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Cyclic di-GMP mediates a histidine kinase/phosphatase switch by noncovalent domain cross-linking

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Science Advances  16 Sep 2016:
Vol. 2, no. 9, e1600823
DOI: 10.1126/sciadv.1600823
  • Fig. 1 CckA crystal structures of the c-di-GMP–complexed CA domain.

    (A) CckA domain arrangement and constructs used. (B to D) Crystal structure of CckA_CA with bound c-di-GMP and AMPPNP/Mg2+. The ATP lid partly covering the mononucleotide site (magenta), the gripper helix (gray, residues 508 to 520), and the CckA-specific insertion (tower, yellow, residues 456 to 471) are distinguished by color. Detailed views of the c-di-GMP and the mononucleotide-protein interactions are shown in (C) and (D), respectively. H-bonds and Mg2+ coordination are shown with green and brown broken lines, respectively.

  • Fig. 2 Crystal structure of CckA_DHp-CA dimer.

    Cartoon representation with one of the subunits colored in green (DHp domain)/orange (CA domain), the other in gray (with asterisked labels); other colors are the same as in Fig. 1. Some important residues and ADP/Mg2+ are shown in full. The Cα atoms of Gly318 and Gly515 are shown as magenta spheres. The domain arrangement is similar as in CpxA/ADP/Mg2+ (17) (fig. S6) but with domains swapped in the dimer. The Phe496 thumb of CA is inserted between DHp helices, and the gripper helix (cyan) mediates most of the interdomain contact.

  • Fig. 3 Ligand binding to various CckA constructs.

    Kd determination was measured by isothermal titration calorimetry (ITC). (A) ADP. (B) AMPPNP in the absence or presence of 100 μM c-di-GMP. (C) c-di-GMP in the presence of 5 mM ADP or AMPPNP. Further information is given in table S2 and fig. S4.

  • Fig. 4 Modeled DHp-CA domain constellations of CckA.

    The crystal structures of the individual DHp helices and the CA domains of CckA (molecular surface representation) are shown as obtained upon superposition onto CpxA structures (17). (A) Phosphatase constellation as in the CpxA/ADP complex (PDB code 4bix). C-di-GMP is cross-linking the primary site (CA domain; Y514, K518, and W523) with the secondary site (DHp domain; G318 shown in magenta, R374 with nitrogen atoms in blue). G515 (magenta) is part of the interface. The full model is shown in fig. S6. This constellation is predicted to be competent for dephosphorylation of cognate P~Rec (represented symbolically) (13). (B) Kinase constellation as in the CpxA/AMPPNP complex (PDB code 4biw). Note that relative to the constellation in (A), the CA domain has turned around by about 70° to bring the AMPPNP substrate analog close to the phospho-acceptor His322 (not shown, buried within the interface). (C) Sequence logo excerpts for CckA orthologs (top) and HKs in general (CckA orthologs plus paralogs; bottom). For the upper logo, only residues with a conservation contrast score (Δcons) in the upper quartile are shown (see also fig. S8A). Residues related to c-di-GMP binding are labeled according to CckA numbering.

  • Fig. 5 Net phosphorylation of CckA variants in response to c-di-GMP and ADP.

    (A) Time course of net CckA_ΔTM~P formation as measured by autoradiography (bottom row). ADP or c-di-GMP was added to aliquots after 30 min of incubation (top three rows). (B) Quantification of the data in (A). Continuous lines have been calculated on the basis of the kinetic model shown in Fig. 7, with parameters (table S3) obtained from the global fit to the data. (C) Net CckA phosphorylation for the indicated variants after 15 min of ATP incubation, in the absence and presence of 75 μM c-di-GMP. (D) Net CckA phosphorylation for the same variants as in (C) but with addition of hexokinase/glucose at t = 15 min and total incubation time of 65 min.

  • Fig. 6 DNA replication activity of cells expressing various CckA variants.

    The indicated cckA(ΔTM) variants were expressed from a low– or high–copy number plasmid in the WT (+) or cdG0 (−) strain, followed by analysis of DNA content using flow cytometry. Representative DNA profiles of the two biological replicates are shown. The fraction of cells bearing more than two chromosomes is indicated and shown as percentage. *Transformed cells did not grow. **Not tested.

  • Fig. 7 Kinetic regulatory model of CckA.

    Autophosphorylation and autodephosphorylation proceed with first-order rates k1 and k2, respectively. For simplicity, no distinction is made between phosphorylation of His322 (DHp), Asp623 (Rec), or both. Red double arrows indicate ADP ↔ ATP equilibration. (A) After autophosphorylation, the ADP product gets efficiently replaced by ATP. The dephosphorylation branch is not effective, and the enzyme can catalyze phosphotransfer to the cognate downstream. (B) C-di-GMP allosterically stabilizes the ADP complex, which is competent for autodephosphorylation. Note that rephosphorylation is impeded because of ADP product inhibition. Phosphoryl groups will flow back along the phosphorelay.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/9/e1600823/DC1

    table S1. Data collection and refinement statics.

    table S2. Thermodynamic parameters of CckA-ligand interactions as measured by ITC.

    table S3. Kinetic parameters of HK/phosphatase CckA_ΔTM.

    fig. S1. CckA crystal packings.

    fig. S2. Detailed views of domain interface of CckA_DHp-CA and comparison with c-di-GMP–bound CckA_CA.

    fig. S3. Sequence alignment between CckA orthologs and paralogs.

    fig. S4. ITC ligand-binding profiles for several CckA.

    fig. S5. Structure prediction for PAS2 domain of CckA.

    fig. S6. Proposed model of noncovalent c-di-GMP–mediated cross-linking of the CA with the DHp domain of CckA.

    fig. S7. Consensus tree analysis for CckA orthologs and paralogs.

    fig. S8. Residue conservation in CckA orthologs and paralogs.

    database S1. Bacterial strains used in this study.

    database S2. Plasmids used in this study.

    database S3. Oligonucleotides used in this study.

    References (4551)

  • Supplementary Materials

    This PDF file includes:

    • table S1. Data collection and refinement statics.
    • table S2. Thermodynamic parameters of CckA-ligand interactions as measured by ITC.
    • table S3. Kinetic parameters of HK/phosphatase CckA_ΔTM.
    • fig. S1. CckA crystal packings.
    • fig. S2. Detailed views of domain interface of CckA_DHp-CA and comparison with c-di-GMP–bound CckA_CA.
    • fig. S3. Sequence alignment between CckA orthologs and paralogs.
    • fig. S4. ITC ligand-binding profiles for several CckA.
    • fig. S5. Structure prediction for PAS2 domain of CckA.
    • fig. S6. Proposed model of noncovalent c-di-GMP–mediated cross-linking of the CA with the DHp domain of CckA.
    • fig. S7. Consensus tree analysis for CckA orthologs and paralogs.
    • fig. S8. Residue conservation in CckA orthologs and paralogs.
    • References (4551)

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    Other Supplementary Material for this manuscript includes the following:

    • database S1 (Microsoft Excel format). Bacterial strains used in this study.
    • database S2 (Microsoft Excel format). Plasmids used in this study.
    • database S3 (Microsoft Excel format). Oligonucleotides used in this study.

    Files in this Data Supplement:

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