Crystal structure of the sensory domain of Escherichia coli CadC, a member of the ToxR‐like protein family

Abstract

The membrane-integral transcriptional activator CadC comprises sensory and transcriptional regulatory functions within one polypeptide chain. Its C-terminal periplasmic domain, CadC pd , is responsible for sensing of environmental pH as well as for binding of the feedback inhibitor cadaverine. Here we describe the crystal structure of CadC pd (residues 188-512) solved at a resolution of 1.8 Å via multiple wavelength anomalous dispersion (MAD) using a ReCl 6 22 derivative. CadC pd reveals a novel fold comprising two subdomains: an N-terminal subdomain dominated by a b-sheet in contact with three a-helices and a C-terminal subdomain formed by an eleven-membered a-helical bundle, which is oriented almost perpendicular to the helices in the first subdomain. Further to the native protein, crystal structures were also solved for its variants D471N and D471E, which show functionally different behavior in pH sensing. Interestingly, in the heavy metal derivative of CadC pd used for MAD phasing a ReCl 6 22 ion was found in a cavity located between the two subdomains. Amino acid side chains that coordinate this complex ion are conserved in CadC homologues from various bacterial species, suggesting a function of the cavity in the binding of cadaverine, which was supported by docking studies. Notably, CadC pd forms a homodimer in solution, which can be explained by an extended, albeit rather polar interface between two symmetry-related monomers in the crystal structure. The occurrence of several acidic residues in this region suggests protonation-dependent changes in the mode of dimerization, which could eventually trigger transcriptional activation by CadC in the bacterial cytoplasm.

Acid Evolution of Escherichia coli K-12 Eliminates Amino Acid Decarboxylases and Reregulates Catabolism

“…The newly identified mutations include IS-mediated knockouts of cadC (20), which encodes the pH-sensing transcriptional activator of cadA (lysine decarboxylase) and cadB (lysine-cadaverine antiporter) (21). The inserted regions consist of an IS5 element, 11 copies of which occur in the reference sequence of strain W3110 (22).…”

Deactivation of the E. coli pH Stress Sensor CadC by Cadaverine

“…10 The crystal structure of the periplasmic domain of CadC, CadC pd , is solved; docking studies identify a putative cadaverine binding site within the central cavity of the periplasmic domain. 11 The side chains of Glu447, Gln421 and Tyr374 are likely candidates to coordinate one amino group of cadaverine while Asp225 and Thr229 could coordinate the second one. To verify the results of these docking studies, we constructed CadC variants with single, double and triple amino acid substitutions at these positions.…”

“…[13][14][15][16][17] Therefore, we also analyzed residues Trp450 and His344, which were found to coordinate the hexachlororhenate ion in the CadC complex that had been prepared for multiwavelength anomalous dispersion phasing and might thus contribute further side chains participating in ligand interactions. 11 Cadaverine-dependent down-regulation of transcription was determined in reporter assays. E. coli strain EP314 lacks the native cadC gene and carries a cadA′-lacZ fusion gene.…”

“…10 Acidification of the external milieu is sensed by protonation of a patch of acidic amino acids at the dimer interface within the periplasmic domain of CadC. 11,12 This neutralization probably enables conformational changes that lead to activation of cadBA transcription. While these studies shed light on the mechanisms of lysine sensing and low pH detection by CadC, the molecular details of feedback inhibition by cadaverine are still unclear.…”

“…Crystal structure analysis of the periplasmic sensor domain of CadC (CadC pd ) reveals a central cavity between the two periplasmic subdomains of each CadC monomer suitable for accommodation of the positively charged cadaverine, both with respect to sterical fit and to electrostatic complementarity. 11 In the present study, we functionally analyzed the molecular details of CadC deactivation by the feedback inhibitor cadaverine. We identified a second, pivotal cadaverine binding site per monomer that overlaps with the patch of pH-responsive amino acids, and we used mathematical simulations to deduce a mechanistic deactivation model for CadC.…”