Structural and Functional Analysis of the Signal-Transducing Linker in the pH-Responsive One-Component System CadC of Escherichia coli

Abstract

The pH-responsive one-component signaling system CadC in Escherichia coli belongs to the family of ToxR-like proteins, whose members share a conserved modular structure, with an N-terminal cytoplasmic winged helix-turn-helix DNA-binding domain being followed by a single transmembrane helix and a C-terminal periplasmic pH-sensing domain. In E. coli CadC, a cytoplasmic linker comprising approximately 50 amino acids is essential for transmission of the signal from the sensor to the DNA-binding domain. However, the mechanism of transduction is poorly understood. Using NMR spectroscopy, we demonstrate here that the linker region is intrinsically disordered in solution. Furthermore, mutational analyses showed that it tolerates a range of amino acid substitutions (altering polarity, rigidity and α-helix-forming propensity), is robust to extension but is sensitive to truncation. Indeed, truncations either reversed the expression profile of the target operon cadBA or decoupled expression from external pH altogether. CadC dimerizes via its periplasmic domain, but light-scattering analysis provided no evidence for dimerization of the isolated DNA-binding domain, with or without the linker region. However, bacterial two-hybrid analysis revealed that CadC forms stable dimers in a stimulus- and linker-dependent manner, interacting only at pH<6.8. Strikingly, a variant with inversed cadBA expression profile, which lacks most of the linker, dimerizes preferentially at higher pH. Thus, we propose that the disordered CadC linker is required for transducing the pH-dependent response of the periplasmic sensor into a structural rearrangement that facilitates dimerization of the cytoplasmic CadC DNA-binding domain.

Transporters as information processors in bacterial signalling pathways

“…Interestingly, signal integration is achieved by the membrane-integrated ToxR-like 1CS CadC, which is able to sense low pH (Tetsch et al, 2011;Buchner et al, 2015) and high cadaverine levels (Eichinger et al, 2011;Haneburger et al, 2012) via its periplasmic domain, while its cytoplasmic domain directly binds to and activates the cadBA promoter (Kuper and Jung, 2005). However, CadC cannot directly sense the presence of lysine (Tetsch et al, 2008), and instead relies on the secondary lysine/H 1 symporter LysP, which is required for lysine uptake from the growth medium.…”

Transcriptional regulation of theNε-fructoselysine metabolism inEscherichia coliby global and substrate-specific cues

“…Reportedly, NagR derepression is not achieved by monomerization upon signal perception (Resch et al , 2010) as e.g. in the case of other one-component systems such CadC (Lindner & White, 2014, Buchner et al , 2015). Instead, substrate binding to the UTRA-domain is transduced into a structural rearrangement of the two wHTH-domains, that hinders proper DNA-binding.…”

Protein Activity Sensing in Bacteria in Regulating Metabolism and Motility

“…The presence of lysine is transduced to CadC via LysP (Tetsch et al, 2008;Rauschmeier et al, 2014). LysP inhibits the dimerization of CadC under non-inducing conditions, but when the external pH drops, CadC undergoes structural modifications that permit its binding and consequent activation of the cadBA operon (Haneburger et al, 2011(Haneburger et al, , 2012Lindner and White, 2014;Buchner et al, 2015).…”