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

Abstract

Intracellular proteases combat proteotoxic stress by degrading damaged proteins, but their activity must be carefully controlled to maintain cellular fitness. The activity of Escherichia coli DegP, a highly conserved periplasmic protease, is regulated by substrate-dependent allosteric transformations between inactive and active trimer conformations and by the formation of polyhedral cages that confine the active sites within a proteolytic chamber. Here, we investigate how these distinct control mechanisms contribute to bacterial fitness under heat stress. We found that mutations that increase or decrease the equilibrium population of active DegP trimers reduce high-temperature fitness, that a mutation that blocks cage formation causes a mild fitness decrease, and that combining mutations that stabilize active DegP and block cage formation generates a lethal rogue protease. This lethality is suppressed by an extragenic mutation that prevents covalent attachment of an abundant outermembrane lipoprotein to peptidoglycan and makes this protein an inhibitor of the rogue protease. Lethality is also suppressed by intragenic mutations that stabilize inactive DegP trimers. In combination, our results suggest that allosteric control of active and inactive conformations is the primary mechanism that regulates DegP proteolysis and fitness, with cage formation providing an additional layer of cellular protection against excessive protease activity.

A Small Periplasmic Protein with a Hydrophobic C-Terminal Residue Enhances DegP Proteolysis as a Suicide Activator

“…Its effect on cells may not be significant for cell viability but still sufficient for providing a selective advantage under stress conditions. Indeed, the small increase or decrease in the level of DegP activity was shown to be accumulated during multiple replication cycles and meaningfully affects the growth fitness of cells at high temperature (17). Therefore, we suggest that the YjfN-mediated fine-tuning of DegP activity can be an additional mechanism for the regulation of PQC proteolysis in the bacterial periplasm.…”

“…To test the effect of YjfN overexpression on DegP function, we inserted either an empty plasmid or a YjfN expression plasmid into three strains containing degP WT , degP A184S , or no degP gene. DegP A184S is a less-active variant of DegP and has less ability to combat misfolded protein stress (17). Under heat shock stress (43°C), cells producing wild-type DegP, but not those producing no DegP, were viable, regardless of whether YjfN was overexpressed (Fig.…”

“…Introduction of two mutations that disrupt the balance of the two molecular transformations can generate a hyperactive DegP variant that can kill bacteria by excessive protein degradation (17). Both transformations are allosterically induced by substrate binding, and thus the DegP cage is proteolytically activated only in the presence of proper substrates.…”

Regulation of Proteolysis in the Gram-Negative Bacterial Envelope

“…Substrate binding by HtrA family proteases not only triggers allosteric conformational changes that alter the active site but also stimulates the formation of higher-order trimer-or hexamer-based oligomeric assemblies (6,7,14,18,19) that are thought to protect the cell from unwarranted degradation of proteins by sequestering the active site (14,19). The oligomeric state of DegP was not studied in relation to YjfN, however, and so we must await future studies to ascertain whether it works through these same principles.…”

“…Stimulation of DegP proteolysis leads to destruction of YjfN itself (1). Since DegP requires allosteric activation by substrate binding (6,14,18,19,41,42), once all of the YjfN is degraded, and no other substrates are available, DegP will return to a resting, less-active state. This is also reminiscent of the periplasmic adaptor/ chaperone CpxP, which itself becomes degraded upon delivery of misfolded pilus subunits to DegP (23).…”