Structures of the PelD Cyclic Diguanylate Effector Involved in Pellicle Formation in Pseudomonas aeruginosa PAO1

Background: Bis-(3′–5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) binding to PelD is required for pellicle formation by Pseudomonas aeruginosa.

Results: The crystal structures of a cytosolic fragment of PelD show the binding mode of c-di-GMP.

Conclusion: PelD has a degenerate active site but binds c-di-GMP through a conserved allosteric site.

Significance: PelD represents a novel c-di-GMP effector that has not been structurally characterized before.

Abstract

The second messenger bis-(3′–5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) plays a vital role in the global regulation in bacteria. Here, we describe structural and biochemical characterization of a novel c-di-GMP effector PelD that is critical to the formation of pellicles by Pseudomonas aeruginosa. We present high-resolution structures of a cytosolic fragment of PelD in apo form and its complex with c-di-GMP. The structure contains a bi-domain architecture composed of a GAF domain (commonly found in cyclic nucleotide receptors) and a GGDEF domain (found in c-di-GMP synthesizing enzymes), with the latter binding to one molecule of c-di-GMP. The GGDEF domain has a degenerate active site but a conserved allosteric site (I-site), which we show binds c-di-GMP with a K_d of 0.5 μm. We identified a series of residues that are crucial for c-di-GMP binding, and confirmed the roles of these residues through biochemical characterization of site-specific variants. The structures of PelD represent a novel class of c-di-GMP effector and expand the knowledge of scaffolds that mediate c-di-GMP recognition.

Introduction

Bis-(3′–5′)-cyclic dimeric guanosine monophosphate (c-di-GMP)⁴ is a central regulator, which functions as an intracellular second messenger. In bacteria, this molecule confers adaptability to various environmental conditions, by coordinating the transition between the motile planktonic state to a sessile state associated with biofilm production (1–3). Specifically, c-di-GMP stimulates the production of adhesins and exopolysaccharide matrix components and leads to biofilm formation to protect bacteria from host-defense, starvation conditions, and antibiotics (4, 5). Additional roles for c-di-GMP include control of cell cycle progression (6), antibiotic biosynthesis (7), and expression of virulence genes (8–11). The bacterial signaling nodes that respond to the c-di-GMP message present targets for therapeutic intervention against pathogens.

Similar to other second messenger pathways, the c-di-GMP control module can be generally divided into four components that govern signal generation, degradation, recognition, and targeting, respectively (2). The level of the signal molecule is dynamically regulated by the opposing activities of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs), which synthesize and degrade c-di-GMP, respectively. DGC activity is attributed to proteins that contain a characteristic GGDEF domain (named for the single letter amino acid nomenclature of essential active site residues), whereas PDE activity is associated with either enzymes that contain either an EAL or a HD-GYP domain (12–15). Interestingly, many DGCs have been shown to be subject to allosteric product inhibition, which is often caused by c-di-GMP binding to the so-called I-site in the GGDEF domain (16–18). The I-site is readily identified by the RXXD (Arg-X-X-Glu; where X is any amino acid) motif, which is connected N terminally to the GGDEF motif through a 5-residue linker. These two motifs are antipodal to each other in the three-dimensional structure as shown by the structures of PleD from Caulobacter vibriodes (16, 19) and WspR from Pseudomonas aeruginosa (18, 20).

Direct recognition of the c-di-GMP signal occurs through an effector component that is often linked to a signal input component that regulates cellular functions at transcriptional, translational, or post-translational levels (6, 21–23). Strikingly, c-di-GMP effectors are highly diverse and are responsible in the diversity of the cellular functions and processes controlled by c-di-GMP in bacteria (2, 3). The effectors identified so far encompass a variety of domains that are capable of recognizing c-di-GMP, including the well characterized PilZ domains (22, 24–26), an unusual receiver domain in VpsT from Vibrio cholera (27), the cyclic nucleotide monophosphate binding domain in Clp from Xanthomonas campestris (28–30), the AAA σ⁵⁴ interaction domain in FleQ from P. aeruginosa (31), and a degenerate (noncanonical) EAL domain in FimX from P. aeruginosa (32) and in LapD from Pseudomononas fluorescens (33, 34), as well as RNA riboswitches (35). Most of the above mentioned types of c-di-GMP effectors have been structurally characterized, including PilZ-domain containing proteins (26, 36–38), VpsT (27), Clp (29), FimX (32), LapD (34), and class I and II c-di-GMP-binding riboswitches (39–41).

A distinct class of c-di-GMP effector consists of molecules that can bind c-di-GMP through an RXXD motif that resembles the I-site of the GGDEF domain of DGCs, but do not show catalytic activity as their active sites lack the requisite GGDEF motif. Examples include PelD from P. aeruginosa (42), CdgG from V. cholera (43), and PopA from C. vibriodes (6). In contrast to the effectors described in the previous paragraph, biochemical data for I-site-containing c-di-GMP effectors is sparse, and there are no crystal structures available for any members of this receptor class.

PelD is found in the Pel pathway involved in the formation of pellicles, one of the major biofilm types formed by P. aeruginosa (44). The Pel pathway synthesizes and exports PEL polysaccharides that play both structural and protective roles in P. aeruginosa biofilms (45). This pathway contains seven proteins, namely PelABCDEFG, which are highly conserved in diverse microbes (44), and have been shown to be functionally conserved (46). Although a few attempts were made to assign functions to the seven proteins (44, 46, 47), the mechanism of polysaccharides production by this pathway remains largely unknown.

