Biology subjects

Ishimoto, N.

Publications and source records attributed to Ishimoto, N..

6 recordsLinked to original sources

bioRxiv2026

Structural basis of polyspecific drug recognition by MRP2

Multidrug resistance-associated protein 2 (MRP2/ABCC2) is a hepatobiliary ATP-binding cassette transporter that mediates the efflux of endogenous conjugates and many therapeutic agents, yet the basis of its broad ligand specificity remains poorly understood. Here we combine cryo-electron microscopy, ATPase measurements and cell-based transport inhibition assays to define how MRP2 recognizes clinically relevant compounds from distinct chemical classes. We find that chemically diverse ligands occupy overlapping regions of a shared transmembrane cavity but adopt different poses, contact networks and stoichiometries. Ligand recognition is mediated predominantly by a permissive environment complemented by ligand-specific polar interactions, rather than by a single conserved pharmacophore. These findings establish a structural framework for MRP2 polyspecificity and provide insight into hepatobiliary drug disposition and transporter-mediated drug-drug interactions.

biochemistry↗

bioRxiv2025

R27 pilus biogenesis is promiscuous and electrostatic perturbations impair functionality

During conjugation, plasmid DNA is transferred from donor to recipient bacteria via the plasmid-encoded mating pilus, formed as thin helical assemblies of polymerised pilin subunits. In the IncHI1 R27 plasmid-encoded pilus, the TrhA pilin undergoes cyclisation (via a peptide bond between Gly1 and Asp69), essential for conjugation. Gly1 and Asp69 are exposed on the pilus surface and conserved in all TrhA pilins in the Plascad database. Substituting Asp69 with Asn, Ala, Gly, or Arg did not prevent cyclisation or pilus formation, which remained structurally indistinguishable from the wild type. Conjugation efficiency of the Asp69 substitutions across multiple recipient species correlated with side chain size, in the order Asp69Asn > Asp69Ala > Asp69Gly. However, Asp69Arg, as well as Asp69Lys and Gly1Lys substitutions abolished conjugation, likely due to the positively charged pilus surface (opposite to the wild-type negative charge) forming unfavourable electrostatic interactions with the recipient outer membranes inner leaflet, composed solely of zwitterionic phosphatidylethanolamine (PE). Consistently, conjugation was rescued in recipients lacking PE. These findings indicate strong selective pressure to maintain Gly1 and Asp69, as efficient DNA transfer depends on precise electrostatic and steric constraints of the pilus surface.

microbiology↗

bioRxiv2025

Phospholipid-independent biogenesis of a functional RP4 conjugation pilus

Conjugation, the process of DNA transfer between bacteria, is initiated universally by the formation of a mating pair formation (MPF) via a conjugative pilus. Conjugation of the IncP RP4 plasmid is mediated by short, non-retractable, rigid mating pili. Here, we report the cryo-EM structure of the RP4 pilus at 2.75 [A] resolution. Uniquely, and consistently with quantitative mass spectral analysis, this revealed that the cyclic TrbC pilin subunit is not lipidated. Consistently, an E. coli pgsA mutant lacking phosphatidylglycerol (PG) can serve as a donor of RP4 but not of R27, encoding the H-pilus consisting of PG-associated cyclic pilin subunits (TrhA). The RP4 is the first example of a lipid-independent functional mating pilus. This discovery not only challenges the long-held assumption that an amphipathic lipid moiety is essential for the construction of conjugative pili and for MPF, but also expanding our understanding of the diverse mechanisms that bacteria employ to transfer genetic material.

microbiology↗

bioRxiv2024

Cryo-EM structure of the conjugation H-pilus reveals the cyclic nature of the TrhA pilin

