Biology subjects

Imada, K.

Publications and source records attributed to Imada, K..

10 recordsLinked to original sources

bioRxiv2026

A single conserved residue in FlhA couples export gate activation to substrate specificity switching

FlhA is a core component of the bacterial flagellar type III secretion system that functions as both an export engine and a switch regulator of substrate specificity. How these two processes are mechanistically coupled remains unclear. Here we identify a single conserved residue in FlhA, Arg-391, as a key molecular node that links export gate activation to substrate specificity switching. The R391A substitution blocks the transition from hook-type to filament-type secretion, resulting in polyhooks lacking filaments. An intermolecular salt-bridge formation of Arg-391 with Glu-351 of an adjacent FlhA subunit stabilizes the filament-type FlhA ring. Suppressor mutations in FlhA and the ruler protein FliK restore this interaction, indicating that FliK promotes the intermolecular salt-bridge formation during export switching. Moreover, Arg-391 is required for efficient ATPase-independent activation of the export gate. Together, these findings reveal an electrostatic mechanism by which FlhA coordinates export gate activation and substrate specificity switching during flagellar assembly.

microbiology↗

bioRxiv2025

Structural basis of flagellar rod assembly on the FliPQR protein-export channel

The FliPQR complex constitutes a channel for export of the bacterial flagellar proteins involved in axial structure assembly. It also serves as a template for flagellar rod assembly. A periplasmic gate, formed by the N-terminal -helices of FliP and FliR, remains closed until FliE assembles onto FliP and FliR. The mechanism by which FliE opens the gate and assembles has remained unclear. Here, we present a cryoEM structure of the FliPQR complex in closed form at 3.0 [A] resolution. A {beta}-cap formed by the N-terminal {beta}-strands of FliP and FliR creates a tight seal in the closed gate. Interaction of FliE with FliP and FliR induces a conformational change in FliP and FliR, with their N-terminal -helices move up and outward. Consequently, the N-terminal {beta}-strands of FliP and FliR start opening the periplasmic gate one after another and form a docking site for FliE to initiate rod assembly.

microbiology↗

bioRxiv2024

Structural insights into the assembly mechanism of the flagellar MS-ring with three different symmetries

The flagellar basal body MS-ring, formed by 34 FliF subunits, is the core of the flagellar motor as well as the base for flagellar assembly. The MS-ring is also a housing for the flagellar protein export gate complex that is required for construction of the flagellum on the cell surface. A large periplasmic region of FliF contains three ring-building motifs named RBM1, RBM2, and RBM3. RBM3 forms the S-ring and {beta}-collar with C34 symmetry. RBM2 forms the inner core ring of the M-ring with C23 symmetry surrounded by 11 cog-like structures formed by RBM1 and RBM2. However, it remains unknown how FliF assembles to generate these three different symmetries within the MS-ring. Here, we report the two cryoEM structures of the MS-ring formed by FliF co-expressed with FliG and transmembrane export gate proteins. Structural comparison of 33-mer and 34-mer MS-rings revealed that a subtle change in the conformation of RBM3 produces the different rotational symmetries. Combination of cryoEM structural and mutational analyses of the MS-ring with C33 symmetry provides evidence that the well-conserved DQxGxxL motif within a flexible loop connecting RBM2 and RBM3 allows RBM2 to take two different orientations relative to RBM3 to form not only 11 cog-like structures just outside the inner core ring with C22 symmetry but also an appropriately sized central pore in the inner core ring to accommodate the export gate complex. IMPORTANCEThe flagellar MS-ring is the core of the flagellar motor and serves not only as an initial template for flagellar assembly but also as a base to accommodate the flagellar protein export complex. The MS-ring is formed by 34 subunits of FliF with two transmembrane helices and a large periplasmic region containing ring-building motifs, RBM1, RBM2, and RBM3. FliF adopts two different conformations in the MS-ring to generate three different rotational symmetries, C34, C23, and C11. However, how FliF assembles to produce these three symmetries remains a mystery. Combination of cryoEM structural and mutational analyses has provided evidence that the well-conserved DQxGxxL motif within a hinge loop connecting RBM2 and RBM3 allows RBM2 to take two different orientations relative to RBM3, allowing 23 RBM2 domains of 34 subunits to form the inner core ring with a properly sized central pore to accommodate the flagellar protein export complex.