Interestingly, the pellicle formation of P. aeruginosa was implied to be stimulated by c-di-GMP (11, 48). Subsequently, PelD was shown to be the receptor of c-di-GMP in the Pel pathway and c-di-GMP binding to PelD is essential to pellicle production (42). In vivo studies showed that c-di-GMP enhanced the transcription level of pel genes and deletion of pelD genes rendered P. aeruginosa PA14 incapable of producing pellicles (42). In vitro biochemical assays showed that PelD is the only protein in the Pel pathway that binds c-di-GMP (42). PelD is predicted to be an inner membrane protein with four transmembrane helices and a large cytosolic region (42, 44). Remarkably, it is proposed to bind c-di-GMP through an I-site-like motif and thus represent a novel family of c-di-GMP-binding proteins (42). Mutation of residues in the I-site-like motif abolished the binding of PelD to c-di-GMP and in vivo assays showed that these PelD mutants lack the ability to restore pellicle formation and Congo red binding by P. aeruginosa PA14, indicating that binding of c-di-GMP to PelD is required for pellicle production.

Here, we present structural, biochemical, and mutational analyses of a soluble, cytosolic domain of PelD from P. aeruginosa PAO1 that harbors all of the necessary elements for c-di-GMP recognition. We have determined the crystal structures of this cytosolic domain both alone and in complex with c-di-GMP (each to 2.0 Å resolution), as well as that of a deletion variant in complex with c-di-GMP (to 1.7-Å resolution). Utilizing the structural data as a guide, we have carried out structure-function analysis of a number of residues at the c-di-GMP binding site and have quantitatively assessed their roles in ligand binding. The combined structural and biochemical data expand upon the current knowledge of c-di-GMP receptors and provide the first structural view of a c-di-GMP effector that recognizes cognate ligand only through the I-site.

EXPERIMENTAL PROCEDURES

Protein Purification and Crystallization

PelD_158-CT was cloned into pET28 vector and expressed in Escherichia coli Rosetta2. Wild type and mutant proteins were purified using an Ni-NTA affinity column followed by size exclusion chromatography in 20 mm HEPES, pH 7.5, 100 mm KCl. Protein was concentrated to 20–30 mg/ml and kept at 4 °C. Protein was precipitated when stored in this condition but became soluble again at room temperature. The protein concentration was determined by UV absorbance at 280 nm using an calculated extinction coefficient, 11,920 m⁻¹ cm⁻¹. PelD_158-CT crystals were grown by the hanging drop vapor diffusion method at room temperature. Each hanging drop contained 1 μl of protein solution and 1 μl of mother liquor. The mother liquor conditions are as follows. (i) PelD_158-CT apo: 100 mm Tris, pH 8, 200 mm MgCl₂, and 10% (v/v) PEG 8000; (ii) PelD_158-CT wild type in complex with c-di-GMP (two molecules in the asymmetric unit): 100 mm Tris, pH 8.5, 200 mm Li₂SO₄, and 1.26 m (NH₄)₂SO₄; (iii) PelD_158-CT wild type or Δ-loop mutant in complex with c-di-GMP (1 molecule in the asymmetric unit): 50 mm sodium cacodylate, pH 6.5, 10 mm MgSO₄, 1.3 m Li₂SO₄. The protein concentration was 2–5 mg/ml in all cases. For co-crystallization, 2 mm c-di-GMP was incubated with the protein at room temperature for 30 min prior to crystallization. Crystals were cryo-protected in 15% ethylene glycol before being flash frozen in liquid nitrogen.

Data Collection and Structure Determination

Initial crystallographic studies were carried out using a construct that spanned residues Ile¹⁴⁴ (PelD_144-CT) through the C terminus. Flash-cooled crystals of PelD_144-CT diffract x-rays beyond a Bragg spacing of 2.5 Å, using an insertion device x-ray beam line (LS-CAT, Sector 21ID, Advanced Photon Source, Argonne, IL). A mercury derivative was prepared by treating crystals with 5 mm ethylmercury bromide for 24 h. Crystallographic phases were determined by single wavelength anomalous diffraction from the mercurial derivative. A 4-fold redundant data set was collected at 100 K to a limiting resolution of 2.6 Å (overall R_merge = 9.2%, I/σ(I) = 1.8 in the highest resolution shell). All diffraction data were integrated and scaled using the HKL2000 package (49). Heavy atom refinement and phase calculation were carried out using PHASER (50) as implemented in the PHENIX software suite (51, 52), followed by density modification using DM (53) and cycles of automated building using ARP/wARP (54) and manual rebuilding using XtalView (55). Continuous electron density could only be observed for two of the four molecules that were expected to be in the crystallographic asymmetric unit, and subsequent refinement of all models using REFMAC5 (56) stalled with a free R factor greater than 40%. As subsequent inspection of the model revealed that electron density for the amino terminus could only be observed starting from Asn¹⁵⁸, all further crystallographic analysis utilized a construct spanning Asn¹⁵⁸ through the C terminus (PelD_158-CT). Structures of ligand complexes and deletion variants were determined using molecular replacement, as implemented in PHENIX. Ramachandran analysis shows that over 90% of the protein main chain dihedral angles are in the most favored regions, and the rest in generously allowed regions. Data collection, phasing, and refinement statistic are summarized in Table 1.

TABLE 1.

