Unlocking non-natural chemistries in ancestral cytochromes P450 - and - Structural Characterisation of Endosomal Trafficking Protein Sorting Nexin 27

Title:  Unlocking non-natural chemistries in ancestral cytochromes P450

Speaker:  Dr. Raine Thomson, SCMB

Abstract:  Over the last decade, the Gillam group has resurrected >40 ancestral Cytochromes P450 from the major mammalian xenobiotic metabolising families; CYP1, CYP2 and CYP3. These ancestors are consistently found to be more thermostable than their respective extant descendants, while also exhibiting substrate promiscuity. This makes them very interesting as scaffolds for further engineering in the pursuit of biocatalysts. While typical P450s are promising oxene transfer biocatalysts, there are many industrially important reactions involving carbene and nitrene transfer for which there are few (if any) known natural precedents. Work by the group of Nobel prize winner, Francis Arnold, found that mutagenesis of a highly conserved cysteine residue, responsible for ligating the P450 haem prosthetic group, can help unlock these non-natural chemistries in an extant bacterial P450. Inspired by this work, we introduced similar mutations into our thermostable and promiscuous ancestral P450s to develop biocatalysts that are not only diverse in their substrate range but are also capable of performing a wide range of chemistries.

Title:  Structural Characterisation of Endosomal Trafficking Protein Sorting Nexin 27

Speaker:  Qian Guo

Abstract:  Maintenance of endocytic trafficking and subsequent sorting of transmembrane ‘cargo’ proteins in endosomes is essential for cellular homeostasis and function. Retromer is an evolutionarily conserved protein complex composed of three ‘vacuolar protein sorting’ subunits VPS35, VPS26, and VPS29, and is a central regulator of endosomal trafficking in all eukaryotes. More specifically, it functions together with a number of accessory proteins and cargo adaptors for membrane recruitment, engagement with cargos, and assembly into tubular or vesicular transport structures. Previous work from our lab and others has established that sorting nexin 27 (SNX27) is an essential cargo adaptor of Retromer, responsible for recycling hundreds of transmembrane cargo proteins from endosomes to the plasma membrane.  SNX27 is a peripheral membrane protein containing PDZ, PX and FERM domains that mediate cargo selection, Retromer recruitment, and protein-lipid interactions; however, the exact molecular mechanism describing how SNX27, Retromer, cargos, and lipids work together is only partially understood. In addition, other Retromer accessory proteins have recently been shown to also interact with SNX27, expanding the network of protein complexes involved. These include the BAR domain-containing sorting nexin proteins SNX1 and SNX2, and Fam21 (also called WASHC2), a subunit of the F-actin-nucleating WASH complex. In this talk, I will focus on investigating the direct interactions among these proteins, and the identification of a novel subset of proteins potentially interacting with SNX27 via noncanonical PDZ-domain binding using AlphaFold2 modelling. These studies provide novel insights into the molecular mechanisms underlying the coordination of endosomal cargo transport by the Retromer complex, with a specific focus on the role of the cargo adaptor protein SNX27 and its interaction with other accessory proteins and the membrane itself. Hopefully, these findings not only improve our understanding of fundamental cellular processes, but also provide a basis for the development of new therapeutic approaches targeting the Retromer pathway in various diseases.