Structure of the T9SS PorKN Ring Complex Reveals Conformational Plasticity Based on the Repurposed FGE Fold

Authors

• Xiangan Liu
• John D Perpich
• Liqiang Song
• Christian Cambillau
• Thierry Doan
• Lei Zheng
• Richard J Lamont
• Eric Cascales
• Bo Hu

Language

English

Publication Date

9-10-2025

Journal

mBio

DOI

10.1128/mbio.01799-25

PMID

40772760

PMCID

PMC12421860

PubMedCentral® Posted Date

8-7-2025

PubMedCentral® Full Text Version

Post-print

Abstract

The type IX secretion system (T9SS) is a protein secretion machinery unique to the Bacteroidetes-Chlorobi-Fibrobacteres superphylum, which plays crucial roles in bacterial pathogenesis and gliding motility. It is composed of >15 proteins, including the proton-motive force-dependent PorLM motor, the PorKN ring anchored to the outer membrane, and the Sov translocon. Here, we present the cryo-electron microscopy (EM) structure of the PorKN ring complex from Porphyromonas gingivalis at 3.2 Å resolution. Our structural analysis reveals that PorK contains a repurposed formylglycine-generating enzyme-like fold, which serves as a structural hub for complex assembly rather than enzymatic activity. The complex exhibits a 33-fold symmetry with PorK and PorN assembling two tightly packed and wedged subrings. The structure reveals previously uncharacterized N- and C-terminal helices in PorN that are crucial for PorK binding and complex stability. By combining our high-resolution structure with in situ cryo-electron tomography data, we propose a mechanism whereby PorKN undergoes conformational changes during substrate transport, transitioning between 50° and 90° states relative to the membrane plane. Finally, structural predictions coupled to site-directed disulfide cross-linking identified contacts between PorM and the PorKN ring. Collectively, these findings provide crucial insights into the molecular architecture and dynamic behavior of the T9SS machinery, advancing our understanding of bacterial protein secretion mechanisms.

IMPORTANCE

The bacterial type IX secretion system (T9SS) is essential for processes such as gliding motility and secretion of virulence factors. In Porphyromonas gingivalis, a major periodontal pathogen, the T9SS transports over 30 virulence-associated proteins, making it central to disease development. The T9SS core is composed of PorLM motors that are thought to energize the PorKN outer membrane-associated ring. However, the molecular architecture of the PorKN ring has remained unresolved. Here, we present its atomic-resolution cryo-EM structure, revealing a formylglycine-generating enzyme-like fold in PorK that mediates PorK-PorN interactions through specific insertion motifs. Our results show that the ring exhibits intrinsic structural plasticity, including dynamic flexibility and variable stoichiometry. AlphaFold models and disulfide cross-linking experiments further provide information on how PorLM motors are connected to the PorKN ring. These insights redefine our understanding of the T9SS mechanism of action and offer a structural framework for the development of targeted antimicrobial strategies.

Keywords

Cryoelectron Microscopy, Bacterial Secretion Systems, Models, Molecular, Porphyromonas gingivalis, Bacterial Proteins, Protein Conformation, Protein Folding, type 9 protein secretion, surface structures, cryo-EM

Published Open-Access

yes

Recommended Citation

Liu, Xiangan; Perpich, John D; Song, Liqiang; et al., "Structure of the T9SS PorKN Ring Complex Reveals Conformational Plasticity Based on the Repurposed FGE Fold" (2025). Faculty, Staff and Student Publications. 3725.
https://digitalcommons.library.tmc.edu/uthmed_docs/3725