Structural basis for two-component system inhibition and pilus sensing by the auxiliary CpxP protein

Abstract

Bacteria are equipped with two-component systems to cope with environmental changes, and auxiliary proteins provide response to additional stimuli. The Cpx two-component system is the global modulator of cell envelope stress in gram-negative bacteria that integrates very different signals and consists of the kinase CpxA, the regulator CpxR, and the dual function auxiliary protein CpxP. CpxP both inhibits activation of CpxA and is indispensable for the quality control system of P pili that are crucial for uropathogenic Escherichia coli during kidney colonization. How these two essential biological functions of CpxP are linked is not known. Here, we report the crystal structure of CpxP at 1.45 Å resolution with two monomers being interdigitated like "left hands" forming a cap-shaped dimer. Our combined structural and functional studies suggest that CpxP inhibits the kinase CpxA through direct interaction between its concave polar surface and the negatively charged sensor domain on CpxA. Moreover, an extended hydrophobic cleft on the convex surface suggests a potent substrate recognition site for misfolded pilus subunits. Altogether, the structural details of CpxP provide a first insight how a periplasmic two-component system inhibitor blocks its cognate kinase and is released from it.

FIGURE 1.

CpxPΔ151 CpxP has two functions. A, steady-state analysis of PapE by immunological determination according to an established protocol (11) with two modifications as described in detail under “Experimental Procedures.” Cells expressing PapE-Strep and indicated CpxP variants were subjected to immunological determination using antiserum to the Strep-tag, the CpxP protein, and the MalE protein (loading control), respectively. B, CpxA autophosphorylation activity was measured by incubating proteoliposomes containing purified CpxA-His6 (1 μm) in phosphorylation buffer containing [γ-³²P]ATP for 20 min. To test the influence of CpxP variants on CpxA autophosphorylation, the experiment was performed as described (12) with wild-type CpxP or CpxPΔ151-loaded proteoliposomes. Samples were separated by SDS-PAGE and, analyzed as phosphorimages, and the amounts of [³²P]ATP were quantified by phospho imaging using [γ-³²P]ATP as a standard. Shown are averages ± S.E. from three different experiments (t test).

FIGURE 2.

Crystal structure of CpxPΔ151. A, ribbon representation of a CpxPΔ151 monomer containing four helices α1 to α4 (helices, orange; loops, green). Two characteristic glutamine residues of the conserved LTXXQ repeat motifs (Leu⁵¹–Gln⁵⁵ and Leu¹²⁴–Gln¹²⁸) and a PP motif (Pro⁷¹–Pro⁷²) are shown as sticks. B, the monomer conformation with a V-shaped structure formed by helices α3 and α4 is stabilized by a double hydrogen bond between the conserved LTXXQ motifs (highlighted in blue) in helices α1 and α4. C, close-up view of the region surrounding the double hydrogen bond between Gln⁵⁵ and Gln¹²⁸ shown with 2F_o − F_c as electron density map (blue mesh) contoured at 1.2σ. D, the asymmetric unit contains a CpxPΔ151 dimer with two monomers that are interdigitated like left hands. Distinct perspectives of the CpxPΔ151 dimer are shown as surface representation (CpxPΔ151 monomer 1, blue; CpxPΔ151 monomer 2′, orange). E, ribbon representation of the CpxPΔ151 dimer using the same color scheme as in D. F, size-exclusion chromatography of CpxP was carried out on a Superdex 200 (HR10/30) column (GE Healthcare) equilibrated in 50 mm Tris/HCl, pH 7.5, containing 150 mm NaCl and 0.5% glycerol (v/v) at 4 °C. Protein samples (100 μl at 0.7 mg ml⁻¹) were applied to the column at a flow rate of 0.5 ml min⁻¹. The elution volumes for the molecular weight standards are shown by dashed lines (F = ferritin, A = aldolase, B = bovine serum albumin, C = carbonic anhydrase). See supplemental Fig. S3 for molecular mass determination of CpxPΔ151 by the combination of size-exclusion chromatography and sucrose-gradient sedimentation analysis.

FIGURE 3.

