Cellular proteomes are distributed in multiple compartments: on DNA, ribosomes, on and inside membranes, or they become secreted. Structural properties that allow polypeptides to occupy subcellular niches, particularly to after crossing membranes, remain unclear. We compared intrinsic and extrinsic features in cytoplasmic and secreted polypeptides of the Escherichia coli K-12 proteome. Structural features between the cytoplasmome and secretome are sharply distinct, such that a signal peptide-agnostic machine learning tool distinguishes cytoplasmic from secreted proteins with 95.5% success. Cytoplasmic polypeptides are enriched in aliphatic, aromatic, charged and hydrophobic residues, unique folds and higher early folding propensities. Secretory polypeptides are enriched in polar/small amino acids, β folds, have higher backbone dynamics, higher disorder and contact order and are more often intrinsically disordered. These non-random distributions and experimental evidence imply that evolutionary pressure selected enhanced secretome flexibility, slow folding and looser structures, placing the secretome in a distinct protein class. These adaptations protect the secretome from premature folding during its cytoplasmic transit, optimize its lipid bilayer crossing and allowed it to acquire cell envelope specific chemistries. The latter may favor promiscuous multi-ligand binding, sensing of stress and cell envelope structure changes. In conclusion, enhanced flexibility, slow folding, looser structures and unique folds differentiate the secretome from the cytoplasmome. These findings have wide implications on the structural diversity and evolution of modern proteomes and the protein folding problem.

No abstract

Polysaccharide export outer membrane proteins of Gram-negative bacteria are involved in the export of polysaccharides across the outer membrane. The mechanisms of polysaccharide export across the outer membrane in Gram-negative bacteria are not yet completely clear. However, the mechanisms of polysaccharide assembly in Escherichia coli have been intensively investigated. Here, we mainly review the current understanding of the assembly mechanisms of group 1 capsular polysaccharide, group 2 capsular polysaccharide and lipopolysaccharide of E. coli, and the current structures and interactions of some polysaccharide export outer membrane proteins with other proteins involved in polysaccharide export in Gram-negative bacteria. In addition, LptD may be targeted by peptidomimetic antibiotics in Gram-negative bacteria. We also give insights into the directions of future research regarding the mechanisms of polysaccharide export.

The Type III protein secretion (T3S) pathway is widespread in bacterial Gram-negative pathogens. It comprises the injectisome with a cytoplasm-facing inner membrane translocase and a surface-exposed needle. The translocase comprises a conical SctR5S4T1 export channel, decorated by SctU, and enveloped by SctV. The large cytoplasmic domain (C-domain) of SctV binds T3S chaperone/exported protein and forms a putative ante-chamber leading to the membrane translocase. Here we probed the mechanism of assembly and function of SctV. Using live cell imaging, SctV was shown to assemble in peripheral oligomeric clusters in both EPEC and a non-T3SS harbouring E.coli strain. Non-ionic detergents extracted SctV homo-nonamers from membranes of both strains. His-SctV9 reconstituted in peptidiscs revealed an elongated, tripartite particle of ~22nm with a membrane domain and a narrower linker connecting to a C-domain. The C-domain assembles in a hollow asymmetric ring with a 5-6 nm-wide inner opening. SctV9 is necessary and sufficient to act as a receptor for two different chaperone/exported protein pairs by binding them at distinct C-domain sites identified by immobilized peptide arrays. Binding sites are not only important for binding but also essential for secretion suggesting a close mechanistic link between the receptor and secretion activities. These findings advance structural understanding of injectisome assembly and reveal that chaperone/exported protein targeting is mechanistically uncoupled from the succeeding translocation step.Author summaryThe export apparatus of the Type III secretion pathway is conserved in flagellar and virulence injectisomes. Its major component SctV, is essential for T3S substrate targeting and translocation. Here, we analysed SctV assembly and function as a receptor for targeting T3S exported proteins. SctV was shown to self-nonamerize, in a structure that is sufficient for functional binding of chaperone/exported protein complexes. Nonameric SctV reconstituted in peptidiscs and its nonameric ring-forming cytoplasmic domain reveal structural features and lay the foundation for high-resolution cryoEM. These tools set the stage for mechanistic dissection of the structural interactions of the export apparatus with the exported proteins, independently of the transmembrane crossing reaction.