Data collection, phasing, and refinement statistics

	Apo-PelD		PelD-c-diGMP		PelD Δ-loop-c-di-GMP
	Native	Hg derivative	Form I	Form II
PDB codes	4ETX		4EUV	4ETZ	4EU0
Data collection
Cell dimensions
a, b, c (Å), β (°)	60.3, 42.4, 60.5	56.1, 102.3, 103.1	59.5, 41.5, 64.4	70.3, 41.3, 110.9	58.1, 41.4, 62.9
	112.8	100.1	110.9	95.5	109.9
Resolution (Å)a	50–2.0 (2.1–2.0)	50–2.6 (2.7–2.6)	50–2.0 (2.07–2.0)	50–2.05 (2.15–2.05)	50–1.7 (1.76–1.7)
Rsym (%)b	4.9 (46.8)	9.2 (74.3)	7.5 (36.5)	6.4 (89.7)	6.0 (30.8)
I/σ(I)	24.7 (4.0)	12.0 (1.8)	23.0 (2.8)	15.6 (2.3)	19.8 (5.3)
Completeness (%)	99.8 (99.7)	99.2 (98.8)	99.1 (95.0)	99.7 (99.8)	97.6 (82.5)
Redundancy	7.4 (6.6)	4.3 (4.2)	6.2 (5.3)	5.8 (5.4)	37.7 (3.1)
Phasing
FOM/DM FOMc		0.417/0.668
Refinement
Resolution (Å)	25.0–2.0		25.0–2.0	25.0–2.05	25.0–1.7
No. reflections	18,333		18,926	38,272	29,209
Rwork/Rfreed	23.2/28.5		22.0/28.1	23.0/27.7	22.9/26.6
Number of atoms
Protein	2,370		2,256	4,536	2,247
c-di-GMP			46	92	46
Water	86		128	140	241
B-factors
Protein	22.5		15.9	28.6	11.7
c-di-GMP			27.8	46.2	24.8
Water	28.0		19.3	34.1	25.4
R.m.s. deviations
Bond lengths (Å)	0.012		0.013	0.013	0.007
Bond angles (°)	1.35		1.52	1.51	1.21

^a Highest resolution shell is shown in parentheses.

^b R_sym = Σ|(I_i − 〈I_i〉|Σ I_i, where I_i = intensity of the ith reflection and 〈I_i〉 = mean intensity.

^c Mean figure of merit before and after density modification.

^d R-factor = Σ(|F_obs|− |F_calc|)/Σ|F_obs| and R-free is the R value for a test set of reflections consisting of a random 5% of the diffraction data not used in refinement.

Analytical Size Exclusion Chromatography

Oligomerization of PelD_158-CT was examined using analytical size exclusion chromatography (Superdex 200 HR 10/30, GE Healthcare) in 20 mm HEPES, pH 7.5, 100 mm KCl. 600 μl of sample with a protein concentration of 0.5 mg/ml was applied to the column. For protein complexed with c-di-GMP or cGMP, 70 μm c-di-GMP or 10 mm cGMP was included in the sample, and 10 μm c-di-GMP or 1 mm cGMP was included in the mobile phase. The molecular weight standards were blue dextran (∼2,000,000 Da), β-amylase (∼200,000 Da), albumin (∼66,200 Da), carbonic anhydrase (∼29,000 Da), and cytochrome c (∼12,400 Da) and purchased from Sigma.

[³²P]c-di-GMP Binding Assay

[³²P]c-di-GMP binding assay was adopted from the method in Ref. 42 with some major modifications. [³²P]c-di-GMP was synthesized by YdeH (57) using [α-³²P]GTP. It was then incubated with purified wild type PelD₁₅₈ or each of the mutants that have N-terminal His tags under the following conditions: 7 nm [³²P]c-di-GMP, 30 μm protein, 50% Ni-NTA-agarose beads (GE Healthcare), 10 mm Tris, pH 7.5, and 50 mm NaCl. The mixture was incubated at room temperature for 30 min and then transferred to a Spin-X 0.22-μm centrifuge tube filter (Costar). The remaining beads in the original tube were washed with 50 μl of wash buffer (10 mm Tris pH 7.5, and 50 mm NaCl) and also transferred to the filter. Free [³²P]c-di-GMP was removed from the mixture by centrifugation. The flow-through was collected in the 2-ml centrifuge tube that held the filter insert. The filter was washed twice with 300 μl of wash buffer and the flow-through was collected in the same centrifuge tube. The filter insert and the flow-through were both counted in the scintillation counter and the fraction of bound [³²P]c-di-GMP was calculated by dividing the filter counts with the sum of both counts.

Isothermal Titration Calorimetry

Measurements were carried out on a Nano ITC (TA Instruments, Waters LLC) with protein protomer typically at 30 μm in the cell and c-di-GMP at 0.5 mm in the syringe. For D370A mutant, the protein concentration was 100 μm and the c-di-GMP concentration was 3 mm. An initial 1-μl injection was followed by 24 injections of 2 μl each at 240-s intervals. c-di-GMP was synthesized by YdeH using GTP and purified according to the method Zähringer et al. (57), and quantitated based on UV absorbance using a extinction coefficient of 26 mm⁻¹ cm⁻¹ at 260 nm (58). Heat of dilution for c-di-GMP was estimated from the last 7–10 injections and subtracted from raw data before fitting the binding isotherm in NanoAnalyze (TA Instruments). Curve fitting was conducted using single independent site binding model. When using the nominal ligand concentration we consistently obtained a stoichiometry of more than 2 c-di-GMP molecules per protomer of PelD. This physically unreasonable value of stoichiometry lead us suspect that the ligand concentration was overestimated due to the presence of UV-absorbing contaminants, a similar situation as reported previously (26). Therefore, the ligand concentration was empirically adjusted by a 2-fold reduction to yield a stoichiometry of approximately 1 when using the single independent site binding model. cGMP or cAMP binding experiments were carried out similarly except that the protein concentration was 100 μm and cNMP (Sigma) concentration was 10 mm. Curve fitting was conducted using a single independent site binding model without any concentration adjustment.

