Structural basis for diguanylate cyclase activation by its binding partner in Pseudomonas aeruginosa
- Key Laboratory of Resources Biology and Biotechnology in Western China, Ministry of Education, College of Life Sciences, Northwest University, China
- Department of Pharmacology and Chemical Biology, Shanghai Jiao Tong University, School of Medicine, China
- School of Medicine, Southern University of Science and Technology, China
Figures
Figure 1 with 3 supplements
SiaC promotes the DGC activity of SiaD through direct interaction.
(A) Analysis of the initiation codon and coding sequence of SiaD. (B) Overexpression of SiaD but not SiaDΔN40 restored the biofilm formation of siaD mutant. The biofilm formation of the indicated strains was stained by crystal violet staining and quantified with optical density measurement. (C) Pull down assay revealed the role of N67/T68/S69 for SiaC to interact with SiaD. Cell lysates of E. coli containing pMMB67EH-siaC-Flag or siaCN67A/T68A/S69A-Flag (siaCM3-Flag) were incubated with GST or GST-SiaD, individually, and protein complexes were captured by glutathione beads. (D) Production of c-di-GMP by SiaD, SiaD with SiaC, and SiaD with SiaCN67A/T68A/S69A (SiaCM3).
Figure 1—figure supplement 1
SiaD coding sequence.
The stability of SiaD was not influenced by deletion of siaC gene in P. aerugionsa.
Figure 1—figure supplement 2
SDS–PAGE for purifed SiaD and SiaDΔN40 proteins.
(A) Sumo-SiaD and Sumo-SiaDΔN40 were furified by Ni affinity column and were digested by ULP1 protease to get SiaD and SiaDΔN40. (B) The retention time of c-di-GMP standard. (C) GTP standard. (D–F) The c-di-GMP levels produced by SiaD (D), SiaD with SiaC (E) and SiaD with SiaCN67A/T68A/S69A (SiaCm3) (F) after 30 min were detected.
Figure 1—figure supplement 3
Production of c-di-GMP by SiaD, SiaDΔN40 determined by HPLC.
To evaluate the allosteric inhibition of c-di-GMP on SiaD protien, 50 μM c-di-GMP was added to reaction mixture.
Figure 2 with 5 supplements
Crystal structure of SiaC–SiaD complex with GTP analog.
Two monomers of SiaD (SiaD-A and SiaD-B) and four SiaC molecules (SiaC-C/D, SiaC-E/F) are colored in green, cyan, yellow, magenta, salmon, and white, respectively. The secondary structural elements of SiaD was labeled. The GTP analog GpCpp and Mg2+ ion are shown in slate stick and lightblue sphere. H-bond network is shown as yellow dashes. (A) Overall crystal structure of SiaC–SiaD complex. The N-terminal domain, middle stalk domain, and C-terminal DGC domain of SiaD are labeled. (B) Overview of the DGC domain dimer. A-site and I-site were labeled, respectively. (C) GpCpp binds to the C-terminal DGC domain of SiaD (A-site). (D) Results of isothermal titration calorimetry (ITC) for GTP binding to SiaD (salmon), SiaC (slate), or SiaC–SiaD complex (black). Plots of molar enthalpy change against GTP-SiaD or SiaC–SiaD complex molar ratio are shown. (E) Divalent ion Mg2+ or Mn2+ is essential for SiaD activity. (F) Amino acid residues involve in GTP and Mg2+ binding are essential for SiaD activity in vivo. The biofilm formation of the indicated strains were displayed with crystal violet staining (up) and quantified with optical density measurement (down). Data represent the means and SDs of three biological replicates. **p<0.01 based on one-way ANOVA test; ns, non-significance.
Figure 2—figure supplement 1
Multiple structural alignment of SiaD homologs.
The structural alignment was generated by Espript online server (http://espript.ibcp.fr/ESPript/ESPript/). Residues of SiaD involved in the interactions with GTP (A-site), c-di-GMP (I-site), Mg2+, and SiaC are labeled with purple, cyan, magenta, brown, and green disks. The identical residues, similar residues, and residues of the stalk domain among the homologs are shadowed in red, yellow, and cyan, respectively. The secondary structure of SiaD are shown and labeled. Structures used in the alignment were obtained from Protein Data Bank (PDB) with pdb codes: 2V0N (PleD), 3BRE (WspR from Pseudomonas aeruginosa), 3I5A (WspR from Pseudomonas syringae), 5XGB (RbdA), 4WXO (SadC), 4ZMU (Dcsbis), 5LLX (PadC), 6HBZ (DgcB), and 4H54 (DgcZ).
