Biology subjects

Namba, K.

Publications and source records attributed to Namba, K..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

FliH and FliI help FlhA bring strict order to flagellar protein export in Salmonella

The flagellar type III secretion system (fT3SS) switches substrate specificity from rod-hook-type to filament-type upon hook completion, terminating hook assembly and initiating filament assembly. The FlhA ring is directly involved in substrate recognition, allowing the fT3SS to coordinate flagellar protein export with assembly, but the mechanism remains a mystery. Here, we report that the highly conserved GYXLI motif of FlhA is important for ordered protein export by the fT3SS. The fT3SS with the flhA(Y369A/R370A/L371A/I372A) (AAAA) or flhA(Y369G/R370G/L371G/I372G) (GGGG) mutation did not switch the substrate specificity at an appropriate timing of hook assembly. The A372V/T and G372V substitutions recovered the export switching function of the AAAA and GGGG mutants, respectively, in the presence but not in the absence of FliH and FliI, components of the flagellar ATPase complex. Interestingly, a filament-type substrate, FlgL, was secreted via the fT3SS with the AAAA or GGGG mutation during hook assembly in the absence of FliH and FliI but not in their presence. These observations suggest that FlhA requires the flagellar ATPase complex not only to efficiently remodel its ring structure responsible for the substrate specificity switching of the fT3SS but also to correct substrate recognition errors that occur during flagellar assembly.

microbiology↗

bioRxiv2023

Antibodies halting motility in Mycoplasma pneumoniae reveal the dynamic nature of the adhesion complex

Mycoplasma pneumoniae is a bacterial wall-less human pathogen and the etiological agent of atypical pneumonia and tracheobronchitis in both adults and children. M. pneumoniae infectivity, gliding motility and adherence to host target respiratory epithelial cells are mediated by adhesin proteins P1 and P40/P90 forming a transmembrane complex that binds to sialylated oligosaccharides human cell ligands. Here we report the cryo-EM structure of P1 bound to the Fab fragment of monoclonal antibody P1/MCA4, which stops gliding and induces detachment of motile M. pneumoniae cells. On the contrary, polyclonal antibodies generated against the N-domain of P1 or against the whole ectodomain of P40/P90 have little or no effects on adhesion or motility. The epitope of P1/MCA4, centred on loop Thr1426-Asp1438 in the small C-terminal domain of P1, is inaccessible to antibodies in the "open" conformation of the adhesion complex, when ready for attachment to sialylated oligosaccharides. Mutations in the highly conserved Engelman motifs found in the transmembrane helix of P40/P90 also alter adhesion and motility. During the attachment/detachment cycle of the adhesion complex, the C-terminal domain of P1 experiences large conformational rearrangements that are hindered by the antibodies against the domain. Interfering with the gliding of mycoplasma cells suggests new ways to confront M. pneumoniae infections.

molecular biology↗

bioRxiv2023

Cryo-EM of cytoskeletal 'fibril' protein involved in Spiroplasma swimming

Spiroplasma, parasitic or commensal bacteria, can swim by switching the handedness of its helical cell body. A helical cell body is formed by an internal ribbon of MreB, an actin superfamily, and Spiroplasma-specific fibril proteins. Here we have solved the structure of the fibril filament by single-particle cryo-electron microscopy at 3.6 [A] resolution and built its atomic structure. The structure is composed of repeated rings and cylinders. The N-terminal cylinder of the fibril protein shows a structure similar to that of S-adenosylhomocysteine nucleosidase, while the C-terminal ring does not show similarity to other proteins. The filament is nonpolar and flexible, possessing a helical pitch of 700 nm, consistent with cell helicity. Cryo-electron tomography revealed aligned several MreB filaments in the center of the ribbon, flanked by membrane-binding fibril filaments through electrostatic interactions. This study discusses the evolution and roles of the fibril filament.

microbiology↗

bioRxiv2022

Development of a 1:1-binding biparatopic anti-TNFR2 antagonist by epitope selection