CpxPΔ151 dimer stability is accomplished by intermolecular contacts. A–C, salt bridges between Glu⁷⁹ and Arg¹⁴⁴ (A) and Glu⁹² and Lys¹¹⁵ (B) and a π-π-stacking between His⁸² and His¹³⁶ (C) stabilize the CpxPΔ151 dimer. D, CpxP-mediated Cpx pathway inhibition was investigated by β-galactosidase activity measurement (upper panel). Shown are averages ± S.E. from four independent determinations each with three replicates (t test). CpxP levels in periplasmic fractions were analyzed by immunoblotting (middle panel). Numbers above bars give the relative inhibition of β-galactosidase activity (given in Miller Units MU) or the relative protein amount, both normalized for wild-type CpxP. The CpxP loss-of-function variants Q55P and D61E were used as functional controls (49) (supplemental Fig. S4), and MalE was used as loading control (lower panel).

FIGURE 4.

Chaperone-like function of CpxP. A, chaperone function of CpxP was investigated by determining the activity of citrate synthase (CS) during thermal stress in the absence of additional protein (control) or in the presence of BSA or CpxP at the indicated concentrations. Shown are averages ± S.E. from three independent determinations (t test). B, hydrophobic surface representation of the CpxPΔ151 dimer. The coloring scheme reflects the hydropathic index at a surface position as calculated by the method of Kyte and Doolittle (61) to analyze the hydrophobic character of the surface of the protein. Regions with a hydropathy index above 0 are hydrophobic in character and depicted in blue. Hydrophilic regions are shown in gray. The hydrophobic cleft on the convex surface (green) of the CpxPΔ151 dimer is highlighted. C, detailed views of the hydrophobic cleft between the two symmetry-related long helices α3 and α3′ as hydrophobic surface and stick representations. D, steady-state analysis of PapE by immunological determination was performed as described for Fig. 1. For control purposes, we used the empty vector (V, lane 1) and wild-type CpxP with 10-fold reduced arabinose concentration (lane 3).

FIGURE 5.

Protein-protein interaction between CpxP and CpxA. A, a peptide array derived from the sequence of CpxP was screened for binding of the ³⁵S-labeled CpxA sensor domain (CpxA28–164-His₆) as described under “Experimental Procedures.” Rows are labeled for peptide numbers and the N-terminal peptide residues. Those spots exhibiting the strongest signal within a serial of increasing signals are highlighted. B, reverse experiment to A. Peptide arrays (15-mer, 20-mer, and 25-mer) derived from the sequence of the CpxA sensor domain were prepared and incubated with ³⁵S-labeled CpxP-His₆. One spot exhibiting the strongest signal within a serial of increasing signals of the 25-mer peptide array that covers sequences of signals of the 15-mer and 20-mer peptide arrays is highlighted. C, complete substitutional and length analysis of the interacting 25-mer peptide of the CpxA-derived peptide recognized by CpxP was generated using the software LISA and subsequently synthesized as described under “Experimental Procedures.” Each amino acid of the peptides (25-mer) corresponding to signal 1 in B is substituted by all other 20 l-amino acids in alphabetical order (shown on top of the membrane) and tested for binding of ³⁵S-labeled CpxP-His₆. All spots in the left column comprise the wild-type sequence (WT) of the peptide. Those spots with increased negative or positive charged amino acids are highlighted by red and blue boxes, respectively. D, surface representation of CpxPΔ151 dimer. The electrostatic surface potentials were calculated using the program APBS (39) with the non-linear Poisson-Boltzmann equation and contoured at ± 3 k * T/e, where k is Boltzmann's constant; T, the temperature in K, and e, the charge of an electron. Negatively and positively charged surface areas are colored in red and blue, respectively. E, CpxP-mediated Cpx pathway inhibition was investigated as described for Fig. 3. F, the salt dependence of CpxP-mediated Cpx pathway inhibition was investigated by β-galactosidase activity measurement using the strain SP594 (16). CpxP was expressed from vector pTrc99A (Invitrogen). Averages ± S.E. from five independent determinations each with three replicates are shown. Numbers above the bars give the relative inhibition of β-galactosidase activity at the indicated NaCl concentrations.

FIGURE 6.

A model of signal integration by CpxP. Direct interaction of the cap-shaped CpxP dimer (ribbon representation) via its concave polar surface with the negatively charged sensor domain of CpxA keeps the kinase in an off mode. Direct interaction of misfolded pilus subunits with the hydrophobic cleft on the convex surface on CpxP results in the release of CpxP from CpxA and switches the kinase in an “on” mode. The release of CpxP from CpxA results in CpxR activation, which acts as inducer (arrow) or repressor (bar) for target gene expression.