Biology subjects

Makino, F.

Publications and source records attributed to Makino, F..

13 recordsLinked to original sources

bioRxiv2026

Reconstitution in a minimal synthetic bacterium reveals distinct Mollicute MreB-based motility systems driven by orthogonal force directions

MreB, a bacterial actin homolog, is widely conserved in bacteria where it functions as a scaffold for a peptidoglycan synthesis. Intriguingly, members of the cell wall-lacking Mollicutes retain multiple mreB genes despite the absence of peptidoglycan. Previous work demonstrated that two MreB isoforms from Spiroplasma can reconstitute swimming motility in the minimal synthetic bacterium syn3B, indicating that MreB was repurposed for motility. Here, we expressed seven MreBs from Haloplasma contractile, an early-diverging Mollicute, in syn3B. Approximately 50% of cells elongated, and 15% exhibited curving and coiling movements that resembled Haloplasma but differed from Spiroplasma motility. Systematic analysis of isoform combinations revealed that movement requires specific MreB pairs, and that additional isoforms enhance motility efficiency. Cryo-electron tomography showed membrane-associated ribbons composed of MreB filaments. Mathematical modeling demonstrated that Haloplasma- and Spiroplasma-type movements can be reproduced by altering only the direction of force generation. Thus, diversification of force orientation within an actin homolog enabled independent evolution of distinct motility systems in Mollicutes.

Synthetic Biology↗

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

From Monomers to Oligomers: Structural Mechanism of Receptor-Triggered MyD88 Assembly in Innate Immune Signaling

MyD88 plays a pivotal role in Toll-like receptor (TLR) and interleukin-1 family signaling through its oligomerization upon receptor activation, leading to downstream protein recruitment. The Toll/interleukin-1 receptor domain of MyD88 (TIRMyD88) is responsible for this receptor-mediated oligomerization, but the detailed mechanism involved remains elusive. We investigated the structure of TIRMyD88 oligomers and their interactions with TLRs. Cryoelectron microscopy revealed that tandemly arrayed TIRMyD88 subunits formed an antiparallel double-stranded filament that could further form rings and cylindrical filaments. Moreover, the self-assembly of TIRMyD88 in vitro was markedly accelerated by dimeric rather than monomeric receptor TIRs, possibly reflecting the signal initiation step in vivo. High-speed atomic force microscopy further captured the dynamic processes of oligomerization of TIRMyD88, in addition to its direct interaction with the receptor TIRs. Based on these results, a novel regulatory mechanism of TIRMyD88 oligomerization underlying the signal initiation step was revealed.

biophysics↗

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↗

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

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

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↗

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↗

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↗