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

Abstract

25Bacterial protein degradation is a regulated process aided by protease adaptors that alter 26 specificity of energy dependent proteases. In Caulobacter crescentus, cell-cycle 27 dependent protein degradation depends on a hierarchy of adaptors, such as the dimeric 28 RcdA adaptor which binds multiple cargo and delivers substrates to the ClpXP protease. 29RcdA itself is degraded in the absence of cargo and how RcdA recognizes its targets is 30 unknown. Here we show that RcdA dimerization and cargo binding compete for a 31 common interface. Cargo binding separates RcdA dimers and a monomeric variant of 32 RcdA fails to be degraded, suggesting that RcdA degradation is a result of self-delivery. 33Based on HDX-MS studies showing that different cargo rely on different regions of the 34 dimerization interface, we generate RcdA variants that are selective for specific cargo and 35 show cellular defects consistent with changes in selectivity. Using the same interface for 36 dimerization and cargo binding offers an ability to limit excess protease adaptors by self-37 degradation, while providing capacity for binding a range of substrates. 38 39 40 Significance Statement: 41 Energy-dependent proteases broadly regulate bacterial physiology and development. 42 Adaptor proteins tune the substrate specificity of proteases to only degrade selective 43 substrates during the bacterial life cycle and during times of cellular stress. In the 44 process of delivering cargo to their respective proteases, adaptor proteins are inherently 45 protected from degradation until the delivery is complete. How protease adaptors can 46 recognize a wide range of cargo while maintaining stringent specificity and how this 47 process results in stabilization of adaptors remains unclear. Here, we show that direct 48 competition for distinct regions of the dimer interface of the RcdA adaptor by its cargo 49 protects RcdA from degradation by the ClpXP protease, and that this interface can be 50 selectively perturbed in a rational manner with biochemical and physiological 51 consequences. 52 53 Highlights: 54 Cargo binding of RcdA cargo competes with dimerization 55 Dimerization of RcdA is necessary for self-degradation by ClpXP 56 RcdA can deliver either cargo or other RcdA subunits to ClpXP 57 Different regions of the dimerization interface are needed for different cargo 58 59

Structure, dynamics and functions of UBQLNs: at the crossroads of protein quality control machinery

“…First, as we discussed above, the oligomerization-driving STI1-II domains of the UBQLN paralogs exhibit different extents of disorder and prion-like characteristics and could determine the propensity for each UBQLN to oligomerize. Second, it has been established that noncovalent interactions between the adaptor protein and its clients could affect the oligomerization propensity of the adaptor, which could apply to UBQLNs as well [104,155]. Third, disease-linked mutations perturb oligomerization and aggregation propensity both in vitro and in vivo (Table 2) [4,11,29,30].…”