Figure 2—figure supplement 2
Superposition of SiaD with its homolog.
The secondary structure of SiaD, A-site (substrate binding site), I-site (product binding/inhibitory site), and the ligands are labeled, respectively. The structure of SiaD is colored in green. Structures used in the superposition were obtained from Protein Data Bank (PDB) with pdb code: 2V0N (PleD, colored in Salmon).
Figure 2—figure supplement 3
Analysis of SiaD A-site with GpCpp.
Structures used in the superposition were obtained from Protein Data Bank (PDB) with pdb codes: 2V0N (PleD from Caulobacter vibrioides, colored in samlon) and 5LLX (PadC form Idiomarina sp. A28L). GpCpp and Mg2+ ion were shown in slate stick and grey sphere. H-bond network was shown as yellow dashes. (A) The structural superposition between SiaD-A (colored in green) and PleD. (B) IsPadC was used as the template for structural superposition. SiaD-A and GpCpp were colored in green (SiaD), cyan (SiaD’), and slate. The distance between the general base E182 and the 3’ hydroxyl group from the GpCpp of opposite monomer was shown as yellow dashes. (C) Comparing the structure of SiaD-A (with GpCpp, colored in green) and SiaD-B(without GpCpp, colored in cyan).
Figure 2—figure supplement 4
The stereo view of omit (fofc) electron density of GpCpp and Mg2+ bound to SiaD at 3.0 σ.
The SiaD structure is colored in green, and the secondary structure are labeled. Two key residues of SiaD that interact with Mg2+ are shown in sticks and labeled. The GTP analog GpCpp is shown in stick and colored in slate. Mg2+ ion is shown in sphere and colored in lightblue. The density around GpCpp and Mg2+ is colored in blue and purple, respectively.
Figure 2—figure supplement 5
Expression of SiaD and its mutants during biofilm formation.
The biofilm formed by the indicated strains were scraped and subject to western blotting assay. The expression levels of SiaD-Flag or its mutants from these strains were determined. RNA polymerase alpha unit was used as an internal control.
Figure 3 with 2 supplements
Feedback inhibition of I-site of SiaD.
(A) Superposition of I-sites between SiaD and PleD dimer. The PleD dimer were colored in yellow and orange, and four c-di-GMP molecules observed in PleD structure were colored in ligtblue, magenta, orange, and red, respectively. (B) Overview of SiaD I-site. Key residues involved in c-di-GMP binding were show in sticks and labeled. (C) SEC analysis of SiaD, SiaDR201A and SiaC–SiaD complex after incubation with c-di-GMP. The retention time of SiaD and the complex was labeled by dotted line. (D) SiaDS82A-Q86A mutation weakened the function of SiaC–SiaD complex during biofilm formation, while SiaDR201A basically eliminated the feedback inhibition. The biofilm formation of the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down).
Figure 3—figure supplement 1
Production of c-di-GMP by the indicated protein samples determined by HPLC.
To evaluate the allosteric inhibition of c-di-GMP on SiaD or SiaDR201 protien, 50 μM c-di-GMP was added to reaction mixture. S82A/Q86A mutation caused reduction of the production of c-di-GMP. Addition of c-di-GMP to preformed SiaC–SiaD complex decreased the DGC acitivity of SiaD (SiaC–SiaD+ cdG). When pre-incubated with c-di-GMP, SiaD could not be activated after addition of SiaC (SiaD-cdG+ SiaC). Mutation R201A abolished the feedback inhibition of c-di-GMP.
Figure 3—figure supplement 2
Effect of c-di-GMP on SiaD protein thermal stability.
After 5 μM protein was incubated with 25 μM c-di-GMP, the inactive SiaD shifted by 7.1℃ and the SiaD in the complex shifted by 1.5℃, respectively.
Figure 4 with 4 supplements
Details of SiaC–SiaD interaction.
(A, D) The interactions of SiaC α2ʹ and α3ʹ helixes with SiaD α–1 stalk helixes. The secondary elements and residues of SiaD and its symmetry related elements and residues are labeled or with an asterisk; The secondary elements and residues of SiaC and its symmetry related elements and residues are labeled with primes “ʹ” or “"”; (B, E) the interactions of SiaC key residues N67ʹ, T68ʹ, and S69ʹ with SiaD α–1 stalk helixes. (C) The phosphorylation model of SiaC T68ʹ abolishes its binding to SiaD by introducing clashes. (F) Sequence alignment of the two SiaC binding motifs of SiaD. The identical residues and similar residues between the two motifs are shadowed in red and yellow. Conserved residues involved in the interactions with SiaC are labeled with darkpurple disks. (G) Results of isothermal titration calorimetry (ITC) for SiaC (black) or SiaC N67A/T68A/S69A mutant (cyan) binding to SiaD. Plots of molar enthalpy change against SiaC–SiaD or SiaC mutant-SiaD molar ratio are shown.
