Hideki Shigematsu

RIKEN | RIKEN AICS · SPring-8 Center (RSC)

Single particle cryo electron microscopy (cryo‐EM) is now the major method for the determination of integral membrane protein structure. For the success of a given project the type of membrane mimetic used for extraction from the native cell membrane, purification to homogeneity and finally cryo‐grid vitrification is crucial. Although small molecul...

The leading cause of bacterial meningitis, Neisseria meningitidis, deploys a quinol-dependent nitric oxide reductase ( Nm qNOR), belonging to the heme-copper oxidase superfamily. By detoxifying NO, an antimicrobial gas produced by host’s immune system, qNOR enables pathogen survival within hosts. Here, we determined cryoEM structures of the less ac...

The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continued, enabling the virus to escape from host immunity by changing its spike antigen, while biased toward the receptor-binding domain and N-terminal domain. Here, we isolated a novel pan-SARS-CoV-2 neutralizing antibody (which we named MO11) for even the recent do...

In response to environmental changes, cells flexibly and rapidly alter gene expression through translational controls. In plants, the translation of NIP5;1, a boric acid diffusion facilitator, is downregulated in response to an excess amount of boric acid in the environment through upstream open reading frames (uORFs) that consist of only AUG and s...

Existing drugs often suffer in their effectiveness due to detrimental side effects, low binding affinity or pharmacokinetic problems. This may be overcome by the development of distinct compounds. Here, we exploit the rich structural basis of drug-bound gastric proton pump to develop compounds with strong inhibitory potency, employing a combinatori...

We identified neutralizing monoclonal antibodies against severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) variants (including Omicron variants BA.5 and BA.2.75) from individuals who received two doses of mRNA vaccination after they had been infected with the D614G virus. We named them MO1, MO2, and MO3. Among them, MO1 showed particular...

Toll-like receptor 3 (TLR3) is a member of the TLR family, which plays an important role in the innate immune system and is responsible for recognizing viral double-stranded RNA (dsRNA). Previous biochemical and structural studies have revealed that a minimum length of approximately 40–50 base pairs of dsRNA is necessary for TLR3 binding and dimeri...

Antimicrobial resistance (AMR) is a global health problem. Despite the enormous efforts made in the last decade, threats from some species, including drug-resistant Neisseria gonorrhoeae, continue to rise and would become untreatable. The development of antibiotics with a different mechanism of action is seriously required. Here, we identified an a...

Microtubules play important roles in biological functions by forming superstructures, such as doublets and branched structures, in vivo. Despite the importance, it is challenging to construct these superstructures in vitro. Here, we designed a tetrameric fluorescent protein Azami-Green (AG) fused with His-tag and Tau-derived peptide (TP), TP-AG, to...

Kinesin superfamily proteins are microtubule-based molecular motors driven by the energy of ATP hydrolysis. Among them, the kinesin-4 family is a unique motor that inhibits microtubule dynamics. Although mutations of kinesin-4 cause several diseases, its molecular mechanism is unclear because of the difficulty of visualizing the high-resolution str...

We identified novel neutralizing monoclonal antibodies against SARS-CoV-2 variants (including Omicron) from individuals received two doses of mRNA vaccination after they had been infected with wildtype. We named them MO1, MO2 and MO3. MO1 shows high neutralizing activity against authentic variants: D614G, Delta, BA.1, BA.1.1, BA.2, and BA.2.75 and...

Chronic infection with hepatitis B virus (HBV), affecting more than 290 million people worldwide, is a major cause of cirrhosis and hepatocellular carcinoma and results in an estimated 820,000 human deaths annually1,2. Establishment of HBV infection requires a molecular interaction between the virus envelope glycoprotein L (LHBs) and the host entry...

Microtubules are dynamic polymers consisting of αβ-tubulin heterodimers. The initial polymerization process, called microtubule nucleation, occurs spontaneously via αβ-tubulin. Since a large energy barrier prevents microtubule nucleation in cells, the γ-tubulin ring complex is recruited to the centrosome to overcome the nucleation barrier. However,...

As specific inhibitors of the gastric proton pump, responsible for gastric acidification, K+-competitive acid blockers (P-CABs) have recently been utilized in the clinical treatment of gastric acid-related diseases in Asia. However, as these compounds have been developed based on phenotypic screening, their detailed binding poses are unknown. We sh...

