Cargo binding of an adaptor directly competes with oligomerization

Abstract

Bacterial protein degradation is a tightly coordinated process aided by protease adaptors that coordinate cell cycle and manages stress responses. In Caulobacter crescentus, cell‐cycle dependent protein degradation depends on the highly conserved ClpXP protease and several ClpXP specific adaptors that dictate hierarchal substrate degradation. While most protease adaptors deliver only a single substrate, the RcdA adaptor directly binds and coordinates the degradation of several cell‐cycle regulated substrates that share no obvious sequence or structural similarities. Here we show that cargo binding uses the same interface as that responsible for dimerization of RcdA. Cargo binding converts RcdA from a homodimer to a monomer and this effect extends to all RcdA cargo. We identify key residues in this interface with hydrogen‐deuterium exchange mass spectrometry and make mutations to generate a monomeric variant incapable of dimerization that also fails to bind cargo. Interestingly, RcdA itself is normally degraded by ClpXP unless bound to cargo, but the monomeric variant fails to be degraded, suggesting that RcdA delivers cargo or delivers itself to ClpXP. Expressing a monomeric RcdA in vivo results in irregular stalk formation and loss of normal cargo turnover. Our work shows how cargo binding competes with self‐dimerization of RcdA through direct competition for a common interface and that disruption of this interface results in defects in normal cell physiology.