Pili on the surface of Sulfolobus islandicus are used for many functions, and serve as receptors for certain archaeal viruses. The cells grow optimally at pH 3 and 80° C, exposing these extracellular appendages to a very harsh environment. These pili, when removed from cells, resist digestion by trypsin or pepsin, and survive boiling in SDS or 5M guanidinium-HCl. We have used cryo-EM to determine the structure of these filaments at 4.1 Å resolution. An atomic model was built by combining the map with bioinformatics without prior knowledge of the pilin sequence, an approach that should prove useful for assemblies where all of the components are not known. The atomic structure of the pilus was unusual, with almost a third of the residues being either threonine or serine, and with many hydrophobic surface residues. While the map showed extra density consistent with glycosylation for only three residues, mass measurements suggested extensive glycosylation. We propose that this extensive glycosylation renders these filaments soluble and provides the remarkable structural stability. We also show that the overall fold of the archaeal pilin is remarkably similar to archaeal flagellin, establishing common evolutionary origins.

Regulated exocytosis of synaptic vesicles is substantially faster than of endocrine dense core vesicles despite similar molecular machineries. The reasons for this difference are unknown and could be due to different regulatory proteins, different spatial arrangements, different vesicle sizes, or other factors. To address these questions, we take a reconstitution approach and compare regulated SNARE-mediated fusion of purified synaptic and dense core chromaffin and insulin vesicles using a single vesicle-supported membrane fusion assay. In all cases, Munc18 and complexin are required to restrict fusion in the absence of calcium. Calcium triggers fusion of all docked vesicles. Munc13 (C1C2MUN domain) is required for synaptic and enhanced insulin vesicle fusion, but not for chromaffin vesicles, correlating inversely with the presence of CAPS protein on purified vesicles. Striking disparities in calcium-triggered fusion rates are observed, increasing with curvature with time constants 0.23 s (synaptic vesicles), 3.3 s (chromaffin vesicles), and 9.1 s (insulin vesicles) and correlating with rate differences in cells.

Daptomycin is an acidic, 13-amino acid, cyclic polypeptide that contains a number of non-proteinogenic residues and is modified at its N-terminus with a decanoyl chain. It has been in clinical use since 2003 against selected drug-resistant Staphylococcus aureus and Enterococcus spp infections. In vitro, daptomycin is active against Gram-positive pathogens at low concentrations but its antibiotic activity depends critically on the presence of calcium ions. This dependence has been thought to arise from binding of one or two Ca2+ ions to daptomycin as a required step in its interaction with the bacterial membrane. Here, we investigated the interaction of daptomycin with giant unilamellar vesicles (GUVs) composed 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG). We used fluorescence confocal microscopy to monitor binding of the peptide to GUVs and follow its effect on the membrane of the vesicle. We found that, in the absence of POPG or Ca2+, daptomycin does not bind measurably to the lipid membrane. In the presence of 20–30% PG in the membrane and 2 mM Ca2+, daptomycin induces the formation of membrane domains rich in acidic lipids. This effect is not induced by Ca2+ alone. In addition, daptomycin causes GUV collapse, but it does not translocate across the membrane to the inside of intact POPC:POPG vesicles. We conclude that pore formation is probably not the mechanism by which the peptide functions. On the other hand, we found that daptomycin coclusters with the anionic phospholipid POPG and the fluorescent probes used, leading to extensive formation of daptomycin–POPG domains in the membrane.

No abstract

Vertebrates, from zebra fish to humans, have an innate immune recognition of many bacterial flagellins. This involves a conserved eight-amino acid epitope in flagellin recognized by the Toll-like receptor 5 (TLR5). Several important human pathogens, such as Helicobacter pylori and Campylobacter jejuni, have escaped TLR5 activation by mutations in this epitope. When such mutations were introduced into Salmonella flagellin, motility was abolished. It was previously argued, using very low-resolution cryoelectron microscopy (cryo-EM), that C. jejuni accommodated these mutations by forming filaments with 7 protofilaments, rather than the 11 found in other bacteria. We have now determined the atomic structure of the C. jejuni G508A flagellar filament from a 3.5-Å-resolution cryo-EM reconstruction, and show that it has 11 protofilaments. The residues in the C. jejuni TLR5 epitope have reduced contacts with the adjacent subunit compared to other bacterial flagellar filament structures. The weakening of the subunit−subunit interface introduced by the mutations in the TLR5 epitope is compensated for by extensive interactions between the outer domains of the flagellin subunits. In other bacteria, these outer domains can be nearly absent or removed without affecting motility. Furthermore, we provide evidence for the stabilization of these outer domain interactions through glycosylation of key residues. These results explain the essential role of glycosylation in C. jejuni motility, and show how the outer domains have evolved to play a role not previously found in other bacteria.