Competing stress-dependent oligomerization pathways regulate self-assembly of the periplasmic protease-chaperone DegP

Abstract

DegP is an oligomeric protein with dual protease and chaperone activity that regulates protein homeostasis and virulence factor trafficking in the periplasm of gram-negative bacteria. A number of oligomeric architectures adopted by DegP are thought to facilitate its function. For example, DegP can form a “resting” hexamer when not engaged to substrates, mitigating undesired proteolysis of cellular proteins. When bound to substrate proteins or lipid membranes, DegP has been shown to populate a variety of cage- or bowl-like oligomeric states that have increased proteolytic activity. Though a number of DegP’s substrate-engaged structures have been robustly characterized, detailed mechanistic information underpinning its remarkable oligomeric plasticity and the corresponding interplay between these dynamics and biological function has remained elusive. Here, we have used a combination of hydrodynamics and NMR spectroscopy methodologies in combination with cryogenic electron microscopy to shed light on the apo-DegP self-assembly mechanism. We find that, in the absence of bound substrates, DegP populates an ensemble of oligomeric states, mediated by self-assembly of trimers, that are distinct from those observed in the presence of substrate. The oligomeric distribution is sensitive to solution ionic strength and temperature and is shifted toward larger oligomeric assemblies under physiological conditions. Substrate proteins may guide DegP toward canonical cage-like structures by binding to these preorganized oligomers, leading to changes in conformation. The properties of DegP self-assembly identified here suggest that apo-DegP can rapidly shift its oligomeric distribution in order to respond to a variety of biological insults.

Flexible client-dependent cages in the assembly landscape of the periplasmic protease-chaperone DegP

“…Consistent with our previous study on DegP oligomerization in the absence of substrate (28), the Dz values for apo DegP under these conditions initially track with those expected for a hexamer at low temperature (~5-15 °C) and then subsequently decrease as the temperature is elevated, reflecting the higher-order apo oligomers that become populated at higher temperatures (~25-40 °C). The Dz,DegP+hTRF1 data were consistent with the values expected for a 12mer cage, in agreement with our previous report (28). Dz values for DegP in the presence of TrpCage-hTRF1 were slightly below the values for hTRF1, possibly the result of the formation of a small amount of 18mer cages in addition to 12mers (28).…”

“…To ensure binding to DegP and provide each substrate with a standard interaction motif, each of these proteins was engineered to contain a known C-terminal DegP affinity tag. Here we selected the DNA binding domain of the human telomere repeat binding factor (hTRF1, 54 residues) (32) since we have previously shown it to interact with DegP with high affinity (i.e., complete binding saturation at ~1:1 protomer:substrate molar ratio) (28). We refer to these engineered chimeric constructs, for example, as TrpCage-hTRF1 to denote their fusion to the C-terminal hTRF1 tag (see SI Appendix, Table S1 for their amino acid sequences).…”

“…(A) Mature DegP monomers contain a serine protease domain (blue) followed by two PDZ domains (PDZ1 in orange and PDZ2 in green, top). Three monomers associate via protease domain interactions to form trimeric building blocks that give rise to higher-order oligomeric architectures through inter-trimer PDZ1 i :PDZ2 j domain interactions (bottom) (18, 25, 28). In the absence of substrates, DegP trimers assemble into a broadly populated ensemble of higher-order structures that are structurally distinct from bound cage conformations (28).…”

Overexpression of the Bam Complex Improves the Production of Chlamydia trachomatis MOMP in the E. coli Outer Membrane

“…Recently, we have shown that co-overexpression of the E. coli Bam complex improves the secretion of difficult-to-secrete recombinant autotransporter chimeras that include an OM-inserted β-barrel domain and are sensitive to degradation when accumulating in the periplasm [28]. To test whether this approach could also relieve a bottleneck in the production of Ct-MOMP, we co-transformed cells carrying pLemo-MOMP with a compatible plasmid, pJH114, that encodes all subunits of the E. coli Bam complex under control of the IPTG-inducible trc promoter [34]. To ensure the availability of additional Bam complexes for Ct-MOMP biogenesis, production of the Bam subunits was induced 1 h prior to induction of Ct-MOMP expression (with 8 mM L-rhamnose).…”

“…The pJH114 plasmid [34] encoding the ABCDE subunits of the Bam complex under control of the IPTG-inducible trc promoter was kindly provided by Harris Bernstein (Bethesda, USA). To generate versions of pJH114 expressing either the BamA or BamBCDE subunits, BamA and BamBCDE encoding DNA sequences were amplified by PCR using pJH114 as the template and primers 3-4 and primers 5-6, respectively (see Supplementary Document 1-Table S1).…”