Kinesin superfamily proteins are microtubule-based molecular motors driven by the energy of ATP hydrolysis. Among them, the kinesin-4 family is a unique motor that inhibits microtubule dynamics. Although mutations of kinesin-4 cause several diseases, its molecular mechanism is unclear because of the difficulty of visualizing the high-resolution str...

Microtubules (MTs) play important roles in biological functions by forming superstructures, such as doublets, triplets, and branched structures, in vivo. Formation of these superstructures by exogenous molecules in vitro will be useful not only for understanding the functions of MTs but also as components of MT-based nanomaterials. Here, we develop...

Effective from 2021B, CryoTEM will be offered in addition to SPring-8 public beamlines for protein crystallography and BioSAXS beamlines. Two CryoTEMs, EM01CT（JEOL CRYO ARM 300）, and EM02CT（JEOL CRYO ARM 200）are provided as auxiliary instruments for evaluating the properties of samples for crystallization, such as monodispersity and structural hete...

Cryo-electron microscopy (cryo-EM) is widely used for structural biology studies and has been developed extensively in recent years. However, its sample vitrification process is a major limitation because it causes severe particle aggregation and/or denaturation. This effect is thought to occur because particles tend to stick to the “deadly” air-wa...

Microtubules are dynamic polymers consisting of αβ-tubulin heterodimers. The initial polymerization process, called microtubule nucleation, occurs spontaneously via αβ-tubulin. Since a large energy barrier prevents microtubule nucleation in cells, the γ-tubulin ring complex is recruited to the centrosome to overcome the nucleation barrier. However,...

Nucleation of microtubules (MTs) is essential for cellular activities, but its mechanism is unknown because of the difficulty involved in capturing rare stochastic events in the early stage of polymerization. Here, combining rapid flush negative stain electron microscopy (EM) and kinetic analysis, we demonstrate that the formation of straight oligo...

Nucleic acid–sensing Toll-like receptors (TLRs) play a pivotal role in innate immunity by recognizing foreign DNA and RNA. Compartmentalization of these TLRs in the endosome limits their activation by self-derived nucleic acids and reduces the possibility of autoimmune reactions. Although chaperone Unc-93 homolog B1, TLR signaling regulator (UNC93B...

Prostaglandin E receptor EP4, a class A G protein-coupled receptor (GPCR), is a common drug target in various disorders, such as acute decompensated heart failure and ulcerative colitis. Here, we report the cryoelectron microscopy (cryo-EM) structure of the EP4-heterotrimeric G protein (Gs) complex with the endogenous ligand at a global resolution...

Toll-like receptor 7 (TLR7) recognizes both microbial and endogenous RNAs and nucleosides. Aberrant activation of TLR7 has been implicated in several autoimmune diseases including systemic lupus erythematosus (SLE). Here, by modifying potent TLR7 agonists, we develop a series of TLR7-specific antagonists as promising therapeutic agents for SLE. The...

Microtubule (MT) nucleation is essential for cellular activities, but its mechanism is not known because of the difficulty involved in capturing rare stochastic events in the early stage of polymerization. In cells, MTs are nucleated at tubulin concentrations significantly lower than those required for spontaneous nucleation in vitro. The high effi...

Background: Recent innovations in the field of electron cryo-microscopy (cryo-EM) have achieved structure determination of target macromolecules at "near-atomic resolution". In addition, cryo-EM has the potential to deal with proteins in multiple conformations in close to physiological conditions. Scope of review: This is an overview of the late...

Translation initiation of hepatitis C virus (HCV) genomic RNA is induced by an internal ribosome entry site (IRES). Our cryoelectron microscopy (cryo-EM) analysis revealed that the HCV IRES binds to the solvent side of the 40S platform of the cap-dependently translating 80S ribosome. Furthermore, we obtained the cryo-EM structures of the HCV IRES c...

The influenza A virus genome consists of eight single-stranded negative-sense RNA segments. The noncoding regions located at the 3′- and 5′- ends of each segment are necessary for genome packaging, and the terminal coding regions are required to precisely bundle the eight segments. However, the nucleotide residues important for genome bundling are...

Kinesin-1, the founding member of the kinesin superfamily of proteins, is known to use only a subset of microtubules for transport in living cells. This biased use of microtubules is proposed as the guidance cue for polarized transport in neurons, but the underlying mechanisms are still poorly understood. Here, we report that kinesin-1 binding chan...

