Cargo competition for a dimerization interface stabilizes a protease adaptor in Caulobacter crescentus

Abstract

AbstractBacterial protein degradation is a regulated process aided by protease adaptors that alter specificity of energy dependent proteases. In Caulobacter crescentus, cell-cycle dependent protein degradation depends on a hierarchy of adaptors, such as the dimeric RcdA adaptor which binds multiple cargo and delivers substrates to the ClpXP protease. RcdA itself is degraded in the absence of cargo and how RcdA recognizes its targets is unknown. Here we show that RcdA dimerization and cargo binding compete for a common interface. Cargo binding separates RcdA dimers and a monomeric variant of RcdA fails to be degraded, suggesting that RcdA degradation is a result of self-delivery. Based on HDX-MS studies showing that different cargo rely on different regions of the dimerization interface, we generate RcdA variants that are selective for specific cargo and show cellular defects consistent with changes in selectivity. Using the same interface for dimerization and cargo binding offers an ability to limit excess protease adaptors by self-degradation, while providing capacity for binding a range of substrates.Significance StatementEnergy-dependent proteases broadly regulate bacterial physiology and development. Adaptor proteins tune the substrate specificity of proteases to only degrade selective substrates during the bacterial life cycle and during times of cellular stress. In the process of delivering cargo to their respective proteases, adaptor proteins are inherently protected from degradation until the delivery is complete. How protease adaptors can recognize a wide range of cargo while maintaining stringent specificity and how this process results in stabilization of adaptors remains unclear. Here, we show that direct competition for distinct regions of the dimer interface of the RcdA adaptor by its cargo protects RcdA from degradation by the ClpXP protease, and that this interface can be selectively perturbed in a rational manner with biochemical and physiological consequences.HighlightsCargo binding of RcdA cargo competes with dimerizationDimerization of RcdA is necessary for self-degradation by ClpXPRcdA can deliver either cargo or other RcdA subunits to ClpXPDifferent regions of the dimerization interface are needed for different cargo