Conventional bivalent antibodies against cell surface receptors often initiate unwanted signal transduction by crosslinking two antigen molecules. Biparatopic antibodies (BpAbs) bind to two different epitopes on the same antigen, thus altering crosslinking ability. In this study, we developed BpAbs against tumor necrosis factor receptor 2 (TNFR2), which is an attractive immune checkpoint target. Using different pairs of variable regions specific to topographically distinct TNFR2 epitopes, we successfully regulated the size of BpAb-TNFR2 immunocomplexes to result in controlled agonistic activities. One particular antagonist BpAb bound TNFR2 in 1:1 ratio without unwanted signal transduction, with its structural basis revealed by cryo-electron microscopy. This antagonist suppressed the proliferation of regulatory T cells expressing TNFR2. Therefore, the BpAb format would be useful in designing specific and distinct antibody functions.

biochemistry↗

bioRxiv2022

High-resolution structure of a microtubule-like tube composed of FtsZ-monobody complexes

FtsZ, a bacterial tubulin homologue, forms protofilaments and the Z-ring, which acts as a scaffold for accessory proteins during cell division. Although various studies have revealed its molecular mechanisms, the lack of high-resolution solution structures has hindered the understanding of the detailed mechanisms. Here, we developed a monobody (Mb) that binds FtsZs from Escherichia coli and Klebsiella pneumoniae (KpFtsZ) without affecting their GTPase activities. When expressed in E. coli cells, the Mb did not inhibit Z-ring formation but did inhibit cell division. The crystal structures of the KpFtsZ-Mb complexes revealed the epitope, and the cryoEM structure at 2.67 [A] resolution showed a double helical tube consisting of two KpFtsZ protofilaments stabilized by the Mb filling interfilament gaps. Our structural analyses highlight the similarity between the microtubule and the FtsZ tube and the importance of the plasticity of FtsZ protofilaments.

molecular biology↗

bioRxiv2022

The cryo-EM structure of the CENP-A nucleosome in complex with ggKNL2

Centromere protein A (CENP-A) nucleosome is an epigenetic marker that specifies centromere position. The Mis18 complex is a licensing factor for new CENP-A deposition via the CENP-A chaperone, Holliday junction recognition protein (HJURP) on the centromere chromatin. Chicken KINETOCHORE NULL2 (KNL2) (ggKNL2), a Mis18 complex component, has a CENP-C-like motif, and our previous study suggested that ggKNL2 directly binds to the CENP-A nucleosome to recruit HJURP/CENP-A to the centromere. However, the molecular basis for CENP-A nucleosome recognition by ggKNK2 remains unclear. Here, we present the cryo-EM structure of the chicken CENP-A nucleosome in complex with a ggKNL2 fragment containing a CENP-C-like motif. Chicken KNL2 distinguishes between CENP-A and histone H3 in the nucleosome using the CENP-C-like motif and its downstream region. Both the C-terminal tail and RG-loop of CENP-A are simultaneously recognized as CENP-A characteristics. The CENP-A nucleosome-ggKNL2 interaction is thus essential for CENP-A deposition. Furthermore, our structural, biochemical, and cell biology data indicate that ggKNL2 alters its binding partner at the centromere during chicken cell cycle progression.

biochemistry↗

bioRxiv2022

Conserved GYXLI motif of FlhA is involved in dynamic domain motions of FlhA required for flagellar protein export

