Distinct regulatory mechanisms balance DegP proteolysis to maintain cellular fitness during heat stress

Protein degradation is an essential cellular function. In the absence of degradation, toxic misfolded proteins or aggregates can accumulate after heat shock or other environmental stresses, resulting in reduced viability or cell death. Protein quality control (PQC) proteases play critical biological roles by degrading diverse misfolded proteins but also require careful regulation because proteolysis of functional proteins can be cytotoxic. Intracellular proteases whose activities are allosterically regulated have evolved to meet these conflicting needs. DegP is a member of the highly conserved HtrA family of proteases, is overexpressed at high temperatures as the major PQC protease in the periplasmic compartment of Escherichia coli and many other Gram-negative bacteria, and is implicated in the virulence of several pathogens (Lipinska et al. 1989; Strauch et al. 1989; Pallen and Wren 1997; Clausen et al. 2011). Each subunit of DegP contains a trypsin-like protease domain and two PDZ domains (Krojer et al. 2002, 2008b; Kim et al. 2011). The protease domains of three subunits pack together to form a stable trimer, which can switch between a proteolytically active conformation with high substrate affinity and a proteolytically inactive conformation with low substrate affinity (Fig. 1A; Kim et al. 2011). DegP trimers display positively cooperative substrate binding to the active enzyme, a hallmark of Monod-Wyman-Changeux (MWC) allostery, which allows proteolysis to be controlled. Substrate binding also triggers the assembly of DegP trimers and hexamers into large polyhedral cages that restrict the access of native proteins to the active sites within these cages (Fig. 1A; Jiang et al. 2008; Krojer et al. 2008b; Kim et al. 2011). Figure 1. The R207P mutation stabilizes the high-affinity active conformation of DegP. (A) Substrate-binding triggers proteolytic activation and cage assembly. The fundamental unit of DegP structure is a trimer, which reversibly transforms between an inactive conformation ... Distinct modes of substrate binding control DegP activation and assembly. In unfolded or misfolded substrates, two degron sequences in a single polypeptide interact with DegP to promote allosteric activation (Kim et al. 2011). Substrate residues flanking scissile peptide bonds bind to the active site cleft and oxyanion hole of the active protease domain, whereas a hydrophobic residue at the substrate C terminus binds to a site in the PDZ1 domain. These linked interactions also result in the assembly of trimers into hollow polyhedral cages with 12, 18, or 24 subunits by stabilizing interactions between the PDZ1 domains of one trimer and the PDZ2′ domains of neighboring trimers (Kim and Sauer 2012). Substrate cleavage eventually generates peptide products that bind too weakly to maintain DegP in the active conformation, resulting in cage disassembly and a return to the inactive conformation. Thus, bivalent substrate binding helps ensure that DegP is activated and assembled only in the presence of appropriate substrates. Several observations demonstrate the coupling of DegP activation and cage assembly. Crystal structures are known for a DegP hexamer in which trimers assume an inactive conformation with a malformed Ser–His–Asp catalytic triad and oxyanion hole and for substrate-bound cages with 12 or 24 subunits in which trimers assume an active conformation (Krojer et al. 2002, 2008b; Kim et al. 2011). In solution, proteolytic activation and cage assembly of DegP also occur synchronously (Krojer et al. 2008b; Kim et al. 2011), which led to the proposal that cage assembly was the molecular switch for activation (Krojer et al. 2008b). We found, however, that cage assembly was not required for either proteolytic activity in vitro or suppression of proteotoxic heat stress in vivo (Kim and Sauer 2012). At present, it is unclear how allosteric activation and cage assembly contribute to DegP function in vivo and cellular fitness. Here, we show that mutations that increase or decrease the equilibrium population of active DegP trimers result in small decreases in high-temperature fitness. Combining a mutation that allosterically activates DegP with one that prevents cage formation converts the enzyme into a lethal rogue protease. This lethality appears to be caused by excessive proteolysis of proteins associated with cell envelope maintenance and can be relieved by an extragenic suppressor that alters the cell envelope and directly inhibits the rogue protease or by intragenic mutations that stabilize inactive DegP trimers. We propose that substrate-dependent allosteric control of the conformation and activity of DegP trimers represents the primary form of proteolytic regulation, whereas cage assembly protects cells from promiscuous proteolysis that would otherwise be triggered under heat stress and other conditions that result in protein misfolding and allosteric activation of DegP.