Asakereh, I. | Search bioRxiv

bioRxiv2025

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

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.

biochemistry↗

bioRxiv2024

The Streptococcus phage protein paratox is an intrinsically disordered protein.

The bacteriophage protein paratox blocks quorum sensing in its streptococcal host by directly binding the signal receptor and transcription factor ComR. This reduces ability of Streptococcus to uptake environmental DNA and protects phage DNA from damage by recombination. Past work characterizing the paratox:ComR molecular interaction revealed that paratox adopts a well-ordered globular fold when bound to ComR. However, solution-state biophysical measurements suggested that paratox may be conformationally dynamic. To address this discrepancy, we investigated the stability and dynamic properties of paratox in solution using circular dichroism, nuclear magnetic resonance, and several fluorescence-based protein folding assays. Our work shows that under dilute buffer conditions paratox is intrinsically disordered. We also show that the addition of kosmotropic salts or protein stabilizing osmolytes induces paratox folding. However, only the addition of ComR was able to induce paratox to adopt its previously characterized globular fold. Furthermore, as we can induce different paratox folding-states we characterize Prx folding thermodynamics and folding kinetics using stopped flow measurements. Based upon the kinetic results, paratox is a highly dynamic protein in dilute solution, folding and refolding within the 10 ms timescale. Overall, our results demonstrate that the streptococcal phage protein paratox is an intrinsically disordered protein in a two-state equilibrium with a solute-stabilized folded form. Furthermore, the solute-stabilized paratox fold is likely the predominant form of paratox in a solute-crowded bacterial cell. Finally, our work suggests that Prx binds and inhibits ComR, and thus quorum sensing in Streptococcus, by a combination of conformational selection and induced-fit binding mechanisms.

biochemistry↗