Topology, Dimerization, and Stability of the Single-Span Membrane Protein CadC

Abstract

Under acid stress, Escherichia coli induce expression of CadA (lysine decarboxylase) and CadB (lysine/cadaverine antiporter) in a lysine-rich environment. The ToxR-like transcriptional activator CadC controls expression of the cadBA operon. Using a novel Signal Peptidase I (SPase I) cleavage assay, we show that CadC is a Type II single-span membrane protein (MP) with a cytoplasmic DNA binding domain and a periplasmic sensor domain. We further show that, as long assumed, dimerization of the sensor domain is required for activating the cadBA operon. We prove this using a chimera in which the periplasmic domain of RodZ—a Type II MP involved in the maintenance of the rod shape of E. coli—replaces the CadC sensor domain. Because the RodZ periplasmic domain cannot dimerize, the chimera cannot activate the operon. However, replacement of the TM domain of the chimera with the glycophorin-A (GpA) TM domain causes intramembrane dimerization and consequently operon activation. Using a low-expression protocol that eliminates extraneous TM-helix dimerization signals arising from protein over-expression, we enhanced dramatically the dynamic range of the β-galactosidase assay for cadBA activation. Consequently, the strength of the intramembrane dimerization of the GpA domain could be compared quantitatively with the strength of the much stronger periplasmic dimerization of CadC. For the signal-peptidase assay, we inserted a SPase I cleavage site (AAA or AQA) at the periplasmic end of the TM helix. Cleavage occured with high efficiency for all TM and periplasmic domains tested, thus eliminating the need for the cumbersome spheroplast-proteinase K method for topology determinations.

SecA mediates cotranslational targeting and translocation of an inner membrane protein

“…An inner membrane protein, AcrB, showed more severe defects in membrane insertion under SecA-depleted, than SRP-depleted, conditions ( Qi and Bernstein, 1999 ). Recently, Rawat et al (2015) explored the insertion requirements of two single-span membrane proteins, RodZ and CadC, and suggested the possibility that RodZ is inserted cotranslationally by SecA ( Lindner and White, 2014 ). A common feature of both proteins is a TMD >100 residues downstream of the N terminus and a strict dependence on SecA, but not SRP or FtsY, for insertion.…”

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).…”