Cage assembly of DegP protease is not required for substrate-dependent regulation of proteolytic activity or high-temperature cell survival

Abstract

DegP, a member of the highly conserved HtrA family, performs quality-control degradation of misfolded proteins in the periplasm of Gram-negative bacteria and is required for high-temperature survival of Escherichia coli. Substrate binding transforms DegP from an inactive oligomer containing two trimers into active polyhedral cages, typically containing four or eight trimers. Although these observations suggest a causal connection, we show that cage assembly and proteolytic activation can be uncoupled. Indeed, DegP variants that remain trimeric, hexameric, or dodecameric in the presence or absence of substrate still display robust and positively cooperative substrate degradation in vitro and, most importantly, sustain high-temperature bacterial growth as well as the wild-type enzyme. Our results support a model in which substrate binding converts inactive trimers into proteolytically active trimers, and simultaneously leads to cage assembly by enhancing binding of PDZ1 domains in one trimer to PDZ2′ domains in neighboring trimers. Thus, both processes depend on substrate binding, but they can be uncoupled without loss of biological function. We discuss potential coupling mechanisms and why cage formation may have evolved if it is not required for DegP proteolysis. macromolecular assembly | periplasmic degradation | protein quality control | stress survival E fficient proteolytic removal of misfolded and/or damaged proteins is essential for intracellular protein-quality control, but degradation of the wrong proteins can waste cellular resources and destroy essential proteins. Thus, controlling the activity and specificity of intracellular proteases is critical for maintaining viability. The DegP protease is a member of the HtrA family, functions in the periplasm of Gram-negative bacteria, is essential for survival of Escherichia coli at high temperatures, and its expression is increased substantially following heat shock or other stresses that result in protein misfolding (1-6).The fundamental structural unit of DegP is a trimer (7), which is stabilized by contacts between trypsin-like protease domains, each of which contains a conventional Ser-His-Asp catalytic triad and has two attached PDZ domains ( Fig. 1 A and B). In the absence of substrate, two DegP trimers stack face-to-face in an inactive hexamer with malformed active sites (Fig. 1C, Left) (7). Intriguingly, when protein substrates are added, DegP assembles into cage-like polyhedrons (Fig. 1C) that can contain 4, 6, 8, or more trimers (8-10). Packing between PDZ1 domains in one trimer and PDZ2′ domains in neighboring trimers stabilize these cages (Fig. 1D). The active sites are inside these cages and can degrade unfolded substrates that were trapped by assembly or subsequently diffuse into the chamber through openings at the cage vertices.The best model substrates for DegP contain a hydrophobic Cterminal residue, which binds in a pocket in the PDZ1 domain, and a hydrophobic residue at the P1 position of the scissile peptide bond, which binds in the active...

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

“…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).…”

“…For DegP, V max = 1.9 6 0.1, K M = 2.1 6 0.2, and h = 1.6 6 0.2 (Kim and Sauer 2012). For DegP R207P , V max = 1.0 6 0.03, K M = 0.45 6 0.03, and h = 1.9 6 0.2.…”

“…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 PDZ29 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.…”

Proteolytic activation of Chlamydia trachomatis HTRA is mediated by PDZ1 domain interactions with protease domain loops L3 and LC and beta strand β5

“…Our models predicted that these interactions are achievable upon the formation of 24-mers, which correlates with our in vitro data where oligomerization to 24-mer was always observed in the presence of full-length protein or peptide activators. This implies a similar mechanism to that reported for DegP, where oligomerization to 12-/24-mer is shown to be the structural mechanism that promotes the critical 'PDZ1 -L3 -LD*' interactions [18,22]. The ability of CtHtrA to form 24-mers with a disruption in 'PDZ1 -L3' is notable as it suggests that CtHtrA oligomerization can occur in the absence of the correct formation of the proteolytic site and that oligomerization may result from an alternate structural mechanism.…”

“…Structurally, HtrA consists of a serine protease domain with a chymotrypsin fold that contains the traditional Ser-His-Asp catalytic triad [15], and two carboxyterminal PDZ (PSD-95/Dics-Large/ZO-1) domains that are involved in substrate sequestration and oligomer formation [16]. The minimum functional structural unit is a trimer held together by inter-protease domain contacts, while two stacked trimers form an inactive resting hexamer form [17,18]. The inactive hexamer dissociates into trimers upon allosteric binding of the C-terminus of the substrate to the PDZ1 domain and reassembles into proteolytically active 12-mers or 24-mers mediated by PDZ1:PDZ2* domain contacts ('*' indicates a neighboring monomer) [19].…”