Flagellar structural subunits are transported via the flagellar type III secretion system (fT3SS) and assemble at the distal end of the growing flagellar structure. The C-terminal cytoplasmic domain of FlhA (FlhAC) serves as a docking platform for export substrates and flagellar chaperones and plays an important role in hierarchical protein targeting and export. FlhAC consists of domains D1, D2, D3, and D4 and adopts open and closed conformations. Gly-368 of Salmonella FlhA is located within the highly conserved GYXLI motif and is critical for the dynamic domain motions of FlhAC. However, it remains unclear how it works. Here, we report that periodic conformational changes of the GYXLI motif induce a remodeling of hydrophobic side-chain interaction networks in FlhAC and promotes the cyclic open-close domain motions of FlhAC. The temperature- sensitive flhA(G368C) mutation stabilized a completely closed conformation at 42{degrees}C through strong hydrophobic interactions between Gln-498 of domain D1 and Pro-667 of domain D4 and between Phe-459 of domain D2 and Pro-646 of domain D4, thereby inhibiting flagellar protein export by the fT3SS. Its intragenic suppressor mutations reorganized the hydrophobic interaction networks in the closed FlhAC structure, restoring the protein export activity of the fT3SS to a significant degree. Furthermore, the conformational flexibility of the GYXLI motif was critical for flagellar protein export. We propose that the conserved GYXLI motif acts as a structural switch to induce the dynamic domain motions of FlhAC required for efficient and rapid protein export by the fT3SS. IMPORTANCEMany motile bacteria employ the flagellar type III secretion system (fT3SS) to construct flagella beyond the cytoplasmic membrane. The C-terminal cytoplasmic domain of FlhA (FlhAC), a transmembrane subunit of the fT3SS, provides binding-sites for export substrates and flagellar export chaperones to coordinate flagellar protein export with assembly. FlhAC undergoes cyclic open- close domain motions. The highly conserved Gly-368 residue of FlhA is postulated to be critical for dynamic domain motions of FlhAC. However, it remains unknown how it works. Here, we carried out mutational analysis of FlhAC combined with molecular dynamics simulation and provide evidence that the conformational flexibility of FlhAC by Gly-368 is important for remodeling hydrophobic side-chain interaction networks in FlhAC to facilitate its cyclic open-close domain motions, allowing the fT3SS to transport flagellar structural subunits for efficient and rapid flagellar assembly.

microbiology↗

bioRxiv2022

Structure of cyanobacterial photosystem I complexed with Cytochrome c6 and Ferredoxin at 1.97 A resolution

Photosystem I (PSI) is a light driven electron pump transferring electrons from Cytochrome c6 (Cyt c6) to Ferredoxin (Fd). An understanding of this electron transfer process is hampered by a paucity of structural detail concerning the binding of Fd and the bound Cyt c6. Here we describe the high resolution cryo-EM structure of Thermosynechococcus elongatus BP-1 PSI in complex with Fd and Cyt c6. In our structure Cyt c6 is loosely bound in a non-productive site on the periphery of the complex. Side chain interactions at the PSI:Fd interface are visualized in detail. The structure explains the properties of mutants of PsaE and PsaC and suggests a molecular switch for the dissociation of Fd upon reduction. Calorimetry-based thermodynamic analyses confirms a single binding site for Fd and demonstrates that PSI:Fd complexation is purely driven by entropy. A reaction cycle for the efficient transfer of electrons from Cyt c6 to Fd via PSI is proposed.

plant biology↗

bioRxiv2021

Epoxidized graphene grid for high-throughput high-resolution cryoEM structural analysis

Many specimens suffer from low particle density and/or preferred orientation in cryoEM specimen grid preparation, making data collection and structure determination time consuming. We developed an epoxidized graphene grid (EG-grid) that effectively immobilizes protein particles by applying an oxidation reaction using photoactivated ClO2* and further chemical modification. The particle density and orientation distribution are both dramatically improved, having enabled us to reconstruct the density map of GroEL and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), at 1.99 and 2.16 [A] resolution from only 504 and 241 micrographs, respectively. A low concentration sample solution of 0.1 mg ml-1 was sufficient to reconstruct a 3.10 [A] resolution density map of SARS-CoV-2 spike protein from 1,163 micrographs. The density maps of V1-ATPase, {beta}-galactosidase, and apoferritin were also reconstructed at 3.03, 1.81, and 1.29 [A] resolution, respectively. These results indicate that the EG-grid will be a powerful tool for high-throughput cryoEM data collection to accelerate high-resolution structural analysis of biological macromolecules.

molecular biology↗

bioRxiv2021

Nanobodies recognizing conserved hidden clefts of all SARS-CoV-2 spike variants

We are in the midst of the historic coronavirus infectious disease 2019 (COVID-19) pandemic caused by severe respiratory syndrome coronavirus 2 (SARS-CoV-2). Although countless efforts to control the pandemic have been attempted--most successfully, vaccination1-3--imbalances in accessibility to vaccines, medicines, and diagnostics among countries, regions, and populations have been problematic. Camelid variable regions of heavy chain-only antibodies (VHHs or nanobodies)4 have unique modalities: they are smaller, more stable, easier to customize, and, importantly, less expensive to produce than conventional antibodies5, 6. We present the sequences of nine alpaca nanobodies that detect the spike proteins of four SARS-CoV-2 variants of concern (VOCs)--namely, the alpha, beta, gamma, and delta variants. We show that they can quantify or detect spike variants via ELISA and lateral flow, kinetic, flow cytometric, microscopy, and Western blotting assays7. The panel of nanobodies broadly neutralized viral infection by pseudotyped SARS-CoV-2 VOCs. Structural analyses showed that a P86 clone targeted epitopes that were conserved yet unclassified on the receptor-binding domain (RBD) and located inside the N-terminal domain (NTD). Human antibodies have hardly accessed both regions; consequently, the clone buries hidden crevasses of SARS-CoV-2 spike proteins undetected by conventional antibodies and maintains activity against spike proteins carrying escape mutations.

