Cross-talk between phospholipid synthesis and peptidoglycan expansion by a cell wall hydrolase.

The Gram-negative bacterial cell envelope is a complex multilayered structure comprising a bilayered phospholipid (PL) membrane that surrounds the cytoplasm (inner membrane or IM) and an asymmetric outer membrane (OM) with PLs in the inner leaflet and lipopolysaccharides in the outer leaflet. Between these two layers is the periplasmic space, which contains a highly cross-linked mesh-like glycan polymer, peptidoglycan (PG). During cell expansion, coordinated synthesis of each of these components is required to maintain the integrity of the cell envelope; however, it is currently not clear how such coordination is achieved. In this study, we show that a cross-link-specific PG hydrolase couples the expansion of PG sacculus with that of PL synthesis in the Gram-negative model bacterium, Escherichia coli. We find that unregulated activity of a PG hydrolytic enzyme, MepS is detrimental for growth of E. coli during fatty acid (FA)-limiting conditions. Further genetic and biochemical analyses revealed that cellular availability of FA or PL alters the post-translational stability of MepS by modulating the proteolytic activity of a periplasmic adaptor-protease complex, NlpI-Prc toward MepS. Our results indicate that loss of OM lipid asymmetry caused by alterations in PL abundance leads to the generation of a signal to the NlpI-Prc complex for the stabilization of MepS, which subsequently cleaves the cross-links to facilitate expansion of PG. In summary, our study shows the existence of a molecular cross-talk that enables coordinated expansion of the PG sacculus with that of membrane synthesis for balanced cell-envelope biogenesis.

Structural Basis for the Differential Regulatory Roles of the PDZ Domain in C-Terminal Processing Proteases.

Carboxyl (C)-terminal processing proteases (CTPs) participate in protective and regulatory proteolysis in bacteria. The PDZ domain is central to the activity of CTPs but plays inherently different regulatory roles. For example, the PDZ domain inhibits the activity of the signaling protease CtpB by blocking the active site but is required for the activation of Prc (or Tsp), a tail-specific protease that degrades SsrA-tagged proteins. Here, by structural and functional analyses, we show that in the unliganded resting state of Prc, the PDZ domain is docked inside the bowl-shaped scaffold without contacting the active site, which is kept in a default misaligned conformation. In Prc, a hydrophobic substrate sensor distinct from CtpB engages substrate binding to the PDZ domain and triggers a structural remodeling to align the active-site residues. Therefore, this work reveals the structural basis for understanding the contrasting roles of the PDZ domain in the regulation of CTPs.IMPORTANCE Prc, also known previously as Tsp, is the founding member of the carboxyl-terminal processing protease (CTP) family of PDZ domain-containing proteases that include CtpA and CtpB. The substrate-binding PDZ domain is responsible for regulating the protease activity of CTP proteases; however, the regulatory role of PDZ domain is stimulatory in Prc but inhibitory in CtpA/B. By determining a series of crystal structures of Prc in the unliganded resting state, this study presents the structural basis for PDZ-dependent activation of Prc, the results of which explain the contrasting roles of the PDZ domain in the regulation of the protease activity of CTPs.

Structural basis of adaptor-mediated protein degradation by the tail-specific PDZ-protease Prc.

Peptidoglycan (PG) is a highly cross-linked, protective mesh-like sacculus that surrounds the bacterial cytoplasmic membrane. Expansion of PG is tightly coupled to growth of a bacterial cell and requires hydrolases to cleave the cross-links for insertion of nascent PG material. In Escherichia coli, a proteolytic system comprising the periplasmic PDZ-protease Prc and the lipoprotein adaptor NlpI contributes to PG enlargement by regulating cellular levels of MepS, a cross-link-specific hydrolase. Here, we demonstrate how NlpI binds Prc to facilitate the degradation of its substrate MepS by structural and mutational analyses. An NlpI homodimer binds two molecules of Prc and forms three-sided MepS-docking cradles using its tetratricopeptide repeats. Prc forms a monomeric bowl-shaped structure with a lid-like PDZ domain connected by a substrate-sensing hinge that recognizes the bound C terminus of the substrate. In summary, our study reveals mechanistic details of protein degradation by the PDZ-protease Prc bound to its cognate adaptor protein.