Ribosomal RNA (rRNA) modifications are essential for ribosome function in all cellular organisms. Box C/D small (nucleolar) ribonucleoproteins [s(no)RNPs] catalyze 2′‐ O ‐methylation, one type of rRNA modification found in Eukarya and Archaea. Negatively stained electron microscopy (EM) models of archaeal box C/D sRNPs from our laboratory have demo...

The Tau family microtubule-associated proteins (MAPs) promote microtubule stabilization and regulate microtubule-based motility. They share the C-terminal microtubule-binding domain, which includes three to five tubulin-binding repeats. Different numbers of repeats formed by alternative splicing have distinct effects on the activities of these prot...

Epilepsy is a common brain disorder throughout history. Epilepsy-related ligand-receptor complex, LGI1-ADAM22, regulates synaptic transmission and has emerged as a determinant of brain excitability, as their mutations and acquired LGI1 autoantibodies cause epileptic disorders in human. Here, we report the crystal structure of human LGI1-ADAM22 comp...

TorsinA is an essential AAA+ ATPase requiring LAP1 or LULL1 as cofactors. The dynamics of the Torsin/cofactor system remain poorly understood, with previous models invoking Torsin/cofactor assemblies with fixed stoichiometries. Here we demonstrate that TorsinA assembles into homotypic oligomers in the presence of ATP. Torsin variants mutated at the...

Microtubule associated protein Collapsin response mediator protein 2 (CRMP2) regulates neuronal polarity in developing neurons through interactions with tubulins or microtubules. However, how CRMP2 promotes axonal formation by affecting microtubule behavior remains unknown. This study aimed to obtain the structural basis for CRMP2–tubulin/microtubu...

Norovirus is a major causative pathogen of nonbacterial acute gastroenteritis. Despite the sequence similarity among various strains, noroviruses of different genotypes show different antigenicities and different binding profiles to histo-blood group antigens (HBGAs). To reveal the relationships between the structure of the capsid and the diversity...

TorsinA is an essential AAA+ ATPase requiring LAP1 or LULL1 as cofactors. The dynamics of the Torsin/cofactor system remain poorly understood, with previous models invoking Torsin/cofactor assemblies with fixed stoichiometries. Here, we demonstrate that TorsinA assembles into homotypic oligomers in the presence of ATP. Torsin variants mutated at th...

Transcription machinery remains steadfast Eukaryotic transcription of mRNA is a multistep process mediated by RNA polymerase II (Pol II). Pol II combines with several other factors to form an elongation complex that promotes transcription elongation. Ehara et al. determined the high-resolution structure of the elongation complex by means of x-ray c...

A model Post-Termination Complex (PoTC) used for the discovery of Ribosome Recycling Factor (RRF) was purified and characterized by cryo-electron microscopic analysis and biochemical methods. We established that the model PoTC has mostly one tRNA, at the P/E or P/P position, together with one mRNA. The structural studies were supported by the bioch...

Scheme of 3D classification of the PoTC and the reaction products of ribosome recycling reaction. (A) This scheme shows how 3D classifications were performed to obtain cryo-EM structures of PoTCs in rotated and unrotated state. The first round of 3D classification was performed in 118,567 particles selected after 2D classification with the sampling...

Lack of IF3 effects on splitting and mRNA release of PoTC by RRF and EF-G. Sedimentation is shown from left to right. (TIF)

Supporting information. Supplementary information contains control experiments and how to convert CPM into pmoles. (DOCX)

Schematic representation of the preparation of PoTC from naturally occurring polysomes. (A) Various forms of ribosomes exist in naturally occurring polysomes. (B) Treating the complexes in (A) with EF-G and GTP results in two possible forms of ribosomes. (C) PoTC was obtained after treatment of (B) with puromycin in vitro. One tRNA is released from...

Basis of estimation for the molar amount of released tRNA from PoTC. (A) tRNA-Met bound to the purified PoTC was released and aminoacylated with [35S]-Methionine as described in the material and method section, except [35S]-Methionine (1175 Ci/mmol) was used in place of the [14C]-amino acid mixture. (B) Time course of aminoacylation of tRNA release...

Cryo-EM structures and their resolution curves. (A) Cryo-EM structural data shown in this study. From left to right: Polysome, 15.6 Å at FSC 0.5 cutoff; PoTC in rotated state, 8.5 Å at FSC 0.143 cutoff (Gold standard FSC); PoTC in unrotated state, 9.4 Å at FSC 0.143 cutoff (Gold standard FSC); Reassociated 70S, 9.2 Å at FSC 0.143 cutoff (Gold stand...

