9K8V

Cryo-EM structure of the Type II secretion system protein from Vibrio cholerae

This is a non-PDB format compatible entry.

Summary for 9K8V

• Entry DOI: 10.2210/pdb9k8v/pdb
• EMDB information: 62181
• Descriptor: General secretion pathway protein D (1 entity in total)
• Functional Keywords: secretion, gspd, nanopore sequencing, protein transport
• Biological source: Vibrio cholerae
• Total number of polymer chains: 15
• Total formula weight: 683719.64

Authors

Liu, R.H.,Feng, Q.S.,Zhang, K.,Dai, X.,Dai, J.,Guo, X.R.,Lin, W.F.,Wang, Z.F.,Fu, Y.,Li, Y. (deposition date: 2024-10-24, release date: 2025-10-29, Last modification date: 2025-12-24)

Primary citation

Liu, R.,Feng, Q.,Zhang, K.,Dai, X.,Dai, J.,Guo, X.,Lin, W.,Wang, Z.,Wu, Q.,Fu, Y.,Li, Y.
Tailoring Vibrio-Type Secretin Channel Protein GspD Toward "One-Take" Dual-Constriction Nanopore Sensors.
Small, 21:e05878-e05878, 2025

PubMed Abstract: Dual-constriction nanopores offer a second sensing region that enhances the interactions with analytes at the single-molecule level. However, existing biological nanopore complexes, i.e., CsgG-CsgF, are prone to dissociation upon high voltages, enforcing the development of robust "one-take" platforms. Here, Type II general secretin protein D from Vibrio cholerae (VcGspD) as a promising scaffold with dual-constrictions is proposed and engineered. Biochemical analysis reveals that truncation of the N0-N2 domains yields stable multimerization, with the N3 domain being essential. Cryo-electron microscopy (Cryo-EM) resolves the truncated VcGspD (N0-N2) as a 15-mer architecture, confirming its structural integrity and determining localizations of P471 and F472. By introducing a point mutation at position 346 (S346C) and conjugating cholesterol-maleimide, stable channel insertion in lipid bilayers is achieved. Electrophysiological characterization demonstrates a predominantly low-conductance dual-constriction architecture with constriction diameters of ≈2 nm both at the cap and central constriction sites. The F472A mutation, together with the mutations on both constrictions, gives rise to convergent open-channel current and confers high-voltage stability, thus enabling efficient sensing of both single-stranded DNA and polypeptides. The findings establish VcGspD as a promising platform toward dual-constriction nanopore sensing, paving the way for advancements in the development and engineering of secretin channels.

PubMed: 41110147
DOI: 10.1002/smll.202505878

Experimental method

ELECTRON MICROSCOPY (2.39 Å)