microbiology↗

bioRxiv2024

Structural insight into sodium ion pathway in the bacterial flagellar stator from marine Vibrio

Many bacteria swim in liquid or swarm on surface using the flagellum rotated by a motor driven by specific ion flow. The motor consists of the rotor and stator, and the stator converts the energy of ion flow to mechanical rotation. However, the ion pathway and the mechanism of stator rotation coupled with specific ion flow are still obscure. Here, we determined the structures of the Na+-driven stator of Vibrio, namely PomAB, in the presence and absence of sodium ions and the structure with its specific inhibitor, phenamil, by cryo-electron microscopy. The structures and following functional analysis revealed the sodium ion pathway, the mechanism of ion selectivity, and the inhibition mechanism by phenamil. We propose a model of sodium ion flow coupled with the stator rotation based on the structures. This work provides insights into the molecular mechanisms of ion specificity and conversion of the electrochemical potential into mechanical functions.

microbiology↗

bioRxiv2024

Structural analysis of S-ring composed of FliFG fusion proteins in marine Vibrio polar flagellar motors

The marine bacterium Vibrio alginolyticus possesses a polar flagellum driven by a sodium ion flow. The main components of the flagellar motor are the stator and rotor. The C-ring and MS-ring which are composed of FliG and FliF, respectively, are parts of the rotor. Here, we purified an MS-ring composed of FliF-FliG fusion proteins and solved the near-atomic resolution structure of the S-ring--the upper part of the MS-ring--using cryo-electron microscopy. This is the first report of an S-ring structure from Vibrio whereas, previously, only those from Salmonella have been reported. The Vibrio S-ring structure reveals novel features compared to that of Salmonella such as tilt angle differences of the core domain and the {beta}-collar region, the decrease of the inter-subunit interaction between core domains, and altered electrostatic inner-surface. The residues potentially interact with other flagellar components, such as FliE and FlgB, are well structurally conserved in Vibrio S-ring. These comparisons clarified the conserved and non-conserved structural features of the MS-ring across different species. IMPORTANCEUnderstanding the structure and function of the flagellar motor in bacterial species is essential for uncovering the mechanisms underlying bacterial motility and pathogenesis. Our study revealed the structure of the Vibrio S-ring, a part of its polar flagellar motor, and highlighted its unique features compared with the well-studied Salmonella S-ring. The observed differences in the inter-subunit interactions and in the tilt angles between the Vibrio and Salmonella S-rings highlighted the species-specific variations in the flagellar assembly. By concentrating on the region where the S-ring and the rod proteins interact, we uncovered conserved residues essential for the interaction. Our research contributes to advancing of bacterial flagellar biology.

biophysics↗

bioRxiv2021

Structure and polymerization dynamics of bacterial actin MreB3 and MreB5 involved in Spiroplasma swimming.

MreB is a bacterial protein belonging to the actin superfamily. It polymerises into an antiparallel double-stranded filament that generally functions in cell shape determination by maintaining cell wall synthesis. Spiroplasma eriocheiris, a helical wall-less bacterium, has five classes of MreB homologs (SpeMreB1-5) that are likely to be involved in swimming motility. Here, we investigated the structure, ATPase activity, and polymerisation dynamics of SpeMreB3 and SpeMreB5. SpeMreB3 polymerised into an antiparallel double-stranded filament, and SpeMreB5 formed sheets, including the antiparallel filament, upon the binding of a nucleotide. SpeMreB3 showed slow Pi release owing to the lack of an amino acid motif conserved in the catalytic centre of MreB family proteins. Our crystal structures of SpeMreB3 and analyses of the mutant variants showed that the amino acid motif most likely plays a role in eliminating the proton of the nucleophilic water for ATP hydrolysis. Our sedimentation assay suggests that SpeMreB3 has a lower polymerisation activity than SpeMreB5, while their polymerisation dynamics are qualitatively similar to those of other actin superfamily proteins, in which ATP hydrolysis stabilises the filament, and Pi release leads to depolymerisation.

biochemistry↗

bioRxiv2020

Native structure of flagellar MS ring is formed by 34 subunits with 23-fold and 11-fold subsymmetries

