A F₄₂₀-dependent single domain chemogenetic tool for protein de-dimerization

Abstract

Protein-protein interactions (PPIs) mediate many fundamental cellular processes and their control through optically or chemically responsive protein domains has a profound impact on basic research and some clinical applications. Most available chemogenetic methods induce the association, i.e., dimerization or oligomerization, 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, which is not present in mammals, as a bioorthogonal monomerization switch. We found that in the absence of F420, MSMEG_2027 forms a unique domain-swapped dimer that occludes the cofactor binding site. Substantial remodelling of the intertwined N-terminal helix upon F420 binding results in the 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 induce and release MAPK/ERK signalling downstream of a chimeric fibroblast growth factor receptor 1 (FGFR1) tyrosine kinase. This F420-dependent chemogenetic de-dimerization tool is stoichiometric, based on a single domain and presents a novel mechanism to investigate protein complexes in situ.

Active site mutations of F420-dependent alkene reductases reverse stereoselectivity

“…Perturbation of dimer formation is of interest for MSMEG_2027 given that it forms the basis of a recently reported chemically dependent protein de-dimerization tool for studying protein-protein interactions. 51 The OYE homologue 12-oxophytodienoate reductase 3 (OPR3) from tomato forms a self-inhibiting dimer through insertion of a loop into the active site that is similar to what is observed in MSMEG_2027 and MSMEG_2850. 52 Interestingly two point mutations were identified that completely abolished OPR3 dimer formation, although the protomers do not undergo the large conformational change seen in MSMEG_2027 upon dissociation.…”

“…The active forms of both MSMEG_2027 and MSMEG_2850 are monomeric, however both enzymes form head-to-head dimers in the absence of cofactor that partially occlude the active site. 10, 51 As a consequence, many of the residues targeted for mutagenesis here are involved in the dimer interface as well as substrate binding. It is unclear what effect these mutations may have on dimer formation and protein stability.…”