Highly stable oligomeric complexes of the monotopic membrane protein caveolin serve as fundamental building blocks of caveolae. Current evidence suggests these complexes are disc shaped, but the details of their structural organization and how they assemble are poorly understood. Here, we address these questions using single particle electron microscopy of negatively stained recombinant 8S complexes of human caveolin 1. We show that 8S complexes are toroidal structures ~15 nm in diameter that consist of an outer ring, an inner ring, and central protruding stalk. Moreover, we map the position of the N and C termini and determine their role in complex assembly, and visualize the 8S complexes in heterologous caveolae. Our findings provide critical insights into the structural features of 8S complexes and allow us to propose a model for how these highly stable membrane-embedded complexes are generated.

Helicobacter pylori colonizes the human stomach and contributes to the development of gastric cancer and peptic ulcer disease. H. pylori secretes a pore-forming toxin called vacuolating cytotoxin A (VacA), which contains two domains (p33 and p55) and assembles into oligomeric structures. Using single particle cryo-electron microscopy, we have determined low-resolution structures of a VacA dodecamer and heptamer, as well as a 3.8 Å structure of the VacA hexamer. These analyses show that VacA p88 consists predominantly of a right-handed beta-helix that extends from the p55 domain into the p33 domain. We map the regions of p33 and p55 involved in hexamer assembly, model how interactions between protomers support heptamer formation, and identify surfaces of VacA that likely contact membrane. This work provides structural insights into the process of VacA oligomerization and identifies regions of VacA protomers that are predicted to contact the host cell surface during channel formation.

No abstract

Short title: Single particle analysis of caveolin-1 complexesOne sentence summary. Single particle electron microscopy reveals the overall structural organization of oligomeric complexes of an essential structural component of caveolae, the monotopic membrane protein caveolin-1.Abbreviations: Caveolin (Cav), electron microscopy (EM), heterologous caveolae (h-caveolae), wild type (WT), Maltose binding protein (MBP), congenital generalized lipodystrophy (CGL), pulmonary arterial hypertension (PAH), Fast protein liquid chromatography (FPLC). AbstractHighly stable oligomeric complexes of the monotopic membrane protein caveolin serve as fundamental building blocks of caveolae. Current evidence suggests these complexes are disc shaped, but the details of their structural organization and how they assemble are poorly understood. Here, we address these questions using single particle electron microscopy of negatively stained recombinant 8S complexes of human Caveolin-1. We show that 8S complexes are toroidal structures ~15 nm in diameter that consist of an outer ring, an inner ring, and central protruding stalk. Moreover, we map the position of the N-and C-termini and determine their role in complex assembly, and visualize the 8S complexes in heterologous caveolae. Our findings provide critical insights into the structural features of 8S complexes and allow us to propose a new model for how these highly stable membrane-embedded complexes are generated.

adsorption of wild-typed HD5 (WT) and T7E21R on a bacterial membrane were conducted. A bacterial membrane models with O-antigen of lipopolysaccharides (LPS) mimicking E. coli membrane was used. Atomistic molecular simulations were performed to obtain an insight into the adsorption of these peptides on the bacterial membrane. Our data highlight the different dimeric conformation between WT and T7E21R. It is found that the parallel b-strands of T7E21R is formed instead of antiparallel in HD5. This conformation of change results in different degrees of binding affinities. Key interactions are also extracted here. However, it is shown that WT can penetrate deeper into the O-antigen than T7E21R. The uncharged residues such as Thr, Ser, Gly, and Tyr are dominated in contacts with O-antigen of LPS as much as the charged residues. An insight obtained here can serve as a guideline for further improvement of defensin-derived antibiotic design.