The Streptococcus phage protein paratox is an intrinsically disordered protein

Abstract

The bacteriophage protein paratox (Prx) blocks quorum sensing in its streptococcal host by directly binding the signal receptor and transcription factor ComR. This reduces the ability of Streptococcus to uptake environmental DNA and protects phage DNA from damage by recombination. Past work characterizing the Prx:ComR molecular interaction revealed that paratox adopts a well‐ordered globular fold when bound to ComR. However, solution‐state biophysical measurements suggested that Prx may be conformationally dynamic. To address this discrepancy, we investigated the stability and dynamic properties of Prx in solution using circular dichroism, nuclear magnetic resonance, and several fluorescence‐based protein folding assays. Our work shows that under dilute buffer conditions Prx is intrinsically disordered. We also show that the addition of kosmotropic salts or protein stabilizing osmolytes induces Prx folding. However, the solute stabilized fold is different from the conformation Prx adopts when it is bound to ComR. Furthermore, we have characterized Prx folding thermodynamics and folding kinetics through steady‐state fluorescence and stopped flow kinetic measurements. Our results show that Prx 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 Prx is an intrinsically disordered protein in a two‐state equilibrium with a solute‐stabilized folded form. Furthermore, the solute‐stabilized fold is likely the predominant form of Prx 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.

The phage protein paratox is a multifunctional metabolic regulator ofStreptococcus

“…Specifically, our previous work has shown that Prx is in equilibrium with a disordered state and a globular fold. Moreover, this unbound Prx globular fold is different from the structure Prx finally adopts when bound to either EndoS1 or ComR 34 . As such, the conformation of Prx bound to JM3 could be different from the known crystal structures and therefore unpredictable by AlphaFold.…”

“…We have previously shown that Prx is a highly dynamic protein and is in equilibrium between an intrinsically disordered ensemble and a compact globular fold. Moreover, this unbound Prx fold is distinct from the fold Prx adopts when it binds ComR or EndoS1 34 . Considering the apparent binding promiscuity of Prx and its dynamic properties, it is tempting to speculate that Prx may adopt a different conformation when it complexes with proteins that lack the RxΦxR binding helix.…”

“…As such, we also tested the binding of EndoS1 to several Prx residue variants created from our previous studies. These variants included PrxE6A, PrxE9A, and PrxD32A, which we show disrupt binding to ComR (Figure 5) 22 , and PrxF31W which we have used as a spectroscopic probe to monitor Prx folding (Figure S7) 34 . Similar to ComR, PrxE6A and PrxD32A disrupt complex formation as assayed by SEC while PrxF31W still binds EndoS1 (Figure 5A and Figure S7).…”

“…To determine the molecular contacts, an experimental structure will be required. Although the AlphaFold prediction of the JM3:Prx complex is of high confidence, Prx is an intrinsically disordered protein 34 . Specifically, our previous work has shown that Prx is in equilibrium with a disordered state and a globular fold.…”