Time course of tRNA release from PoTC by RRF/EF-G. Analysis by UREA-PAGE. (A) The densities of RNA bands from lanes 1 to 8. (B) Densities were measured using ImageJ. The numbers next to each peak indicate the area of the peak. The preparation of RNA from PoTC is described in the material and method section. (C) Absence of tRNA on PoTC after dissoci...

Ice-cold temperature completely stops all RRF reactions, mRNA/tRNA release and ribosomal splitting. The release of tRNA and mRNA from PoTC and ribosome splitting were analyzed at 0 and 15 min after incubation on ice. (TIF)

The data and analysis for 353WT and L2 mutants.Sheet F3D: The data and analysis of dose-response MT depolymerization curve for different concentrations of 353WT and L2 mutants.DOI: http://dx.doi.org/10.7554/eLife.18101.010

The data and analysis for 353WT, L2 and swap mutants.Sheet F4A_F4FS1: The data and analysis of ATPase activity of 353WT and L2 mutants. Sheet F4B: The data and analysis of MT binding assay of 353WT and L2 mutants. Sheet F4C: Motility Velocity of 353WT and L55A mutant. Sheet F4E: Motility Velocity of KIF8A WT and KIF18A swap. Sheet F4G Motility Velo...

Crystal structure statistics for KIF19A motor domain 353WT. Data collection and refinement statistic table. DOI: http://dx.doi.org/10.7554/eLife.18101.025

The data and analysis for 353WT and L12 mutants.Sheet F5E: The data and analysis of dose-response MT depolymerization curve for different concentrations of 353WT and L12 mutants. Sheet F5H_F5FS1: The data and analysis of ATPase activity of 353WT and L12 mutants. Sheet F5I: The data and analysis of MT binding assay of 353WT and L12 mutants.DOI: http...

The data and analysis for 353WT and N297P mutant.Sheet F6C The data and analysis of dose-response MT depolymerization curve for different concentrations of 353WT and N297P mutant. Sheet F6F: Motility Velocity of 353WT and N297P mutant. Sheet F6H_F6FS2: The data and analysis of ATPase activity of 353WT and N297P mutant.DOI: http://dx.doi.org/10.7554...

The data and analysis for 353WT.Sheet F1B: Motility Velocity of 353WT. Sheet F1E: The data and analysis of dose-response MT depolymerization curve for different concentrations of 353WT.DOI: http://dx.doi.org/10.7554/eLife.18101.004

Kinesin-14 is a unique minus-end-directed microtubule-based motor. A swinging motion of a class-specific N-terminal neck helix has been proposed to produce minus-end directionality. However, it is unclear how swinging of the neck helix is driven by ATP hydrolysis utilizing the highly conserved catalytic core among all kinesins. Here, using a motili...

Ribosomal RNA (rRNA) modifications are essential for ribosome function in all cellular organisms. Box C/D small (nucleolar) ribonucleoproteins [s(no)RNPs] catalyze 2′-O-methylation, one rRNA modification type in Eukarya and Archaea. Negatively stained electron microscopy (EM) models of archaeal box C/D sRNPs have demonstrated the dimeric sRNP (di-s...

Bacterial group II introns are large catalytic RNAs related to nuclear spliceosomal introns and eukaryotic retrotransposons. They self-splice, yielding mature RNA, and integrate into DNA as retroelements. A fully active group II intron forms a ribonucleoprotein complex comprising the intron ribozyme and an intron-encoded protein that performs multi...

Artificial lipid-bilayer membranes are valuable tools for the study of membrane structure and dynamics. For applications such as the study of vesicular transport and drug delivery, there is a pressing need for artificial vesicles with controlled size. However, controlling vesicle size and shape with nanometre precision is challenging, and approache...

This paper describes steps in the single-particle cryo-EM 3D structure determination of membrane proteins in their membrane environment. Using images of the Kv1.2 potassium-channel complex reconstituted into lipid vesicles, we describe procedures for the merging of focal-pairs of exposures and the removal of the vesicle-membrane signal from the mic...

The resistance-nodulation-division type tripartite pump AcrAB-TolC and its homologs are responsible for multidrug resistance in Gram-negative bacteria by expelling a wide variety of toxic substrates. The three essential components, AcrA, AcrB, and TolC, must function in concert with each respective binding partner within the complex. In this study,...