The bacterial flagellar MS ring is a transmembrane complex acting as the core of the flagellar motor. It not only acts as the template for rod and C ring assembly but also houses the type III protein export gate for assembly of the rod, hook and filament. The cytoplasmic C ring, involved in torque generation and rotation switch, is directly attached to the MS ring, and a symmetry mismatch between 26-fold MS ring and 34-fold C ring had been a long puzzle as to whether this would play some role in motor function. Although this puzzle seemed to have been resolved by the recent high-resolution structure of the MS ring with 33-fold symmetry with a variation from 32-fold to 35-fold because the C ring also shows a similar symmetry variation, it still remained ambiguous whether their symmetries are matched in the native motor structure. Here we show that the native MS ring structure formed by full-length FliF is 34-fold with no symmetry variation whereas the C ring has a small symmetry variation, indicating a flexibility in C ring assembly to generate small symmetry mismatches. We also show two conformations of FliF in part of its periplasmic region to form the 34-subunit ring with 23-fold and 11-fold subsymmetries in the inner and middle M ring, respectively, to accommodate the export gate at the center of the M ring.

microbiology↗

bioRxiv2020

The FlhA linker mediates flagellar protein export switching during flagellar assembly

The flagellar protein export apparatus switches export specificity from hook-type to filament-type upon completion of hook assembly, thereby initiating filament assembly at the hook tip. The C-terminal cytoplasmic domain of FlhA (FlhAC) forms a homo-nonameric ring structure that serves as a docking platform for flagellar export chaperones in complex with their cognate filament-type substrates. Interactions of the flexible linker of FlhA (FlhAL) with its nearest FlhAC subunit in the ring allow the chaperones to bind to FlhAC to facilitate filament-type protein export, but it remains unclear how it occurs. Here, we report that FlhAL acts as a switch that brings the order to flagellar assembly. The crystal structure of FlhAC(E351A/D356A) showed that Trp-354 in FlhAL bound to the chaperone-binding site of its neighboring subunit. We propose that FlhAL binds to the chaperon-binding site of FlhAC to suppress the interaction between FlhAC and the chaperones until hook assembly is completed.

microbiology↗

bioRxiv2019

Thick perirenal fat predicts the growth pattern of renal cell carcinoma

ObjectiveTo examine the relationship between the direction of renal cell carcinoma growth and the visceral/perirenal fat volume.\n\nPatients and Methods: We retrospectively reviewed computed tomography scans of 153 patients with stage 1 renal cell carcinoma who underwent radical or partial nephrectomy in our hospital between January 2013 and July 2016. We calculated the visceral/subcutaneous/perirenal fat volumes using SYNAPSE VINCENT(R). Of the 60 patients, the perirenal fat was immunohistochemically stained for leptin, adiponectin, COX-2 and UCP-1, and the association with outward tumor protrusion was evaluated.\n\nResultsOf the 153 cases, 88 had confirmed outward expansion (57.5%), 110 were classed as pT1a (52 and 58 with outer and inner expansion, respectively), 43 were classed as pT1b (36 and 7 with outer and inner expansion, respectively; P<0.0001). Multivariate logistic regression model showed a trend toward significance in pT1b (vs pT1a, [OR] 6.033, 95%CI=2.409-15.108, P=0.0001), perirenal fat percentage >1.0 (vs [≤]1.0, [OR] 2.596, 95%CI=1.205-5.591, P=0.014). as independent predictors for outer protrusion. Immunohistochemical staining was positive for UCP-1 expression in 31 out of 41 outgrowth types (75.6%), and all 19 endogenous types (100%; P=0.003).\n\nConclusionsRenal cell carcinoma with thick perirenal fat correlates with an increased likelihood of developing outward tumor protrusion; therefore, fat distribution may affect the development of renal cell carcinoma.

cancer biology↗

bioRxiv2018

Second harmonic generation polarization microscopy as a tool for protein structure analysis

Second-harmonic generation (SHG) is a nonlinear coherent scattering process that is sensitive to molecular structures in illuminated materials. We report SHG polarization measurement for the detection of protein conformational changes in solutions of macromolecular protein assemblies such as microtubules and protein crystals. The results illustrate the potential of this method for protein structural analysis in physiological solutions at room temperature without labelling.

biophysics↗