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 …
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) …
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 …
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+, …
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. …
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). …
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 …
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 …
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, …
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. …
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 …
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 …
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 …
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 …
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 …
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 …
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 …
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 …
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. …
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 …
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 …
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 …
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