Figure 4—figure supplement 1
Superposition of SiaC structure with four SiaC molecules from the SiaC–SiaD complex.
The SiaC alone structure is colored in green, and the SiaC structures from the SiaC–SiaD complex are colored in magenta, yellow, white, and salmon, respectively. The secondary structures of SiaC are also labeled.
Figure 4—figure supplement 2
The stereo view of omit (fofc) electron density of the key residues N67, T68, and S69 of SiaC at 3.0 σ.
The two SiaD monomers and SiaC are colored in green, cyan, and salmon. The secondary structures are also labeled. The three key residues of SiaC are shown in sticks and labeled, and the density around the residues is colored in blue.
Figure 4—figure supplement 3
Multiple structural alignment and superposition of SiaC with the CheY homolog.
Structures used in the alignment and superposition were obtained from Protein Data Bank (PDB) with pdb code: 1FQW (CheY from Escherichia coli, colored in cyan). (A) The structural alignment was generated by Espript online server (http://espript.ibcp.fr/ESPript/ESPript/). The identical residues and similar residues among the homologs are shadowed in red and yellow. The secondary structure of SiaC is shown and labeled. Residues of SiaC involved in the interactions with SiaD are labeled with magenta disks. The key residue T68 of SiaC involved in phosphorylation is shadowed in pink and labeled with purple disks. Residues of CheY homologs involved in phosphorylation are labeled with brown disks. (B) The SiaC structure is colored in salmon, and the secondary structures are also labeled. The key residue of SiaC and CheY homolog are shown in sticks and labeled, respectively.
Figure 4—figure supplement 4
Multiple structural alignment and superposition of SiaC with the STAS domains of stressosome homologs.
Structures used in the alignment and superposition were obtained from Protein Data Bank (PDB) with pdb code: 1H4Z (SpoIIAA from Lysinibacillus sphaericus, colored in cyan). (A) The structural alignment was generated by Espript online server (http://espript.ibcp.fr/ESPript/ESPript/). The similar residues among the homologs are shadowed in yellow. The secondary structure of SiaC is shown and labeled. Residues of SiaC involved in the interactions with SiaD are labeled with magenta disks. The key residue T68 of SiaC or the key residues of the STAS domain homologs involved in phosphorylation are labeled with purple disks. (B) The SiaC structure is colored in salmon, and the secondary structures are also labeled. The key residue of SiaC and stressosome homolog are shown in sticks and labeled, respectively.
Figure 5 with 2 supplements
Mutation of SiaC–SiaD interface.
(A) SEC analysis of SiaC-SiaDS50A/Q54A and SiaC-SiaDS82A/Q86A complex. After overnight placement, part of the SiaC-SiaDS82A/Q86A dissociated. The dissociation peaks were marked by black arrow and the retention time of SiaC-SiaD complex was marked by dotted line. (B, C) SEC-MALS analysis of SiaC-SiaDS50A/Q54A and SiaC-SiaDS82A/Q86A complex. The protein was separated using a Wyatt Technology WTC-030S5 column. The running buffer contains 20 mM HEPES (pH 7.0), 150 mM NaCl and 1 mM DTT. The linear curve indicates the calculated molecular masses of the samples throughout the peaks. (D) N67A/T68A/S69A triple mutation abolished the function of SiaC during biofilm formation. The biofilm formation of the indicated strains were displayed with crystal violet staining (up) and quantified with optical density measurement (down). (E) SiaCN67A/T68A/S69A triple mutation (SiaCM3) was unable to interact with SiaD. SPR measurements of SiaC or SiaCM3 binding at varying concentrations to SiaD. SiaC-His specifically interacted with SiaD with a Kd of 11.8 nM. Shown are measured binding responses (black) and curve fits to a 2:1 interaction (red). Plots are representative from two experiments with similar results. RU, response units; Kd, dissociation constant.
Figure 5—figure supplement 1
SEC-MALS analysis of SiaC–SiaD complex, SiaC and SiaD protein under 2 mg/ml, 2 mg/ml, and 8 mg/ml.
The protein was separated using a Wyatt Technology WTC-030S5 column. The running buffer contains 20 mM HEPES (pH 7.0), 150 mM NaCl, and 1 mM DTT. The linear curve indicates the calculated molecular masses of protein throughout the peaks.
