# Structural analysis of inner membrane platform in the type 2 secretion system

> **NIH NIH R21** · YALE UNIVERSITY · 2022 · $224,079

## Abstract

ABSTRACT
The type II secretion system (T2SS) exists widely in gram-negative bacteria and exports a
variety of folded protein substrates, such as cholera toxin (CT) from Vibrio cholerae and heat-
labile enterotoxin (LT) from enterotoxigenic Escherichia coli (ETEC). The T2SS machinery
spans the entire cell envelope and consists of three subassemblies: the mysterious inner
membrane platform (IMP), the dynamic pseudopilus, and the channel-forming outer membrane
complex. In current mechanism models, protein substrate binding to the T2SS in the periplasm
triggers the cytoplasmic ATPase (GspE) to hydrolyze ATP, which energizes the incorporation of
the major pseudopilin subunit (GspG) into a pilus-like structure. This growing pseudopilus acts
as a piston or Archimedes screw, pushing the folded protein substrates through the outer
membrane channel to the cell surface or extracellular milieu. Despite decades of research, the
structure of IMP is not yet available; mainly for this reason, it is still unknown how the inner
membrane assembly platform converts energy from ATP hydrolysis in the cytoplasm to extend
the pseudopilus and push the substrates across the outer membrane. It is also unknown how
the inner membrane proteins regulate the GspE ATPase activity. Our proposal exploits
fluorescence size exclusion chromatography and coevolution analysis to identify interactions
among components of IMP. We will use an assistant-multimer strategy and unnatural amino
acid crosslinking to purify stable inner membrane protein complexes, followed by single particle
cryo-electron microscopy to determine complex structures. We will analysis the ATPase activity
of GspE with different inner membrane protein complexes. This proposal will substantially
increase the fundamental scientific knowledge about the architecture and mechanisms of the
T2SS, and thereby provide a new basis for developing future therapeutic interventions against a
broad range of bacterial infections.

## Key facts

- **NIH application ID:** 10308683
- **Project number:** 5R21AI156595-02
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** Wei Mi
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $224,079
- **Award type:** 5
- **Project period:** 2020-12-01 → 2022-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10308683

## Citation

> US National Institutes of Health, RePORTER application 10308683, Structural analysis of inner membrane platform in the type 2 secretion system (5R21AI156595-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10308683. Licensed CC0.

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