PDBsum entry: 3i5a

Signaling protein

PDB id: 3i5a

Contents

Protein chain

319 a.a. *

Ligands

C2E ×2

Metals

_SR

Waters ×30

* Residue conservation analysis

PDB id:

3i5a

Name:

Signaling protein

Title:

Crystal structure of full-length wpsr from pseudomonas syrin

Structure:

Response regulator/ggdef domain protein. Chain: a. Synonym: response regulator wspr. Engineered: yes

Source:

Pseudomonas syringae pv. Tomato. Organism_taxid: 223283. Strain: dc3000. Gene: pspto1499, pspto_1499, wspr. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.80Å R-factor: 0.239 R-free: 0.266

Authors:

M.V.A.S.Navarro,N.De,H.Sondermann

Key ref:

N.De et al. (2009). Determinants for the activation and autoinhibition of the diguanylate cyclase response regulator WspR. J Mol Biol, 393, 619-633. PubMed id: 19695263 DOI: 10.1016/j.jmb.2009.08.030

Date:

03-Jul-09 Release date: 18-Aug-09

Protein chain

Q886S7  (Q886S7_PSESM) -  Response regulator/GGDEF domain protein

Seq: 334 a.a.

Struc: 319 a.a.

 Gene Ontology (GO) functional annotation 

• Cellular component: intracellular (1 term)
• Biological process: intracellular signal transduction (4 terms)
• Biochemical function: two-component response regulator activity (2 terms)

DOI no: 10.1016/j.jmb.2009.08.030

J Mol Biol 393:619-633 (2009)

PubMed id: 19695263

Determinants for the activation and autoinhibition of the diguanylate cyclase response regulator WspR.

N.De, M.V.Navarro, R.V.Raghavan, H.Sondermann.

ABSTRACT

The bacterial second messenger bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) controls secretion, cell adhesion, and motility, leading to biofilm formation and increased cytotoxicity. Diguanylate cyclases containing GGDEF and phosphodiesterases containing EAL or HD-GYP domains have been identified as the enzymes controlling cellular c-di-GMP levels, yet less is known regarding the molecular mechanisms governing regulation and signaling specificity. We recently determined a product-inhibition pathway for the diguanylate cyclase response regulator WspR from Pseudomonas, a potent molecular switch that controls biofilm formation. In WspR, catalytic activity is modulated by a helical stalk motif that connects its phospho-receiver and GGDEF domains. The stalks facilitate the formation of distinct oligomeric states that contribute to both activation and autoinhibition. Here, we provide novel insights into the regulation of diguanylate cyclase activity in WspR based on the crystal structures of full-length WspR, the isolated GGDEF domain, and an artificially dimerized catalytic domain. The structures highlight that inhibition is achieved by restricting the mobility of rigid GGDEF domains, mediated by c-di-GMP binding to an inhibitory site at the GGDEF domain. Kinetic measurements and biochemical characterization corroborate a model in which the activation of WspR requires the formation of a tetrameric species. Tetramerization occurs spontaneously at high protein concentration or upon addition of the phosphomimetic compound beryllium fluoride. Our analyses elucidate common and WspR-specific mechanisms for the fine-tuning of diguanylate cyclase activity.

Selected figure(s)

Figure 8.
Fig. 8. Structure of WspR^GCN4-GGDEF. (a) Crystal structure of WspR^GCN4-GGDEF. The crystals contain two molecules of WspR^GCN4-GGDEF in the asymmetric unit with two c-di-GMP dimers bound to the I-sites. The GGDEF domains of WspR are shown in green and gray, with their corresponding GCN4 stalks in olive and gray, respectively. A close-up view of an I-site bound to c-di-GMP (shown in stick presentation) is shown in the right panel. (b) Flexible linkage between the GGDEF domains and the stalk units. The two crystallographic chains of full-length WspR from P. aeruginosa and of WspRGCN4-GGDEF were superimposed in their GGDEF domains. The REC domains of full-length WspR are not shown for clarity. (c) Comparison of the structure of the WspR tetramer with activated PleD. The crystal structure of dimeric PleD activated by beryllium fluoride is shown in a c-di-GMP-mediated, inhibited conformation (PDB code: 2V0N)^9 (left panel). The REC domains are shown in violet and blue (chain A) and in light gray and dark gray (chain B). The GGDEF motif at the active site is shown in yellow. Beryllium fluoride and magnesium ions are shown as spheres, and c-di-GMP is shown as stick presentation. The right panel depicts a WspR tetramer in a c-di-GMP-mediated, inhibited conformation, highlighting the stalk-GGDEF domain modules that are coordinated by c-di-GMP binding to the I-sites of adjacent domains. (d) Crystallographic, parallel WspR dimer. The ribbon presentation depicts a model for parallel dimer of WspR from P. aeruginosa (PDB code: 3BRE)^5 in colors according to Fig. 1.

Figure 9.
Fig. 9. Crystal structure of nucleotide-free WspR^GGDEF. (a) Comparison of nucleotide-free and c-di-GMP-bound GGDEF domains of WspR. The crystal structures of the isolated GGDEF domain of WspR (WspR^GGDEF; two molecules per asymmetric unit), full-length WspR (PDB code: 3BRE), and WspR^GCN4-GGDEF were superimposed using their GGDEF domains as reference. The structures are shown as C^α traces. The position of the N- and C-termini and that of the active and the inhibitory site are indicated. A coloring key distinguishing the separate chains is provided (right panel). (b) The GGEEF motif. A close-up view of the GGDEF motif is shown. (c) Comparison of rotamer conformations within the GGEEF motif. A similar view of the active site as in (b) is shown. Residues of the GGEEF motifs are shown in stick presentation and colored as listed in (a).

The above figures are reprinted by permission from Elsevier: J Mol Biol (2009, 393, 619-633) copyright 2009.

Figures were selected by an automated process.