Language：English   Publisher：Nature Communications  

Divalent cation block is observed in various tetrameric ion channels. For blocking, a divalent cation is thought to bind in the ion pathway of the channel, but such block has not yet been directly observed. So, the behaviour of these blocking divalent cations remains still uncertain. Here, we elucidated the mechanism of the divalent cation block by reproducing the blocking effect into NavAb, a well-studied tetrameric sodium channel. Our crystal structures of NavAb mutants show that the mutations increasing the hydrophilicity of the inner vestibule of the pore domain enable a divalent cation to stack on the ion pathway. Furthermore, non-equilibrium molecular dynamics simulation showed that the stacking calcium ion repel sodium ion at the bottom of the selectivity filter. These results suggest the primary process of the divalent cation block mechanism in tetrameric cation channels.

DOI： 10.1038/s41467-023-39987-0

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Language：English   Publisher：Journal of Molecular Biology  

Mirogabalin is a novel gabapentinoid drug with a hydrophobic bicyclo substituent on the γ-aminobutyric acid moiety that targets the voltage-gated calcium channel subunit α2δ1. Here, to reveal the mirogabalin recognition mechanisms of α2δ1, we present structures of recombinant human α2δ1 with and without mirogabalin analyzed by cryo-electron microscopy. These structures show the binding of mirogabalin to the previously reported gabapentinoid binding site, which is the extracellular dCache_1 domain containing a conserved amino acid binding motif. A slight conformational change occurs around the residues positioned close to the hydrophobic group of mirogabalin. Mutagenesis binding assays identified that residues in the hydrophobic interaction region, in addition to several amino acid binding motif residues around the amino and carboxyl groups of mirogabalin, are critical for mirogabalin binding. The A215L mutation introduced to decrease the hydrophobic pocket volume predictably suppressed mirogabalin binding and promoted the binding of another ligand, L-Leu, with a smaller hydrophobic substituent than mirogabalin. Alterations of residues in the hydrophobic interaction region of α2δ1 to those of the α2δ2, α2δ3, and α2δ4 isoforms, of which α2δ3 and α2δ4 are gabapentin-insensitive, suppressed the binding of mirogabalin. These results support the importance of hydrophobic interactions in α2δ1 ligand recognition.

DOI： 10.1016/j.jmb.2023.168049

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Language：English   Publisher：Nature Communications  

Ion-transport mechanisms evolve by changing ion-selectivity, such as switching from Na+ to H+ selectivity in secondary-active transporters or P-type-ATPases. Here we study primary-active transport via P-type ATPases using functional and structural analyses to demonstrate that four simultaneous residue substitutions transform the non-gastric H+/K+ pump, a strict H+-dependent electroneutral P-type ATPase, into a bona fide Na+-dependent electrogenic Na+/K+ pump. Conversion of a H+-dependent primary-active transporter into a Na+-dependent one provides a prototype for similar studies of ion-transport proteins. Moreover, we solve the structures of the wild-type non-gastric H+/K+ pump, a suitable drug target to treat cystic fibrosis, and of its Na+/K+ pump-mimicking mutant in two major conformations, providing insight on how Na+ binding drives a concerted mechanism leading to Na+/K+ pump phosphorylation.

DOI： 10.1038/s41467-022-32793-0

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Language：English   Publisher：Communications Biology  

MrgD, a member of the Mas-related G protein-coupled receptor (MRGPR) family, has high basal activity for Gi activation. It recognizes endogenous ligands, such as β-alanine, and is involved in pain and itch signaling. The lack of a high-resolution structure for MrgD hinders our understanding of whether its activation is ligand-dependent or constitutive. Here, we report two cryo-EM structures of the MrgD-Gi complex in the β-alanine-bound and apo states at 3.1 Å and 2.8 Å resolution, respectively. These structures show that β-alanine is bound to a shallow pocket at the extracellular domains. The extracellular half of the sixth transmembrane helix undergoes a significant movement and is tightly packed into the third transmembrane helix through hydrophobic residues, creating the active form. Our structures demonstrate a structural basis for the characteristic ligand recognition of MrgD. These findings provide a framework to guide drug designs targeting the MrgD receptor.

DOI： 10.1038/s42003-022-03668-3

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Language：English   Publisher：Journal of Medicinal Chemistry  

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 show crystal and cryo-EM structures of the gastric proton pump in complex with four different P-CABs, tegoprazan, soraprazan, PF-03716556 and revaprazan, at resolutions reaching 2.8 Å. The structures describe molecular details of their interactions and are supported by functional analyses of mutations and molecular dynamics simulations. We reveal that revaprazan has a novel binding mode in which its tetrahydroisoquinoline moiety binds deep in the cation transport conduit. The mechanism of action of these P-CABs can now be evaluated at the molecular level, which will facilitate the rational development and improvement of currently available P-CABs to provide better treatment of acid-related gastrointestinal diseases.

DOI： 10.1021/acs.jmedchem.2c00338

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1126/sciadv.abm2225

Language：Japanese  

DOI： 10.1016/j.jbc.2021.101498

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Language：English   Publisher：Current Research in Structural Biology  

Human stomatin (hSTOM) is a component of the membrane skeleton of erythrocytes that maintains the membrane's shape and stiffness through interconnecting spectrin and actin. hSTOM is a member of the protein family that possesses a single stomatin/prohibitin/flotillin/HflK (SPFH) domain at the center of the molecule. Although SPFH domain proteins are widely distributed from archaea to mammals, the detailed function of the domain remains unclear. In this study, we first determined the solution structure of the SPFH domain of hSTOM (hSTOM(SPFH)) via NMR. The solution structure of hSTOM(SPFH) is essentially identical to the already reported crystal structure of the STOM SPFH domain (mSTOM(SPFH)) of mice, except for the existence of a small hydrophilic pocket on the surface. We identified this pocket as a phosphate-binding site by comparing its NMR spectra with and without phosphate ions. Meanwhile, during the conventional process of protein NMR analysis, we eventually discovered that hSTOM(SPFH) formed a unique solid material after lyophilization. This lyophilized hSTOM(SPFH) sample was moderately slowly dissolved in a physiological buffer. Interestingly, it was resistant to dissolution against the phosphate buffer. We then found that the lyophilized hSTOM(SPFH) formed a fibril-like assembly under electron microscopy. Finally, we succeeded in reproducing this fibril-like assembly of hSTOM(SPFH) using a centrifugal ultrafiltration device, thus demonstrating that the increased protein concentration may promote self-assembly of hSTOM(SPFH) into fibril forms. Our observations may help understand the molecular function of the SPFH domain and its involvement in protein oligomerization as a component of the membrane skeleton. (245 words).

DOI： 10.1016/j.crstbi.2022.05.002

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Language：Japanese  

DOI： 10.1038/s41467-021-26024-1

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Authorship：Lead author   Language：Japanese  

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/s41598-020-69129-1

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DOI： 10.1016/j.sbi.2020.03.008

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.sbi.2019.01.005

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Authorship：Lead author   Language：Japanese  

Authorship：Lead author   Language：English  

DOI： 10.1093/jmicro/dfx035

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.jmb.2016.02.011.

Authorship：Lead author   Language：Japanese   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Lead author   Language：English  

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.jmb.2011.07.013

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.jmb.2010.10.032

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.bpj.2010.01.019

Language：Japanese   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.jsb.2009.02.004

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/nature07869

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DOI： 10.1080/15419060802013588

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1073/pnas.0703704104

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1074/jbc.M506533200

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1074/jbc.M207713200

Language：English   Publishing type：Research paper (scientific journal)