complex with reverse transcriptase function named telomerase. The process by which telomerase extends telomeres must be tightly regulated. Human telomerase is inactive in normal human somatic cells but activated by various mechanisms in 85~90% of cancer cells.Mutations in telomerase binding proteins that cause reduced telomerase activity in vivo lead to developmental disorders such as Dyskeratosis Congenita. Although many studies were carried out to study human telomerase, the molecular mechanisms behind telomerase biogenesis, recruitment and elongation process are still not fully understood. Thus, a highresolution structure of human telomerase would provide molecular details of human telomerase catalytic cycle and recruitment. Historically, overexpression of full-length human telomerase has been shown to be difficult and does not give sufficient material for structural studies and biochemical analysis. In this thesis, I present optimised human telomerase expression and purification methods from mammalian cells, which routinely yield around 500μg of active human telomerase sample, allowing further studies of human telomerase structure by cryo-EM. The purified human telomerase sample was biochemically characterised to contain only the catalytic subunit hTERT and RNA subunit hTERC and is shown to be fully active and processive. Previously, Daniel Rhodes' group had determined the structure of an active full-length human telomerase dimer to 25Å resolution by negative stain EM. More recently the Collins group determined the structure of monomeric human telomerase with other binding factors complex to 8Å resolution by cryo-EM. In my studies, I