Gram-negative bacteria have an outer membrane (OM) that functions as a barrier to protect the cell from toxic compounds such as antibiotics and detergents. The OM is a highly asymmetric bilayer composed of phospholipids, glycolipids, and proteins. Assembly of this essential organelle occurs outside the cytoplasm in an environment that lacks obvious energy sources such as ATP, and the mechanisms involved are poorly understood. We describe the identification of a multiprotein complex required for the assembly of proteins in the OM of Escherichia coli. We also demonstrate genetic interactions between genes encoding components of this protein assembly complex and imp, which encodes a protein involved in the assembly of lipopolysaccharides (LPS) in the OM. These genetic interactions suggest a role for YfgL, one of the lipoprotein components of the protein assembly complex, in a homeostatic control mechanism that coordinates the overall OM assembly process.

This review provides a comprehensive update of the advances in discovery, biosynthesis, and engineering of ribosomally-synthesized and post-translationally modified peptides (RiPPs).

Integral β-barrel proteins are found in the outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria. The machine that assembles these proteins contains an integral membrane protein, called YaeT in Escherichia coli , which has one or more polypeptide transport–associated (POTRA) domains. The crystal structure of a periplasmic fragment of YaeT reveals the POTRA domain fold and suggests a model for how POTRA domains can bind different peptide sequences, as required for a machine that handles numerous β-barrel protein precursors. Analysis of POTRA domain deletions shows which are essential and provides a view of the spatial organization of this assembly machine.

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Abstractβ-barrel membrane proteins in Gram-negative bacteria, mitochondria, and chloroplasts are assembled by highly conserved multi-protein complexes. The mechanism by which these molecular machines fold and insert their substrates is poorly understood. It has not been possible to dissect the folding and insertion pathway because the process has not been reproduced in a biochemical system. We purified the components that fold and insert E. coli outer membrane proteins and reconstituted β-barrel protein assembly in proteoliposomes using the enzymatic activity of a protein substrate to report on its folding state. The assembly of this protein occurred without an energy source but required a soluble chaperone in addition to the multi-protein assembly complex.The outer membranes of Gram-negative bacteria and the mitochondria and chloroplasts of higher eukaryotes contain proteins with β-barrel structure, which are assembled in their respective membranes by multi-protein machines (1-6). The folding and insertion of these β-barrels must be coordinated because they would have many unsatisfied hydrogen bonds in the membrane if they were inserted in an unfolded state, but, conversely, they would be "inside out" if they folded first in the aqueous environment and were subsequently inserted. In order to understand how β-barrel proteins assemble into membranes, we purified the proteins comprising the E. coli outer membrane protein (OMP) folding machinery and established a reconstituted system to monitor the activity of this machinery.The β-barrel assembly machine (Bam) in E. coli consists of an integral β-barrel protein, BamA (formerly YaeT), and four lipoproteins, BamB, C, D, and E (formerly YfgL, NlpB, YfiO, and SmpA, respectively). Only BamA and BamD are essential for cell survival, but deleting or depleting any member of the complex causes defects in OMP assembly (6-9). BamA has homologs in prokaryotes and eukaryotes and contains five periplasmic polypeptide transport associated (POTRA) domains, which scaffold the Bam lipoproteins (2-5,10). Unfolded OMPs are delivered to this complex following their synthesis in the cytoplasm and translocation across the inner membrane by the secretion machinery (SecYEG) ( Figure 1A) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript that are released from the Sec machine from aggregating and misfolding as they transit the periplasm. These chaperones are thought to transport OMPs in two parallel, but separate pathways-one that relies on SurA and one that involves both Skp and DegP (12,13). Most OMPs can be handled by either pathway, but SurA delivers the bulk of OMPs to the OM (14).The process of OMP assembly can be monitored in isolated mitochondria (15) and in vivo in E. coli (16). We sought to develop an in vitro system to study the function of the Bam proteins. Expressing all five bam genes in a single strain produced a mixture of complexes and sub-complexes that could not be easily separated. Previous lipoprotein and POTRA domain deletion experiments indicat...

Protein quality control requires careful regulation of intracellular proteolysis. For DegP, a periplasmic protease, substrates promote assembly of inactive hexamers into proteolytically active cages with 12, 18, 24, or 30 subunits. Here, we show that sensitive activation and cage assembly require covalent linkage of distinct substrate sequences that affect degradation (degrons). One degron binds the DegP active site and another degron binds a separate tethering site in PDZ1 in the crystal structure of a substrate-bound DegP dodecamer. FRET experiments demonstrate that active cages assemble rapidly in a reaction that is positively cooperative in substrate concentration, remain stably assembled while uncleaved substrate is present, and dissociate once degradation is complete. Thus, the energy of binding of linked substrate degrons drives assembly of the proteolytic machine responsible for subsequent degradation. Substrate cleavage and depletion results in disassembly, ensuring that DegP is proteolytically active only when sufficient quantities of protein substrates are present.

ω-Ester-containing peptides (OEPs) are a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) containing intramolecular ω-ester or ω-amide bonds. Although their distinct side-to-side connections may create considerable topological diversity of multicyclic peptides, it is largely unknown how diverse ring patterns have been developed in nature. Here, using genome mining of biosynthetic enzymes of OEPs, we identified genes encoding nine new groups of putative OEPs with novel core consensus sequences, disclosing a total of ∼1500 candidate OEPs in 12 groups. Connectivity analysis revealed that OEPs from three different groups contain novel tricyclic structures, one of which has a distinct biosynthetic pathway where a single ATP-grasp enzyme produces both ω-ester and ω-amide linkages. Analysis of the enzyme cross-reactivity showed that, while enzymes are promiscuous to nonconserved regions of the core peptide, they have high specificity to the cognate core consensus sequence, suggesting that the enzyme–core pair has coevolved to create a unique ring topology within the same group and has sufficiently diversified across different groups. Collectively, our results demonstrate that the diverse ring topologies, in addition to diverse sequences, have been developed in nature with multiple ω-ester or ω-amide linkages in the OEP family of RiPPs.

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