molecular biology↗

bioRxiv2021

Chained structure of dimeric F1-like ATPase in Mycoplasma mobile gliding machinery

Mycoplasma mobile, a fish pathogen, exhibits gliding motility using ATP hydrolysis on solid surfaces, including animal cells. The gliding machinery can be divided into surface and internal structures. The internal structure of the motor is composed of 28 so-called chains that are each comprised of 17 repeating protein units called particles. These proteins include homologs of the catalytic - and {beta}-subunits of F1-ATPase. In this study, we isolated the particles and determined their structures using negative-staining electron microscopy and high-speed atomic force microscopy. The isolated particles were composed of five proteins, MMOBs 1660 (-subunit homolog), 1670 ({beta}-subunit homolog), 1630, 1620, and 4530, and showed ATP hydrolyzing activity. The 2D structure, with dimensions of 35 and 26 nm, showed a hexameric ring dimer about 12 nm in diameter, resembling F1-ATPase catalytic ({beta})3. We isolated the F1-like ATPase unit, which is composed of MMOBs 1660, 1670, and 1630. Furthermore, we isolated the chain and analyzed the 3D structure, showing that dimers of mushroom-like structures resembling F1-ATPase were connected and aligned along the dimer axis at 31 nm intervals. An atomic model of F1-ATPase catalytic ({beta})3 from Bacillus PS3 was successfully fitted to each hexameric ring of the mushroom-like structure. These results suggest that the motor for M. mobile gliding shares an evolutionary origin with F1-ATPase. Based on the obtained structure, we propose possible force transmission processes in the gliding mechanism. IMPORTANCEF1Fo- ATPase, a rotary ATPase, is widespread in the membranes of mitochondria, chloroplasts, and bacteria, and converts ATP energy with a proton motive force across the membrane by its physical rotation. Homologous protein complexes play roles in ion and protein transport. Mycoplasma mobile, a pathogenic bacterium, was recently suggested to have a special motility system evolutionarily derived from F1-ATPase. The present study isolated the protein complex from Mycoplasma cells and supported this conclusion by clarifying the detailed structures containing common and novel features as F1-ATPase relatives.

microbiology↗

bioRxiv2021

Elucidation of fibril structure responsible for swimming in Spiroplasma using electron microscopy

Spiroplasma, which are known pathogens and commensals of arthropods and plants, are helical-shaped bacteria that lack a peptidoglycan layer. Spiroplasma swim by alternating between left- and right-handed helicity. Of note, this system is not related to flagellar motility, which is widespread in bacteria. A helical ribbon running along the inner side of the helical cell should be responsible for cell helicity and comprises the bacterial actin homolog, MreB, and a protein specific to Spiroplasma, fibril. Here, we isolated the ribbon and its major component, fibril filament, for electron microscopy (EM) analysis. Single-particle analysis of the fibril filaments using the negative-staining EM revealed a three-dimensional chain structure composed of rings with a size of 11 nm wide and 6 nm long, connected by a backbone cylinder with an 8.7 nm interval with a twist along the filament axis. This structure was verified through EM tomography of quick-freeze deep-etch replica sample, with a focus on its handedness. The handedness and pitch of the helix for the isolated ribbon and fibril filament agreed with those of the cell in the resting state. Structures corresponding to the alternative state were not identified. These results suggest that the helical cell structure is supported by fibril filaments; however, the helical switch is caused by the force generated by the MreB proteins. The isolation and structural outline of the fibril filaments provide crucial information for an in-depth clarification of the unique swimming mechanism of Spiroplasma.