RESULTS

Overall Structures of PelD_158-CT

A series of soluble constructs encompassing the cytoplasmic region of PelD were purified and crystallized, and these constructs were started from Met¹⁰⁵, Leu¹²³, Asp¹³³, or Ile¹⁴⁴ (PelD_144-CT) through the C terminus, respectively. Crystals of PelD_144-CT diffracted beyond 2.5-Å resolution and crystallographic phases were determined to 2.6 Å using a mercurial derivative. Clear electron density could only be observed for two of the four molecules in the crystallographic asymmetric unit and while the quality of the experimental map was sufficient to allow building of an initial, near complete model, the structure could not be satisfactorily refined. Subsequent inspection of the model revealed that electron density for the amino terminus could only be observed starting from Asn¹⁵⁸. A new construct encompassing Asn¹⁵⁸ through the C terminus (PelD_158-CT) was generated and structural analysis was carried out on crystals of both PelD_158-CT (2.0-Å resolution) and its complex with c-di-GMP in two different crystal forms (form I, 2.0-Å resolution; form II, 2.05-Å resolution) (see Table 1 for data collection and refinement statistics). The structures all occupy the same space group of P2₁, but with different unit cell dimensions and, consequently, different packing. There is one molecule in the asymmetric unit of the apo structure and in the form I co-crystal structure, whereas the form II co-crystal structure contacts two molecules in the asymmetric unit that appear to be the result of crystal packing and are biologically irrelevant, as illustrated by both the small contact area between protomers (818 Ų) (59) and the solution behavior of PelD_158-CT as a monomer, both in the presence or absence of c-di-GMP ligand (Fig. 1).

FIGURE 1.

Analytical size exclusion chromatography of PelD_158-CT. A, chromatographic traces of PelD_158-CT in the absence or presence of c-di-GMP or cGMP are shown as indicated. B, molecular weight standard curve. The molecular weight of each standard is indicated. The positions and calculated molecular weight of protein samples in A are indicated.

Although the apo structure is well ordered throughout its entirety, the region between Glu²⁵¹ and Val²⁶³ is poorly defined in both c-di-GMP complex structures. Hence, description of the overall-fold will be based on the apo structure unless otherwise stated. The structure of PelD_158-CT can be clearly divided into two domains of similar size, an N-terminal domain (composed of Gln¹⁵⁸ through Ser³⁰⁹) and a C-terminal domain (encompassing Asp³¹⁸ through Ala⁴⁵⁴), which are connected by a loop composed of residues Asp³¹⁰–Ala³¹⁷ (Fig. 2A). Binding of the ligand does not induce any local or global changes in the structure (Fig. 2B) and the two structures can be aligned with an average r.m.s. deviation of 1.0 Å over 285 C_α atoms.

FIGURE 2.

Overall structures of PelD_158-CT. A, ribbon diagram of the overall structure of PelD_158-CT illustrating the overall architecture and the secondary structural elements are numbered as illustrated. The disposition of the GGDEF (blue) and GAF (pink) domains are shown with the I-site colored in green and the A-site GGDEF motif colored in cyan. B, close-up view of the ligand-binding site in the PelD_158-CT-c-di-GMP co-crystal structure using the same color and numbering scheme as in panel A. The c-di-GMP ligand is shown as a ball-and-stick with yellow carbon atoms. Binding of the ligand does not result in any significant changes in the structure of the protein.

A DALI search (60) against the Protein Data Bank using the complete structure of PelD_158-CT failed to identify any structure with significant similarities over the entire polypeptide. However, a search using either the N- or C-terminal domains identified several candidates that show significant structural homology to each of the individual domains (supplemental Tables S1 and S2). The PelD N terminus consists of a GAF domain found in various cyclic nucleotide receptors including the cyclic GMP-regulated phosphodiesterases, adenylyl cyclases. The closest structural homolog is the GAF-A of human cyclic GMP (cGMP)-specific 3′,5′-cyclic phosphodiesterase PDE5A1 (61) with a r.m.s. deviation of 2.7 Å over 128 C_α atoms (sequence identity: 15%; PDB code 3MF0). The C terminus of PelD shows an architecture similar to the GGDEF domain found in diguanylate cyclases and the closest homolog is the GGDEF domain of P. aeruginosa c-di-GMP receptor FimX (32), with an r.m.s. deviation of 3.9 Å over 124 aligned C_α atoms (sequence identity: 15%; PDB code 3HVA). Consequently, we refer to the N- and C-terminal domains of PelD_158-CT as the GAF and GGDEF domains, respectively.

The GGDEF Domain of PelD_158-CT Shows Significant Differences to Canonical GGDEF Domains

The GGDEF domain of PelD_158-CT is composed of a central four-stranded β-sheet, sandwiched between two pairs of α-helices (Fig. 3A) and is topologically similar to other GGDEF domains such as those noted above (supplemental Table S1). However, in PelD the GGDEF domain is ∼20–25 residues shorter than the canonical equivalents found in these other polypeptides and lacks some of the highly conserved secondary structure features (Fig. 3, A and B). For example, a comparison of the GGDEF domain of PelD_158-CT with that of PleD reveals that the canonical central β-sheet in the PleD GGDEF domain possesses one extra strand (βi3). Additionally, canonical GGDEF domains contain an additional helix (αi1), as well as two additional anti-parallel β-strands (βi1 and βi2) that are peripheral to the core structure (Fig. 3, B and C). PelD also lacks the catalytically requisite GGDEF sequence characteristic of active DGCs, and instead contains RNDEG (Arg³⁷⁶–Gly³⁸⁰) at the equivalent position (Fig. 3C). In active DGCs such as PleD, this motif is located on a loop between two β-strands and is involved in binding to GTP and metal ion. However, in PelD, both β-strands are extended and thus occlude the GTP binding pocket. Even though the two catalytically requisite metal ion-coordinating residues (Asp³⁷⁸ and Glu³⁷⁹) are conserved in PelD, they point away from the position of the GTP binding pocket and thus cannot contribute to either substrate binding or catalysis. The absence of both the requisite active site residues, as well as the additional secondary structural elements found in all active DGCs contribute to the lack of a competent active site in the GGDEF domain of PelD.

