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)(2-) derivative. CadC(pd) reveals a novel fold comprising two subdomains: an N-terminal subdomain dominated by a β-sheet in contact with three α-helices and a C-terminal subdomain formed by an eleven-membered α-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)(2-) 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 homo-dimer 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.

Figure 1

Overview of the CadC_pd crystal structure. (A) Cartoon representation of the apo-protein solved at 1.8 Å. The protein consists of two subdomains. Secondary structure elements are depicted in yellow (β-strands), blue (α-helices of the N-terminal subdomain), and salmon (α-helices of the C-terminal subdomain). The two short 3₁₀-helices 334–336 and 411–413 are also shown in salmon. The disordered loop 207–214 has been modelled and is depicted in light gray. The disulfide bond connecting residues Cys208 and Cys272 is shown as red sticks and labeled. (B) Superposition of the CadC_pd apo-protein (yellow-orange) with the ReCl₆²⁻ complex (raspberry) and the variants D471E (green) and D471N (blue). Disordered loops were modelled with plausible geometry and are depicted in light gray. (C) Electrostatic surface representation of the CadC_pd apo-structure in an orientation identical to (B). Negatively charged areas are colored red (−10 k_BT/e), positively charged areas are colored blue (10 k_BT/e). (D) The molecule in the same representation rotated vertically by around 180° with respect to (C), illustrating a narrow entrance to the central cavity indicated by a dashed ellipse. Residues mentioned in the text are labeled. An interactive view is available in the electronic version of the article.

Figure 2

Analysis of CadC_pd dimerization. (A) Stereo view of one CadC_pd dimer related by a crystallographic symmetry axis. One monomer is colored as in Figure 1(A), the other one is uniformly colored cyan. The N- and C-termini are both directed towards the lipid bilayer indicated at the bottom. (B) Hydrophobic surface representation of the two CadC_pd monomers rotated by 90° about a horizontal axis (with the N-termini directed toward the front) and then rotated by ±90° apart to reveal the dimer interface. The interface forming residues are colored according to increasing hydrophobicity from green (hydrophilic) over white (neutral, including the polypeptide backbone) to brown (hydrophobic). Residues Asp471 and Lys255, which form a salt bridge, are labeled. (C) The same view as in (B) with electrostatic surface representation as in Figure 1(C and D). (D) Analytical size exclusion chromatography of the recombinant CadC_pd at pH 7.5. The protein eluted with a retention volume of 14.4 mL, which corresponds to a molecular size of 64.8 kDa (left) as determined from a plot of log MW of calibration proteins against their elution volumes (right): cytochrome c (12.4 kDa), carbonic anhydrase (29 kDa), ovalbumin (43 kDa), bovine serum albumin (66.3 kDa), alcohol dehydrogenase (150 kDa), β-amylase (200 kDa), apoferritin (443 kDa). The apparent size of CadC_pd is considerably larger than the calculated mass of the proteolytic product after thrombin cleavage (41.8 kDa), indicating stable dimer formation in solution.

Figure 3

Analysis of the cavity and the interface between the two subdomains of CadC_pd. (A) Electrostatic surface properties (cf. Fig. 1) of the C-terminal α-helical subdomain (residues 333–509; left) and the N-terminal predominantly β-sheet subdomain (residues 190–332; right). Both moieties were rotated by ±90° about a vertical axis to visualize the internal cavity surfaces. Residues that contact the bound ReCl₆²⁻ ion are labeled. (B) Hydrophobic surface representation colored as in Figure 2B. (C) Complexation of ReCl₆²⁻ within the central cavity. Interacting side chains of CadC within a distance of 4 Å to the rhenate central ion are indicated. (D) Results from a docking simulation with cadaverine. The three cadaverine conformations with highest scores as well as the putative interacting residues in the cavity are depicted as sticks. Cadaverine carbon atoms are colored differently according to scoring rank order: 1, yellow; 2, salmon; 3, green. Oxygen atoms are shown in red, nitrogen atoms are shown in blue, hydrogen atoms are shown in white, and hydrogen bonds are indicated as dark green dashed lines.

Figure 4

Comparison of the crystal structure and secondary structure topology of CadC_pd with its most closely related α-helical bundle proteins. Subdomains to be considered for the comparison are colored whereas other structural elements are shown in gray. (A) CadC_pd with its C-terminal subdomain (residues 333–512) comprising 11 α-helices (salmon). (B) The peroxisomal targeting signal 1 binding domain of Trypanosoma brucei peroxin 5 (PDB entry 3CVN ) forms a twisted 16 α-helix bundle; helices 3–12 are colored green. (C) The transcription factor MalT domain III protein of Escherichia coli (1HZ4 ) forms another twisted 18 α-helix bundle; helices 6–15 are colored blue. (D) The tetratricopeptide repeat protein NlpI of Escherichia coli (1XNF ) with α-helices 4–13 colored yellow.

Figure 5

Sequence alignment of CadC_pd orthologs (residues 188–512) from a representative set of γ-proteobacteria. Amino acids lining the characteristic cavity of CadC_pd are labeled as small vertical arrows above the sequences while residues involved in the dimer interface are indicated by arrows underneath, many of which involve fully conserved positions. Secondary structure elements are shown as horizontal arrows (β-strands) and rods (α-helices), respectively, above the alignment according to the crystal structure of the Escherichia coli CadC_pd protein determined in this study. Amino acid Asp471, which is important for pH-sensing, is marked by an asterisk. GI numbers (http://www.ncbi.nlm.nih.gov/Sitemap/sequenceIDs.html) are given in parentheses: Escherichia coli MG1655 (16131959), Escherichia fergusonii ATCC 35469 (218549242), Salmonella enterica serovar typhimurium LT2 (16765877), Serratia proteamaculans 568 (157323768), Yersinia ruckeri ATCC 29473 (238756479), Klebsiella pneumoniae NTUH-K2044 (218117876), Edwardsiella tarda EIB202 (267983771).