Biology subjects

Namba, K.

Publications and source records attributed to Namba, K..

40 records · Page 3Linked to original sources

bioRxiv2020

The FlgN chaperone activates the Na+-driven engine of the flagellar protein export apparatus

The bacterial flagellar protein export machinery promotes H+-coupled translocation of flagellar proteins to the cell exterior. When the cytoplasmic ATPase complex does not function, the transmembrane export gate complex opens its Na+ channel and continues protein transport. However, it remains unknown how. Here we report that the FlgN chaperone acts as a switch to activate a backup export mechanism for the ATPase complex by activating the Na+-driven engine. Impaired interaction of FlhA with the FliJ subunit of the ATPase complex increased Na+-dependence of flagellar protein export. Deletion of FlgN inhibited protein export in the absence of the ATPase complex but not in its presence. Gain-of-function mutations in FlhA restored not only the FlgN defect but also the FliJ defect. We propose that the interaction of FlgN with FlhA opens the Na+ channel in the export engine, thereby maintaining the protein export activity in the absence of the active ATPase complex.

microbiology↗

bioRxiv2020

The mechanical inhibition of the isolated Vo from V-ATPase for proton conductance

V-ATPase is an energy converting enzyme, coupling ATP hydrolysis/synthesis in the hydrophilic V1 moiety, with proton flow through the Vo membrane moiety, via rotation of the central rotor complex relative to the surrounding stator apparatus. Upon dissociation from the V1 domain, the Vo of eukaryotic V-ATPase can adopt a physiologically relevant auto-inhibited form in which proton conductance through the Vo is prevented, however the molecular mechanism of this inhibition is not fully understood. Using cryo-electron microscopy, we determined the structure of both the holo V/A-ATPase and the isolated Vo at near-atomic resolution, respectively. These structures clarify how the isolated Vo adopts the auto-inhibited form and how the holo complex prevents the formation of this inhibited Vo form. One Sentence SummaryCryo-EM structures of rotary V-ATPase reveal the ON-OFF switching mechanism of H+ translocation in the Vo membrane domain.

biophysics↗

bioRxiv2019

Refined mechanism of Mycoplasma mobile gliding based on structure, ATPase activity, and sialic acid binding of machinery

Mycoplasma mobile, a fish pathogen, glides on solid surfaces by repeated catch, pull, and release of sialylated oligosaccharides by a unique mechanism based on ATP energy. The gliding machinery is composed of huge surface proteins and an internal jellyfish -like structure. Here, we elucidated the detailed three-dimensional structures of the machinery by electron cryotomography. The internal tentacle -like structure hydrolyzed ATP, which was consistent with the fact that the paralogs of the - and {beta}-subunits of F1-ATPase are at the tentacle structure. The electron microscopy suggested conformational changes of the tentacle structure depending on the presence of ATP analogs. The gliding machinery was isolated and shown that the binding activity to sialylated oligosaccharide was higher in the presence of ADP than in the presence of ATP. Based on these results, we proposed a model to explain the mechanism of M. mobile gliding.\n\nIMPORTANCEThe genus Mycoplasma is made up of the smallest parasitic and sometimes commensal bacteria; Mycoplasma pneumoniae, which causes human walking pneumonia, is representative. More than ten Mycoplasma species glide on host tissues by novel mechanisms always in the direction of the distal side of the machinery. Mycoplasma mobile, the fastest species in the genus, catches, pulls, and releases sialylated oligosaccharides (SOs), the carbohydrate molecules also targeted by influenza viruses, by means of a specific receptor and using ATP hydrolysis for energy. Here, the architecture of the gliding machinery was visualized three-dimensionally by electron cryotomography (ECT), and changes in the structure and binding activity coupled to ATP hydrolysis were discovered. Based on the results, a refined mechanism was proposed for this unique motility.

microbiology↗

bioRxiv2019

Structure of the native supercoiled flagellar hook as a universal joint

Bacteria swim in viscous liquid environments by using the flagellum1-3. The flagellum is composed of about 30 different proteins and can be roughly divided into three parts: the basal body, the hook and the filament. The basal body acts as a rotary motor powered by ion motive force across the cytoplasmic membrane as well as a protein export apparatus to construct the axial structure of the flagellum. The filament is as a helical propeller, and it is a supercoiled form of a helical tubular assembly consisting of a few tens of thousands of flagellin molecules4. The hook is a relatively short axial segment working as a universal joint connecting the basal body and the filament for smooth transmission of motor torque to the filament5,6. The structure of hook has been studied by combining X-ray crystal structure of a core fragment of hook protein FlgE and electron cryomicroscopy (cryoEM) helical image analysis of the polyhook in the straight form and has given a deep insight into the universal joint mechanism7. However, the supercoiled structure of the hook was an approximate model based on the atomic model of the straight hook without its inner core domain7 and EM observations of supercoiled polyhook by freeze-dry and Pt/Pd shadow cast8. Here we report the native supercoiled hook structure at 3.1 [A] resolution by cryoEM single particle image analysis of the polyhook. The atomic model built on the three-dimensional (3D) density map show the actual changes in subunit conformation and intersubunit interactions upon compression and extension of the 11 protofilaments that occur during their smoke ring-like rotation and allow the hook to function as a dynamic molecular universal joint with high bending flexibility and twisting rigidity.

biophysics↗