FIGURE 3.

GGDEF domains of PelD_158-CT and C. vibriodes PleD. A and B, a comparison of the GGDEF domain of (A) PelD_158-CT (shown in pink with secondary structure numbered as in Fig. 1) with that of (B) the active diguanylate cyclase C. vibriodes PleD (shown in gray), with the secondary structural elements that are found in most GGDEF domains but are lacking in PelD_158-CT, shown in magenta. The A-site is colored in cyan and the I-site is colored in green. In the PleD co-crystal structure, two molecules of the c-di-GMP product stack and occupy the autoinhibitory I-site. C, comparison of the secondary structural elements between PelD and PleD near the ligand binding sites. The I-site is highlighted in green, the A-site is colored in cyan, and the additional secondary structure elements in PleD (αi1, βi1, and βi2) are colored in magenta.

PelD_158-CT Binds to c-di-GMP Molecule through the I-site

In the PelD_158-CT-c-di-GMP co-crystal structure, one molecule of c-di-GMP molecule is bound to the GGDEF domain through the I-site, in an open, shallow pocket, with only one guanine ring (Gua-1) in the pocket and the other guanine ring (Gua-2) completely exposed to the solvent (Fig. 4A). The two adenine rings are parallel to each other and both vertical to the 12-membered macrocycle formed by two phosphodiester bonds between the two GMP molecules, engaging in a 2-fold symmetrical clip-shaped manner. This binding configuration of c-di-GMP is similar to the one observed in co-crystal structures of PleD (16, 19) and WspR (18, 20), and the PilZ domain VCA0042 from V. cholera (26), but distinct from that in FimX (32) and LapD (34), where the c-di-GMP ligand adopts an extended conformation and is inserted in a deep binding pocket.

FIGURE 4.

Cyclic di-GMP binding site in PelD_158-CT. A, stereo view of electron density maps calculated using Fourier coefficients F_obs − F_calc with phases derived from the final refined model of the 1.7-Å resolution co-crystal structure of Δ-loop PelD_158-CT with c-di-GMP. The map was calculated by omitting the coordinates of the cyclic di-GMP prior to one round of crystallographic refinement and is contoured at 2.3 σ (blue mesh) and 7 σ (red mesh). A ribbon diagram of the co-crystal structure is superimposed, and the ligand is shown as yellow ball-and-stick and I-site residues Arg³⁶⁷ and Asp³⁷⁰ (of the RXXD motif) are shown in green. B, stereo diagram showing the interactions between the cyclic di-GMP (in yellow) and PelD residues (in green) that are critical for ligand binding (as confirmed by biochemical analysis of site-specific variants; see text for further details). Note that Arg³⁶⁷ wedges between the two guanines, which helps to engage the ligand in a clip-like fashion. C and D, comparison of the interactions between cyclic di-GMP and the I-site in the co-crystal structures of C. vibriodes PelD (C) and P. aeruginosa WspR (D). Note that in both of these co-crystal structures, two molecules of c-di-GMP stack in order and engage their respective protein effectors in a manner analogous to the interactions provided by Arg³⁶⁷ in PelD.

The interactions of PelD_158-CT with c-di-GMP occur mainly through two segments: a loop composed of residues Arg³⁶⁷ through Asp³⁷⁰, and a β-loop-α segment encompassing Leu³⁸⁷ to Arg⁴⁰². Within these regions, Arg³⁶⁷, Asp³⁷⁰, and Arg⁴⁰² are responsible for the majority of the interactions with c-di-GMP (Fig. 4B). One of the carboxylate oxygens of Asp³⁷⁰ forms a hydrogen bond with N-2 of Gua-1, and the second forms a hydrogen bond with N-1 of Gua-1. The guanidinium nitrogen of Arg⁴⁰² forms hydrogen bonds with O-6 and N-7 of Gua-1. The guanidinium group of Arg³⁶⁷ inserts under the Gua-2 ring and forms hydrogen bonds with the diphosphate backbone, and is in electrostatic proximity with both purine rings (Fig. 4B). Importantly, Arg³⁶⁷ and Asp³⁷⁰ belong to the conserved RXXD motif and are equivalent to the I-sites in the GGDEF domain of many DGCs like PleD (19) and WspR (18), where the product c-di-GMP binds and allosterically inhibits DGC activity. In both of these DGCs, two c-di-GMP molecules are bound as intercalated dimers (Fig. 4, C and D). The interactions between the RXXD motif and one c-di-GMP are similar to those observed in PelD, but intercalated Gua rings stabilize each other in a manner similar to the interactions observed between Arg³⁶⁷ and c-di-GMP in the co-crystal structure of PelD (Fig. 4, C and D).

