Layered entrenchment maintains essentiality in protein-protein interactions.

Protein complexes composed of strictly essential subunits are abundant in nature and arise through the gradual complexification of ancestral precursor proteins followed by their co-evolution with the newly recruited components. Essentiality arises during co-evolution by the accumulation of changes that are tolerated in the complex state but would be deleterious for the standalone complex components. While this theoretical framework to explain how essentiality arises has been proposed long ago, it is unclear which factors cause essentiality to persist over evolutionary timescales. In this work we show that the central enzyme of photosynthesis, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), rapidly started to depend on a newly recruited interaction partner through multiple, genetically distinct mechanisms that affect stability, solubility, and catalysis. We further demonstrate that layering multiple mechanisms of essentiality can lead to the persistence of essentiality, even if any given mechanism reverts through chance or selection. More broadly, our work highlights that new interaction partners can drastically re-shape which substitutions are tolerated in the proteins they are recruited into. This can lead to the rapid evolution of multi-layered essentiality through the exploration of areas of sequence space that are only accessible in the complex state.