Poster Presentation 11th Annual Conference of the International Chemical Biology Society 2022

A cofactor F420-dependent single-domain chemogenetic tool for protein de-dimerization (#104)

James P. Antoney 1 2 , Stephanie Kainrath 3 4 5 , F. Hafna Ahmed 6 , Suk Woo Kang 6 , Emily R. R. Mackie 7 8 , Tatiana P. Soares da Costa 7 8 , Colin J. Jackson 6 , Harald Janovjak 3 4 9
  1. Australian National University, Canberra, ACT, Australia
  2. ARC Centre of Excellence in Synthetic Biology, Queensland University of Technology, Brisbane, Queensland, Australia
  3. Australian Regenerative Medicine Institute, Monash University, Melbourne, Victoria, Australia
  4. European Molecular Biology Laboratory (EMBL) Australia, Monash University, Melbourne, Victoria, Australia
  5. Institute of Science and Technology Austria, Klosterneuburg, Austria
  6. Research School of Chemistry, Australian National University, Canberra, ACT, Australia
  7. Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, Melbourne, Victoria, Australia
  8. School of Agriculture, Food and Wine, Waite Research Institute, The University of Adelaide, Adelaide, South Australia, Australia
  9. Flinder Health and Medical Research Institute, College of Medicine and Public Health, Flinders University, Adelaide, South Australia, Australia

Protein-protein interactions (PPIs) mediate many fundamental cellular processes and their control through optically or chemically responsive protein domains has had a profound impact on basic research and some clinical applications. Most available chemogenetic tools induce the association, i.e., dimerization or oligomerisation, of target proteins, and the few available dissociation approaches either break large oligomeric protein clusters or heteromeric complexes. Here, we have exploited the controlled dissociation of a dimeric oxidoreductase from mycobacteria (MSMEG_2027) by its native cofactor, F420, as a bioorthogonal monomerization switch in mammalian cells. We found that in the absence of F420, MSMEG_2027 forms a tight domain-swapped dimer that occludes the cofactor binding site and undergoes substantial remodelling of the N-terminal helix upon cofactor binding and associated dissolution of the dimer. We then show that MSMEG_2027 can be expressed as fusion proteins in human cells and apply it as a tool to inhibit MAPK/ERK cell signalling from a dimeric chimeric fibroblast growth factor receptor 1 (FGFR1) tyrosine kinase. This F420-dependent chemogenetic de-dimerization tool (F420-CDD) is stoichiometric, based a single domain and presents a novel molecular mechanism complementing existing methods to investigate the functionality of protein complexes in situ.