Binding Affinity of PelD Variants for c-di-GMP

To characterize the importance of the residues implicated in c-di-GMP binding in our co-crystal structure, we carried out mutagenesis studies and tested the binding affinity of wild type and variant PelD proteins to c-di-GMP. Residues Arg³⁶⁷, Asp³⁷⁰, and Arg⁴⁰², which form extensive hydrogen bonds with the ligand, and Tyr³⁹⁹, which provides a hydrophobic floor of the binding pocket, were individually mutated to Ala. Gly³⁹⁵, which also lines the floor of the binding pocket, was mutated to Pro to introduce steric hindrance within the pocket. All these mutants, along with the wild type protein, were purified with poly-His tag. The protein variants were immobilized on Ni-NTA-agarose beads, and their ability to retain [³²P]c-di-GMP was tested (Fig. 5A). R367A, Y399A, and R402A failed to retain c-di-GMP, whereas the binding ability of D370A was reduced by more than 60%. G395P bound c-di-GMP at a similar level to the wild type.

FIGURE 5.

Binding affinity of PelD_158-CT and variants to c-di-GMP. A, filter binding analysis measuring the relative affinity of wild-type and variant PelD_158-CT for [³²P]c-di-GMP. Experiments were conducted in triplicate and the black bars represent average values with associated error bars. K_d values (μm) obtained from isothermal titration calorimetric experiments are shown on top of the corresponding columns. B–E, isothermal titration calorimetric analysis of the binding of c-di-GMP to wild-type PelD_158-CT, D370A, G395P, and Δ-loop mutants, respectively. The binding isotherms for the titration experiment (top) and fitted curves based on single independent site binding model (bottom) are shown.

To more accurately quantitatively assess the c-di-GMP binding affinities of D370A and G395P variants, we conducted isothermal calorimetry titration (ITC) analysis (Fig. 5B) of these two mutants as well as on the wild type. Wild type PelD_158-CT binds c-di-GMP at a K_d of ∼0.5 μm (see “Experimental Procedures”). These results are comparable with previously reported values (42). The K_d for D370A and G395P PelD_158-CT are 28.6 and 3.4 μm, a 60- and 7-fold decrease in the affinity, respectively (Fig. 5, C and D). These results confirmed the importance of both Asp³⁷⁰ and Gly³⁹⁵ in c-di-GMP binding, which could not be determined directly from the [³²P]c-di-GMP binding assay.

As noted, the only significant difference between the structure of unliganded PelD_158-CT and the two c-di-GMP co-crystal structures is the lack of ordered electron density in the region spanning Glu²⁵¹ and Val²⁶³ in the latter. In both crystal forms of the ligand bound structure, residues in this loop from the unliganded would clash with a bound c-di-GMP from a symmetry related molecule. To demonstrate that the crystal contacts are not physiologically relevant, we generated a deletion variant in which 10 residues from this loop (Leu²⁴⁹–His²⁵⁸) were replaced with a Gly-Gly linker (Δ-loop). The binding affinity of this mutant was tested using both [³²P]c-di-GMP binding assay and ITC (Fig. 5, A and E). The Δ-loop variant showed a binding affinity similar to the wild type in the [³²P]c-di-GMP binding assay, and ITC yielded a K_d of 0.4 μm, which is similar to that of the wild type. These results demonstrate that this loop does not interfere with c-di-GMP binding and the symmetry-related interactions are a consequence of crystal packing. To further corroborate these results, we solved the co-crystal structure of the Δ-loop-di-c-GMP complex to 1.7-Å resolution and showed that the binding mode of the ligand is identical to that observed in the wild type structure.

GAF Domain of PelD_158-CT

The GAF domain is part of various multidomain proteins that participate in numerous signal transduction processes (62, 63). The PelD_158-CT GAF domain displays structural similarity to those of cAMP- or cGMP-specific PDE, including PDE2A (64, 65), 5A(61), 6C (66), and 10A (67), as well as the adenylyl cyclase CyaB2 (68) (supplemental Table S2). The GAF domains in PDEs and adenylyl cyclases have the capacity to bind cyclic nucleotide (cNMP), including cAMP and cGMP, which allosterically regulate the catalytic activity of these enzymes. Several PDEs are shown to bind cGMP or cAMP with nanomolar affinities (10–200 nm) (69–74) and the cyanobacterial adenylyl cyclases are shown to be activated exclusively by cAMP at submicromolar concentrations (68).

To determine whether the GAF domain of PelD_158-CT is functional in nucleotide binding, we carried out ITC analysis using either cAMP or cGMP. Calorimetric analysis demonstrated that PelD_158-CT does not bind cAMP with any appreciable affinity (data not shown), and only binds cGMP weakly, with a K_d of 221.7 μm that is several orders of magnitude higher than those reported previously for other GAF domains (Fig. 6A). Given the low intracellular concentration of cGMP in bacteria (below 100 nm) (75) and the experimentally determined high K_d value of PelD, the GAF-like domain of PelD_158-CT likely does not bind cyclic nucleotides.

FIGURE 6.

Calorimetric analysis of binding affinities of PelD_158-CT and the Δ-loop variant for cyclic GMP. A and B, binding isotherms and fitted curves from isothermal titration calorimetric analysis of the binding affinity of wild type PelD_158-CT (A) and Δ-loop for cyclic GMP (B).

A structure-based comparison of the PelD_158-CT GAF domain with those of nucleotide-activated GAF domains reveal several important features that may result in the low affinity of PelD_158-CT-GAF for cGMP (Fig. 7). First, in PelD_158-CT-GAF a loop that connects strand β2 and helix α3 travels through the potential cyclic nucleotide-binding pocket and leaves very little room to accommodate any ligands (Fig. 7A). Second, strand βi1 in the GAF domains of PDEs undergoes a significant movement toward the ligand pocket upon cGMP binding (61, 71). However, this strand is absent in the PelD_158-CT-GAF domain (Fig. 7, A and B). Last, several residues that are shown to be required for cyclic nucleotide binding are not conserved in the PelD_158-CT-GAF domain (Fig. 7C). For example, in PDE10A, residues Cys²⁸⁷, Phe³⁰⁴, Asp³⁰⁵, Phe³⁵², Thr³⁶⁴, and Gln³⁸³ are involved in cAMP binding (67), but none of these residues are conserved in the PelD_158-CT-GAF domain. In addition, a conserved NKFDE motif (64, 65, 76) is largely degenerate in the PelD_158-CT-GAF domain.