microbiology↗

bioRxiv2020

Structure of the molecular bushing of the bacterial flagellar motor

The bacterial flagellum is a motility organelle, consisting of the basal body acting as a rotary motor, the filament as a helical propeller and the hook connecting these two as a universal joint1,2. The basal body contains three rings: the MS ring as the transmembrane core of the rotor; the C ring essential for torque generation and switching regulation; and the LP ring as a bushing supporting the distal rod for its rapid, stable rotation without much friction. The negatively charged surface of the distal rod suggested electrostatic repulsive force in supporting high-speed rotation of the rod as a drive shaft3, but the LP ring structure was needed to see the actual mechanisms of its bushing function and assembly against the repulsive force. Here we report the LP ring structure by electron cryomicroscopy at 3.5 [A] resolution, showing 26-fold rotational symmetry and intricate intersubunit interactions of each subunit with up to six partners that explains the structural stability. The inner surface is charged both positively and negatively, and positive charges on the P ring presumably play important roles in its initial assembly around the rod in the peptidoglycan layer followed by the L ring assembly in the outer membrane.

molecular biology↗

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↗

bioRxiv2020

Cryo-EM structure of the MgtE Mg2+ channel pore domain in Mg2+-free conditions reveals cytoplasmic pore opening

MgtE is a Mg2+ channel conserved in organisms ranging from prokaryotes to eukaryotes, including humans, and plays an important role in Mg2+ homeostasis. The previously determined MgtE structures in the Mg2+-bound, closed state and structure-based functional analyses of MgtE revealed that the binding of Mg2+ ions to the MgtE cytoplasmic domain induces channel inactivation to maintain Mg2+ homeostasis. However, due to the lack of a structure of the MgtE channel, including its transmembrane domain in Mg2+-free conditions, the pore-opening mechanism of MgtE has remained unclear. Here, we determined the cryoelectron microscopy (cryo-EM) structure of the MgtE-Fab complex in the absence of Mg2+ ions. The Mg2+-free MgtE transmembrane domain structure and its comparison with the Mg2+-bound, closed-state structure, together with functional analyses, showed the Mg2+-dependent pore opening of MgtE on the cytoplasmic side and revealed the kink motions of the TM2 and TM5 helices at the glycine residues, which are important for channel activity. Overall, our work provides structure-based mechanistic insights into the channel gating of MgtE.

biophysics↗

bioRxiv2020

Membrane voltage-dependent activation of the flagellar protein export engine

Ion motive force (IMF) consists of the electric potential difference ({Delta}{Psi}) and the ion concentration difference ({Delta}pI) across the cytoplasmic membrane. The flagellar protein export machinery is an ion/protein antiporter utilizing IMF to drive ion-coupled protein export, but it remains unknown how. Here, we report a {Delta}{Psi}-dependent activation mechanism of the transmembrane export gate complex. Depletions of both H+ and Na+ gradients nearly diminished flagellar protein export in the absence of the cytoplasmic ATPase complex, but an increase in {Delta}{Psi} by an upward shift of external pH from 7.5 to 8.5 dramatically recovered it. An increase in the cytoplasmic level of export substrates and gain-of-function mutations in FlhA enhanced protein export at external pH 7.5 in the absence of Na+ in a similar manner to {Delta}{Psi} increase. We propose that the export gate complex has a voltage-gated mechanism to activate the ion/protein antiporter of the flagellar protein export engine.

microbiology↗

bioRxiv2020

Stator remodeling mechanism of Bacillus subtilis flagellar motor during biofilm development

Bacillus subtilis possesses two distinct types of stator protein complexes for the flagellar motor: H+-type MotAB and Na+-type MotPS. The MotPS complex is used when both the external Na+ concentration and viscosity are high. Because deletion of the motPS genes does not affect swimming motility of cells, a physiological role of MotPS-dependent motility remains unclear. Here, we report that the MotPS stator complex is required for efficient biofilm maturation. Depletion of the MotPS complex did not cause a significant delay in the initiation of biofilm formation but reduced the number of viable cells in the biofilm. The MotAB stator units were efficiently replaced by the MotPS complexes with an increase in the viscosity of the environments. Therefore, we propose that MotPS-dependent motility of motile cells in the biofilm structure is required to efficiently keep the bacterial society in the biofilm healthy.

microbiology↗