Figure 5—figure supplement 2
Synchrotron solution small angle X-ray Scattering (SAXS) measurements of SiaC–SiaD complex.
Scattering profile (A), PDDFs (B), and dimensionless Kratky plots (C) of SiaC–SiaD Complex. The inset in (A) is the guinier region with fitting line of the scattering profile. The back-calculated scattering profile of the ab initio model (red line) and atomic model (black dash line) was fitted to the experimental SAXS data of SiaC–SiaD complex (dot line). (D) SAXS modeling: low-resolution envelope for the complex was shown as a particle model and was superposed with the complex crystal structure.
Figure 6 with 4 supplements
SEC-MALS and SAXS measurements of SiaD.
(A) SEC-MALS analysis of SiaD protein under 4 mg/ml. The protein was separated using a Wyatt Technology WTC-030S5 column. The running buffer contains 20 mM HEPES (pH 7.0), 150 mM NaCl, and 1 mM DTT. The linear curve indicates the calculated molecular masses of SiaD throughout the peaks. (B–E) SAXS analysis of SiaD protein. Scattering profile (B), PDDFs (C), and dimensionless Kratky plots (D) of SiaD. The inset in (B) is the guinier region with fitting line of the scattering profile. The back-calculated scattering profile of the ab initio model (red line) and atomic model (black dash line) was fitted to the experimental SAXS data of SiaD (dot line). (E) SAXS modeling: low-resolution envelope for SiaD protein was shown as a particle model and was superposed with the SiaD model pentamer. (F) Modeled structure of SiaD monomer. Superposition of SiaD from SiaC–SiaD complex with the modeled SiaD monomer were shown. The N-terminal domain and C-terminal DGC domain of SiaD are labeled.
Figure 6—figure supplement 1
The N-terminal stalk is essencial for SiaD–SiaD interaction.
Cell lysates of E. coli containing pMMB67EH-siaD-flag (A) or pMMB67EH-siaDΔN95-flag (B) were incubated with GST or GST-SiaD, individually, and protein complexes were captured by glutathione beads.
Figure 6—figure supplement 2
Blue native PAGE for SiaD and SiaC–SiaD complex.
3× loading buffer (20% glycerol, 1.05% Coomassie G250, and 1.5 M 6-aminohexanoic acid) was mixed with SiaD (25 μg) alone or SiaC–SiaD mixture (SiaD mixed with excess SiaC [35 μg] for 30 minutes before electrophoresis). Protein samples were loaded onto 8% BN-PAGE. After 50 min electrophoresis at 15 mA, the gel was stained by Coomassie blue R250.
Figure 6—figure supplement 3
Analysis of secondary structures of SiaD by cirular dichroism spectrum.
Protein samples were tested in 10 mM Tris–HCl, pH 7.5 and 500 mM NaCl in 0.1-cm-pathlength quartz cuvettes. The far-UV CD spectrum of protein samples were recorded in the range of 250–200 nm at 25°C, with 1 s/point scanning speed and 1 nm step. Three biological repeats were set for each sample. Three scans were averaged to obtain the final spectra for each sample. As SiaC conformation has no significant change in SiaC–SiaD complex compared to its native structure, the CD signal of SiaC control was substracted from that of the corresponding SiaC–SiaD mixture sample to obtain the CD spectra of SiaD in mixture sample.
Figure 6—figure supplement 4
Feedback inhibition model of SiaC–SiaD complex.
Predicted model of SiaC–SiaD complex inhibited by intercalated c-di-GMP.
Figure 7
Proposed model for SiaC-mediated activation of SiaD.
Without SiaC binding (depletion or phosphorylation of SiaC), SiaD alone forms an inactive pentamer conformation in solution. To activate SiaD, four SiaC binds to the stalks of two SiaD and promotes the formation of SiaD dimer. High level c-di-GMP then represses the SiaD activity via I-site through two distinct modes.
Additional files
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Supplementary file 1
Supplementary tables.
(a) Strains and plasmids used in this study. (b) Primers used in this study. (c) Data collection and refinement statistics. (d) Data collection and refinement statistics. (e) Estimated secondary structure content of SiaD.
- https://cdn.elifesciences.org/articles/67289/elife-67289-supp1-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/67289/elife-67289-transrepform1-v2.docx
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Source data 1
Source data for figures.
- https://cdn.elifesciences.org/articles/67289/elife-67289-supp2-v2.pdf
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Structural basis for diguanylate cyclase activation by its binding partner in Pseudomonas aeruginosa
eLife 10:e67289.
https://doi.org/10.7554/eLife.67289