FIGURE 7.

GAF domains of PelD_158-CT and phosphodiesterase 10A. A and B, comparison of the GAF domains of (A) PelD_158-CT (shown in cyan with secondary structure numbered as in Fig. 1) with the (B) human PDE10A-cyclic GMP co-crystal structure (shown in brown with ligand colored in green ball-and-stick), with the secondary structural elements that are found in most GAF domains but are lacking in PelD_158-CT, shown in magenta. The binding pocket for cyclic GMP is partially occluded in PelD_158-CT by the loop that joins β2 and α3. C, comparison of secondary structural elements between PelD and PDE10A near the cyclic GMP binding sites. The I-site is highlighted in green, the A-site is colored in cyan, and the additional secondary structure elements in PleD (αi1 and βi1) are colored in magenta. Residues in PDE10A that involved in direct contact with the cyclic nucleotide are highlighted in yellow and the NKFDE motif is highlighted in green.

Of particular note, the region in the PelD_158-CT-GAF domain that is disordered in the c-di-GMP co-crystal structure (Glu²⁵¹–Val²⁶³) corresponds to a portion of the cyclic nucleotide binding pocket in the PDEs. The structure of cAMP-bound PDE10A GAF-B showed that the cAMP molecule is deeply buried in a pocket that uses the antiparallel β-sheet as the floor and a short helix (αi1, Asn³⁵³–Gly³⁶¹ in PDE10A) as the roof (67) (Fig. 7B). To test whether the region between Glu²⁵¹–Val²⁶³ plays a role in the inability of PelD_158-CT to bind cyclic nucleotides, we carried out ITC analysis on the Δ-loop mutant with cGMP (Fig. 6B). The result showed that deleting this region led to only a modest increase of the K_d (415.8 μm). Hence, this loop region does not play any role in the inability of PelD_158-CT GAF to bind ligands.

We also examined the possibility that PelD_158-CT-GAF may mediate homodimerization, a typical feature of GAF domains (74). Analytical size exclusion chromatographic analysis failed to identify any changes in the elution profile of PelD_158-CT in the presence of a high concentration of cGMP (Fig. 1). Although the dimerization interface and the domain orientation differ for many GAF domains, they all involve the two or three helices (α1, 2, and 4) located on the opposite side of the ligand binding pocket (64, 65, 67, 77). However, the corresponding helices in the PelD_158-CT-GAF domain, consisting of residues Gln¹⁵⁸–Glu¹⁷⁴ (α1), Leu²³¹–Gly²⁴⁰ (α2), and Glu²⁹⁰–Leu³⁰⁷ (α4), are oriented toward the GGDEF domain and partly buried in the domain interface (Fig. 2). Thus, the conformation of PelD observed in our structures is not competent to mediate dimerization, consistent with the results from our analytical size exclusion data (Fig. 1).

DISCUSSION

The GGDEF domain of PelD_158-CT represents a novel class of c-di-GMP receptor that binds c-di-GMP through a conserved I-site. GGDEF domains constitute the active sites of DGCs, such as PleD from C. vibriodes (19), WspR from P. aeruginosa (18), and XCC4471 from X. campestris (78), but are also found in enzymatically inactive c-di-GMP receptors, such as FimX (32). The GGDEF domains of PleD and WspR both have an active GGEEF motif (A-site) that binds to substrate and catalyzes the cyclization of two molecules of GTP into one molecule of cyclic di-GMP. Both PleD and WspR contain a conserved RXXD motif (I-site) located amino-terminal to the A-site, and this I-site binds to c-di-GMP and accounts for allosteric product inhibition (18, 19). In contrast, XCC4471 has a conserved A-site but a degenerate I-site, and (competitive) product inhibition is achieved by c-di-GMP binding directly to the A-site (78). Last, the GGDEF domain of FimX is degenerate at both the A-site and I-site, and lacks both DGC activity and c-di-GMP-binding capability (32). A similar feature is also observed in another c-di-GMP receptor LapD from P. fluorescens (34). Remarkably, distinct from all these domains, the GGDEF domain of PelD_158-CT has a degenerate A-site (³⁷⁵RNDEG) but a conserved I-site (³⁶⁷RGLD) (Fig. 8). This combination is also conserved in some other potential c-di-GMP receptors, for example, CdgG from V. cholera (43) and PopA from C. vibriodes (6) (Fig. 8). Thus the PelD_158-CT-GGDEF domain is representative of the class of c-di-GMP receptors that contain a degenerate GGDEF active site but a conserved I-site that can engage c-di-GMP.

FIGURE 8.

Multiple sequence alignment of the GGDEF domains discussed in this study (see text for further details). The I-site RXXD motif (highlighted in green) and the A-site GGDEF motif (in cyan) are shown in bold. The nomenclature of the secondary structural elements is based on the PelD structure.

Our structures showed that PelD binds to one molecule of c-di-GMP through the conserved I-site in the GGDEF domain. Mutations of the conserved residues in this site, Arg³⁶⁷ and Asp³⁷⁰, abolished the binding of PelD to c-di-GMP. Importantly, it was shown in previous in vivo studies (42) that such mutants failed to restore the ability of P. aeruginosa PA14 to form pellicles and bind Congo red. Combining these functional studies and our structural data, a strong correlation may be concluded between the binding of PelD to c-di-GMP and the formation of pellicles in vivo.

