The periplasmic domains of Vibriocholerae ToxR and ToxS are forming a strong heterodimeric complex independent on the redox state of ToxR cysteines

Abstract

The transmembrane protein ToxR plays a key role in the virulence expression system of Vibrio cholerae. The activity of ToxR is dependent on its periplasmic sensor domain (ToxRp) and on the inner membrane protein ToxS. Herein, we present the Nuclear Magnetic Resonance NMR solution structure of the sensory ToxRp containing an intramolecular disulfide bond. The presented structural and dynamic experiments with reduced and oxidized ToxRp propose an explanation for the increased proteolytic sensitivity of reduced ToxR. Additionally, for the first time, we could identify the formation of a strong heterodimer complex between the periplasmic domains of ToxR and ToxS in solution. NMR interaction studies reveal that binding of ToxS is not dependent on the redox state of ToxR cysteines, and formed complexes are structurally similar. By monitoring the proteolytic cleavage of ToxRp with NMR, we additionally provide a direct evidence of ToxS protective function. Taken together our results suggest that ToxR activity is regulated by its stability which is, on the one hand, dependent on the redox states of its cysteines, influencing the stability of its fold, and on the other hand, on its interaction with ToxS, which binds independent on the cysteines and acts as a protection against proteases.

Structural and DNA-binding properties of the cytoplasmic domain of Vibrio cholerae transcription factor ToxR

“…Following our recently published structural and functional investigation of the periplasmic domain of ToxR (36), we are aiming to shed light on the versatile functionality of its cytoplasmic DNA binding domain. To adress this question we were interested in solving the structure of the cytoplasmic domain of ToxR (cToxR) as well as studying its binding behaviour to different DNA motifs of V. cholerae operons, which are either activated, repressed, or co-activated by ToxR.…”

Co-Component Signal Transduction Systems: fast-evolving virulence regulation cassettes discovered in enteric bacteria

“…The VtrA/VtrC co-components function as an obligate heterodimer, where the incomplete barrel for VtrC is unstable. Similarly, the ToxR periplasmic domain is regulated by its stability, which is influenced both by interaction with ToxS and by formation of a disulfide (18, 22, 24). Thus, ToxS is thought to activate ToxR by protecting it from degradation(53).…”

“…The membrane tethered ToxR transcription factor that activates the Vibrio cholerae virulence regulatory cascade has long been described as a one-component system (15)(16)(17). The domain architecture of ToxR includes an N-terminal DNA-binding HTH, a TMH and a periplasmic domain with a recently determined structure (18,19). The ToxR HTH belongs to the CadC superfamily of transcription factor DNA-binding domains, while the periplasmic sensor combines an N-terminal -sandwich with a C-terminal -helical ARM repeat (9,11).…”

“…However, the ToxR periplasmic domain structure lacks the CadC sensor subdomain organization and instead requires a second unrelated periplasmic protein ToxS for activity (20). The ToxS co-regulator is encoded by a neighboring gene from the ToxR operon (20,21), and the two gene products form a complex that stabilizes ToxR and renders it resistant to proteolytic cleavage (18,22). Thus, instead of ToxR serving as a traditionally recognized one-component system, we suggest the periplasmic ToxR/ToxS heterodimer serves as a sensor in a "co-component" signal transduction system.…”

Investigations on Vibrio cholerae sensory regulator ToxR

“…The NMR structure of the periplasmic domain of ToxR (ToxRp) reveals the formation of a four stranded β sheet stacked against a long α-helix [1]. C236, in the middle of the helix forms a disulphide bond with C293 at the C-terminal end.…”