Biology subjects

Remaut, H.

Publications and source records attributed to Remaut, H..

22 records · Page 2Linked to original sources

bioRxiv2020

Efficient long-range conduction in cable bacteria through nickel protein wires

Filamentous cable bacteria display unrivalled long-range electron transport, generating electrical currents over centimeter distances through a highly ordered network of fibers embedded in their cell envelope. The conductivity of these periplasmic wires is exceptionally high for a biological material, but their chemical structure and underlying electron transport mechanism remain unresolved. Here, we combine high-resolution microscopy, spectroscopy, and chemical imaging on individual cable bacterium filaments to demonstrate that the periplasmic wires consist of a conductive protein core surrounded by an insulating shell layer. The core proteins contain a sulfur-ligated nickel cofactor, and conductivity decreases when nickel is oxidized or selectively removed. The involvement of nickel as the active metal in biological conduction is remarkable, and suggests a hitherto unknown form of electron transport that enables efficient conduction in centimeter-long protein structures.

microbiology↗

bioRxiv2020

Bacillus endospore appendages form a novel family of disulfide-linked pili

Bacillus cereus sensu lato is a group of Gram-positive endospore-forming bacteria with high ecological diversity. Their endospores are decorated with micrometer-long appendages of unknown identity and function. Here we isolate endospore appendages (Enas) from the food poisoning outbreak strain B. cereus NVH 0075-95 and find proteinaceous fibers of two main morphologies. By using cryo-EM and 3D helical reconstruction we show that Bacillus Enas form a novel class of Gram-positive pili. Enas consist of single domain subunits with jellyroll topology that are laterally stacked by {beta}-sheet augmentation. Enas are longitudinally stabilized by disulfide bonding through N-terminal connector peptides that bridge the helical turns. Together, this results in flexible pili that are highly resistant to heat, drought and chemical damage. Phylogenomic analysis reveals the presence of defined ena clades amongst different eco- and pathotypes. We propose Enas to represent a novel class of pili specifically adapted to the harsh conditions encountered by bacterial spores.

microbiology↗

bioRxiv2019

Structural insight into the formation of lipoprotein-β-barrel complexes by the β-barrel assembly machinery

The {beta}-barrel assembly machinery (BAM) inserts outer membrane {beta}-barrel proteins (OMPs) in the outer membrane of Gram-negative bacteria. In Enterobacteriacea, BAM also mediates export of the stress sensor lipoprotein RcsF to the cell surface by assembling RcsF-OMP complexes. Although BAM has been the focus of intense research due to its essential activity in generating and maintaining the outer membrane, how it functions remains poorly understood. Here, we report the crystal structure of the key BAM component BamA in complex with RcsF. BamA adopts an inward-open conformation, with the lateral gate to the membrane closed. The globular domain of RcsF is lodged deep inside the lumen of the BamA barrel, binding regions proposed to undergo an outward and lateral opening during OMP insertion. Our structural and biochemical data indicate a push-and-pull mechanism for RcsF export upon conformational cycling of BamA and provide a mechanistic explanation for how RcsF uses its interaction with BamA to detect envelope stress. Our data also suggest that the flux of incoming OMP substrates is involved in the control of BAM activity. Overall, the structural insights gleaned here elucidate a fundamental biological process and suggest a new avenue for antibiotic development.

microbiology↗

bioRxiv2019

Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM

Nanobodies (Nbs) are popular and versatile tools for structural biology because they have a compact single immunoglobulin domain organization. Nbs bind their target proteins with high affinities while reducing their conformational heterogeneity, and they stabilize multi-protein complexes. Here we demonstrate that engineered Nbs can also help overcome two major obstacles that limit the resolution of single-particle cryo-EM reconstructions: particle size and preferential orientation at the water-air interface. We have developed and characterised novel constructs, termed megabodies, by grafting Nbs into selected protein scaffolds to increase their molecular weight while retaining the full antigen binding specificity and affinity. We show that the megabody design principles are applicable to different scaffold proteins and recognition domains of compatible geometries and are amenable for efficient selection from yeast display libraries. Moreover, we used a megabody to solve the 2.5 [A] resolution cryo-EM structure of a membrane protein that suffers from severe preferential orientation, the human GABAA {beta}3 homopentameric receptor bound to its small-molecule agonist histamine.

biochemistry↗