# Biophysical characterization of Eag chaperones suggests the mechanism of effector transmembrane domain release

## TL;DR

This study explores how Eag chaperones release toxic effector proteins in bacteria, revealing the molecular mechanism behind their dissociation.

## Contribution

The study identifies the Eag conformational change and residue contacts that enable TMD release from chaperones.

## Key findings

- Eag chaperones bind effector TMDs with high stability.
- A subtle conformational change in Eag initiates rapid TMD release.
- An X-ray structure captures an intermediate state of TMD release.

## Abstract

The type VI secretion system (T6SS) is a dynamic protein nanomachine found in Gram-negative bacteria that secretes toxic effectors into prey-cells. For secretion, effectors require chaperones or adaptors for proper loading onto the T6SS. Effector associated genes (Eags) are a family of T6SS chaperones that stabilize N-terminal transmembrane domains (TMDs) found in thousands of effectors. Eags are essential for secretion and inhibit effector TMDs from prematurely adopting a membrane-penetrative conformation. However, the mechanism of TMD release from its cognate Eag chaperone is unknown. Here, we take a biochemical and biophysical approach to probe the mechanism of TMD binding and dissociation from Eag chaperones. Using steady-state fluorescence, stopped-flow measurements, and bacterial competition assays, we compare the thermodynamics, kinetics, and in vivo chaperone function of wild-type and point variant Eag-TMD complexes. Additionally, we solve an X-ray crystal structure of an Eag-TMD point variant complex that captures an intermediate state of TMD release. Our data reveals the molecular features and specific residue contacts necessary for TMD binding and demonstrates the Eag conformational change required to initiate rapid release of the TMD. Overall, our work details the stability of Eag-TMD complexes and the energetic pathway for the dissociation of effector TMDs from their Eag chaperones.

The type VI secretion system (T6SS) and Eag chaperones deliver toxic membrane protein effectors into rival cells. However, it is unknown how Eags and their effectors dissociate. Here, the authors show Eag chaperones bind effectors tightly and may release them via a subtle conformational change.