Although the GAF domain of PelD_158-CT is topologically similar to canonical GAF domains that can bind to cNMP molecules, our calorimetric studies show that PelD_158-CT does not bind either cAMP or cGMP with affinities that are physiologically meaningful. A number of GAF-containing proteins have tandem GAF domains and the two GAF domains are proposed to have distinct functions (74). For example, only one of the two GAF domains in PDE2A (GAF-B) binds a cyclic nucleotide, whereas the second domain (GAF-A) lacks ligand binding ability but is proposed to function as a dimerization locus (64, 65). cGMP binding to the GAF-B domain has been shown to allosterically increase the PDE activity at the catalytic domain (79). A second example is that cGMP binding to the GAF-A domain of PDE5A stimulates phosphorylation through a cGMP-dependent protein kinase, which in turn increases the catalytic activity of PDE5A and cGMP binding affinity of its GAF-A domain (80–82).

Our results showed that the GAF domain of PelD has only very weak affinity to cyclic nucleotides, thus it is unlikely regulated by these molecules. On the other hand, the location of the PelD_158-CT GAF domain between the C-terminal c-di-GMP-binding GGDEF domain and an N-terminal transmembrane region implies that it might serve as a c-di-GMP signal relay between these two domains. Sequence-based genome analysis demonstrates that the association of a GAF domain with a GGDEF domain occurs in many diguanylate cyclases (3). However, only a few of these proteins have been biochemically characterized. The diguanylate cyclases DgcA from Rhodobacter sphaeroides (83), and MSDGC-1 from Mycobacterium smegmatis (84) have been shown to largely lose their DGC activity after partial or complete removal of the GAF domain. However, the rationale behind this remains elusive, as a ligand-dependent function of the GAF domains in these proteins has not yet been established. In the case of DgcA, neither cAMP nor cGMP stimulated DGC activity (83).

To date, all PDEs that have been structurally characterized form dimers, although the functional significance of PDE dimerization remains unclear. The regulatory N-terminal region of these proteins, including the tandem GAF domains, are suggested to provide dimerization contacts as the isolated catalytic domains from PDE2A (85), PDE5A (86, 87), and PDE10A are monomeric (88). Unlike the GAF domains found in PDEs, the PelD_158-CT-GAF domain does not form a dimer, neither in the crystal nor in solution. The helices that provide the dimerization interface in other GAF domains are partially buried between the GAF and GGDEF domains of PelD_158-CT, and dimerization through the GAF domain would require significant conformational movements.

In most of the characterized receptors, c-di-GMP binding usually induces large conformational changes, and these changes have been proposed to propagate signal transduction. For example, binding of c-di-GMP to the PilZ domain of VCA0042 from V. cholerae induces a 123° rotation, resulting in a more compact overall structure and drastically different accessible protein surface, which is proposed to interact directly with downstream effectors (26). Another example is a membrane-bound effector LapD from P. fluorescens in which autoinhibitory interactions between the degenerate EAL and HAMP domains are relieved upon c-di-GMP binding to the EAL domain, promoting the interaction of the HAMP domain with other effectors (33, 34). Last, c-di-GMP binding to the I-site of two DGCs, PleD (16, 19) and WspR (18, 20), allosterically inhibits the enzyme activity by forcing the protein dimer into a nonproductive conformation.

Unlike these other c-di-GMP binding targets, c-di-GMP binding to PelD_158-CT does not result in any structural changes, either globally or local to the ligand-binding site. The relative orientation of the GAF and GGDEF domains are retained upon c-di-GMP binding, and PelD_158-CT remains as a monomer regardless of the absence and presence of the ligand. Similarly, the c-di-GMP binding EAL domain of FimX from P. aeruginosa is monomeric, and does not undergo any structural changes upon ligand binding (32). It was proposed that c-di-GMP binding might facilitate complex formation between the EAL domain and an unidentified binding partner (32). A similarly plausible model can also be considered for PelD, and this is further strengthened by the fact that c-di-GMP in the complex structure is significantly surface exposed. Binding of the ligand might either interrupt the interaction between the PelD GGDEF domain and an unidentified binding partner, or bridge the GGDEF domain to its binding partner. An example of protein-protein interactions triggered by c-di-GMP was reported recently on a PilZ domain-containing protein (36).

There are a large number of GGDEF domain-containing proteins in bacteria, which have diverse functions and thus account in part for the complexity of c-di-GMP signaling. Although studies have begun to investigate the hierarchy and specificity of GGDEF domain-mediated signaling pathways (89, 90), the knowledge of protein binding partners of GGDEF domains is still limited. The binding or dissociation of a protein partner to the GGDEF domain might cause a conformational change in the N-terminal transmembrane domain of PelD, using the GAF domain as a signal relay. A BLAST search (91) reveals that the N-terminal transmembrane domain of PelD (N terminus, Ala¹⁰⁴) is categorized as a domain of unknown function under the DUF4118 superfamily (Pfam 13493). Domains in this superfamily exist in a wide variety of bacterial signaling proteins, and these may play a role in signal transduction. Along with two other proteins in the Pel pathway, PelE and PelG (44), which are predicted to contain transmembrane helices, PelD could facilitate the export of carbohydrate-containing substances. Such export processes might require interactions between these transmembrane proteins and PelC, an outer-membrane lipoprotein that has been shown to facilitate exopolysaccharide transport (47). We are continuing additional biochemical, microbiological, and structural studies aimed at identifying the effectors downstream of PelD that mediate pellicle formation.