• Cryo-EM structure of the Shigella type III needle complex.

      Lunelli, Michele; Kamprad, Antje; Bürger, Jörg; Mielke, Thorsten; Spahn, Christian M T; Kolbe, Michael; CSSB, Centre for Structural Systems Biology, Notkestraße 85, 22607 Hamburg, Germany. (PLOS, 2020-02-24)
      The Type III Secretion Systems (T3SS) needle complex is a conserved syringe-shaped protein translocation nanomachine with a mass of about 3.5 MDa essential for the survival and virulence of many Gram-negative bacterial pathogens. This system is composed of a membrane-embedded basal body and an extracellular needle that deliver effector proteins into host cells. High-resolution structures of the T3SS from different organisms and infection stages are needed to understand the underlying molecular mechanisms of effector translocation. Here, we present the cryo-electron microscopy structure of the isolated Shigella T3SS needle complex. The inner membrane (IM) region of the basal body adopts 24-fold rotational symmetry and forms a channel system that connects the bacterial periplasm with the export apparatus cage. The secretin oligomer adopts a heterogeneous architecture with 16- and 15-fold cyclic symmetry in the periplasmic N-terminal connector and C-terminal outer membrane ring, respectively. Two out of three IM subunits bind the secretin connector via a β-sheet augmentation. The cryo-EM map also reveals the helical architecture of the export apparatus core, the inner rod, the needle and their intervening interfaces.
    • Role of flagellar hydrogen bonding in Salmonella motility and flagellar polymorphic transition.

      Wang, Chu; Lunelli, Michele; Zschieschang, Erik; Bosse, Jens Bernhard; Thuenauer, Roland; Kolbe, Michael; CSSB, Centre for Structural Systembiologie, Notkestr.85, 22607 Hamburg. Germany. (Wiley, 2019-08-23)
      Bacterial flagellar filaments are assembled by tens of thousands flagellin subunits, forming 11 helically arranged protofilaments. Each protofilament can take either of the two bistable forms L-type or R-type, having slightly different conformations and inter-protofilaments interactions. By mixing different ratios of L-type and R-type protofilaments, flagella adopt multiple filament polymorphs and promote bacterial motility. In this study, we investigated the hydrogen bonding networks at the flagellin crystal packing interface in Salmonella enterica serovar typhimurium (S. typhimurium) by site-directed mutagenesis of each hydrogen bonded residue. We identified three flagellin mutants D108A, N133A and D152A that were non-motile despite their fully assembled flagella. Mutants D108A and D152A trapped their flagellar filament into inflexible right-handed polymorphs, which resemble the previously predicted 3L/8R and 4L/7R helical forms in Calladine's model but have never been reported in vivo. Mutant N133A produces floppy flagella that transform flagellar polymorphs in a disordered manner, preventing the formation of flagellar bundles. Further, we found that the hydrogen bonding interactions around these residues are conserved and coupled to flagellin L/R transition. Therefore, we demonstrate that the hydrogen bonding networks formed around flagellin residues D108, N133 and D152 greatly contribute to flagellar bending, flexibility, polymorphisms and bacterial motility.
    • Structural analysis of ligand-bound states of the Salmonella type III secretion system ATPase InvC.

      Bernal, Ivonne; Römermann, Jonas; Flacht, Lara; Lunelli, Michele; Uetrecht, Charlotte; Kolbe, Michael; CSSB, Centre for Structural Systembiologie, Notkestr.85, 22607 Hamburg. Germany. (Wiley, 2019-10-01)
      Translocation of virulence effector proteins through the type III secretion system (T3SS) is essential for the virulence of many medically relevant Gram‐negative bacteria. The T3SS ATPases are conserved components that specifically recognize chaperone–effector complexes and energize effector secretion through the system. It is thought that functional T3SS ATPases assemble into a cylindrical structure maintained by their N‐terminal domains. Using size‐exclusion chromatography coupled to multi‐angle light scattering and native mass spectrometry, we show that in the absence of the N‐terminal oligomerization domain the Salmonella T3SS ATPase InvC can form monomers and dimers in solution. We also present for the first time a 2.05 å resolution crystal structure of InvC lacking the oligomerization domain (InvCΔ79) and map the amino acids suggested for ATPase intersubunit interaction, binding to other T3SS proteins and chaperone–effector recognition. Furthermore, we validate the InvC ATP‐binding site by co‐crystallization of InvCΔ79 with ATPγS (2.65 å) and ADP (2.80 å). Upon ATP‐analogue recognition, these structures reveal remodeling of the ATP‐binding site and conformational changes of two loops located outside of the catalytic site. Both loops face the central pore of the predicted InvC cylinder and are essential for the function of the T3SS ATPase. Our results present a fine functional and structural correlation of InvC and provide further details of the homo‐oligomerization process and ATP‐dependent conformational changes underlying the T3SS ATPase activity.