Conjugation, the major driver of the spread of antimicrobial resistance genes, relies on a conjugation pilus for DNA transfer. Conjugative pili, such as the F-pilus, are dynamic tubular structures, composed of a polymerized pilin, that mediate the initial donor-recipient interactions, a process known as mating pair formation (MPF). IncH are low-copy-number plasmids, traditionally considered broad host range, which are found in bacteria infecting both humans and animals. The reference IncHI1 plasmid R27, isolated from Salmonella enterica serovar Typhi, encodes the conjugative H-pilus subunit TrhA containing 74 residues after cleavage of the signal sequence. Here, we show that the H-pilus forms long filamentous structures that mediate MPF, and describe its cryo electron-microscopic (cryo-EM) structure at 2.2 [A] resolution. Like the F pilus, the H-pilin subunits form helical assemblies with phospholipid molecules at a stochiometric ratio of 1:1. While there were previous reports that the T-pilus from Agrobacterium tumefaciens was composed of cyclic subunits, three recent cryo-EM structures of the T-pilus found no such cyclization. Here, we report that the H-pilin is cyclic, with a covalent bond connecting the peptide backbone between the N- and C-termini. Both the cryo-EM map and mass spectrometry revealed cleavage of the last five residues of the pilin, followed by cyclization via condensation of the amine and carboxylate residues. The cyclic nature of the pilin could stabilize the pilus and may explain the high incidence of IncH plasmid dissemination. SignificanceA major medical challenge is the spread of bacteria which are resistant to antibiotics. The resistance genes are spread via mobilized DNA, mainly via a process named conjugation. During conjugation, a resistant bacterium (donor), transfers the resistance DNA to another bacterium (recipient) in a contact-dependent manner. The initial donor-recipient interaction is mediated by a hollow filament expressed by the donor, named the conjugation pilus, that binds the recipient. This pilus is built via polymerization of a small protein subunit, pilin. Here, we report the atomic structure of the H-pilus, whose pilin subunit has an unusual cyclic structure where the N- and C-termini of the protein are covalently linked by a peptide bond.

microbiology↗

bioRxiv2024

Cryo-EM structure and evolutionary history of the conjugation surface exclusion protein TraT

Conjugation plays a major role in dissemination of antimicrobial resistance genes. Following transfer of IncF-like plasmids, recipients become refractory to a second wave of conjugation with the same plasmid via entry (TraS) and surface (TraT) exclusion mechanisms. Here, we show that TraT from the pKpQIL and F plasmids (TraTpKpQIL and TraTF) exhibits plasmid surface exclusion specificity. The cryo-EM structures of TraTpKpQIL and TraTF revealed that they oligomerise into decameric champagne bottle cork-like structures, which are anchored to the outer membrane via a diacylglycerol modified -helical barrel domain. Unexpectedly, we identified chromosomal TraT homologues from multiple Gram-negative phyla which formed numerous deep-branching lineages in a phylogenetic tree of TraT sequences. Plasmid-associated TraT sequences largely cluster into two separate lineages that have more recently evolved, incorporating TraT from Enterobacterales IncF and Legionellaceae F-like plasmids. These findings suggest that different plasmid backbones have acquired and co-opted TraT on independent occasions.

microbiology↗

bioRxiv2022

Cryo-EM structure of human somatostatin receptor 2 complex with its agonist somatostatin delineates the ligand binding specificity

Somatostatin is a peptide hormone regulating endocrine systems through binding to G-protein-coupled somatostatin receptors. somatostatin receptor 2 (SSTR2) is one of the human somatostatin receptors and highly implicated in cancers and neurological disorders. Here, we report the high resolution cryo-EM structure of full-length human SSTR2 bound to the agonist somatostatin (SST-14) complex with inhibitory G (Gi) proteins. Our structure shows that seven transmembrane helices form a deep pocket for ligand binding and that the highly conserved Trp-Lys motif of SST-14 positions at the bottom of the pocket. Furthermore, our sequence analysis combined with AlphaFold modeled structures of other SSTR isoforms provide how SSTR family proteins specifically interact with their cognate ligands. This work provides the first glimpse into the molecular recognition of somatostatin receptor and crucial resource to develop therapeutics targeting somatostatin receptors.

biophysics↗