Insights into Biofilm Dispersal Regulation from the Crystal Structure of the PAS-GGDEF-EAL Region of RbdA from Pseudomonas aeruginosa

Abstract

RbdA is a positive regulator of biofilm dispersal of Pseudomonas aeruginosa Its cytoplasmic region (cRbdA) comprises an N-terminal Per-ARNT-Sim (PAS) domain followed by a diguanylate cyclase (GGDEF) domain and an EAL domain, whose phosphodiesterase activity is allosterically stimulated by GTP binding to the GGDEF domain. We report crystal structures of cRbdA and of two binary complexes: one with GTP/Mg²⁺ bound to the GGDEF active site and one with the EAL domain bound to the c-di-GMP substrate. These structures unveil a 2-fold symmetric dimer stabilized by a closely packed N-terminal PAS domain and a noncanonical EAL dimer. The autoinhibitory switch is formed by an α-helix (S-helix) immediately N-terminal to the GGDEF domain that interacts with the EAL dimerization helix (α_6-E) of the other EAL monomer and maintains the protein in a locked conformation. We propose that local conformational changes in cRbdA upon GTP binding lead to a structure with the PAS domain and S-helix shifted away from the GGDEF-EAL domains, as suggested by small-angle X-ray scattering (SAXS) experiments. Domain reorientation should be facilitated by the presence of an α-helical lever (H-helix) that tethers the GGDEF and EAL regions, allowing the EAL domain to rearrange into an active dimeric conformation.IMPORTANCE Biofilm formation by bacterial pathogens increases resistance to antibiotics. RbdA positively regulates biofilm dispersal of Pseudomonas aeruginosa The crystal structures of the cytoplasmic region of the RbdA protein presented here reveal that two evolutionarily conserved helices play an important role in regulating the activity of RbdA, with implications for other GGDEF-EAL dual domains that are abundant in the proteomes of several bacterial pathogens. Thus, this work may assist in the development of small molecules that promote bacterial biofilm dispersal.

Keywords: GGDEF domain; GGDEF-EAL domain; PAS domain; Pseudomonas aeruginosa; allosteric; allosteric control; biofilm; crystal structure; cyclic di-GMP; diguanylate cyclase; phosphodiesterase.

FIG 1

Activity of the RbdA protein. (A) Bacterial colony morphologies and biofilm phenotypes of P. aeruginosa PAO1 and the rbdA^{E585A,L586A,L587A} chromosomal mutant. (B) Visualization of biofilm formation on polystyrene tubes. (C) Quantitative comparison of biofilm formation levels. The data shown are means of triplicate values. Standard deviations are shown by error bars (*, P < 0.05; two-tailed t test). (D) Comparative growth curves of P. aeruginosa PAO1 and the rbdA^{E585A,L586A,L587A} chromosomal mutant (RbdA mutant). (E) Allosteric activation of PDE enzymatic activity of the cRbdA protein at 25°C in the presence of increasing concentrations of GMPPNP (ranging from 5 to 500 μM). (F) DGC enzymatic activity of RbdA. The PDE mutant bears the 585-ELL-587 → ALL single mutation, the PDE + I site double mutant has both 585-ELL-587 → ALL and 444-REGD-447 → AEGD mutations, and the A site mutant (453-GGDEF-457 → GGAAF) is the negative control. Reaction conditions are described in Materials and Methods.

FIG 2

Structure of the RbdA protein. (A) Domain organization of RbdA along its primary structure. Amino acid catalytic motifs and key structural features are indicated. (B) cRbdA monomer with each domain colored and labeled. The N and C termini are labeled. The PAS domain is colored green, the GGDEF domain cyan, the EAL domain red, and connecting segments crucial for protein dynamics (S-helix and H-helix) light blue and yellow, respectively. (C) cRbdA dimer. The position of the single dyad that runs through the crystallographic dimer is indicated. The region from positions 1 to 232 (not included in the cRbdA construct), leading to the periplasmic membrane, is represented schematically with dashed lines. (D) Magnified view of interactions between the S-helix and the dimerization helix α_6-E′ from the EAL′ domain. (E) The cRbdA dimer is shown from a perpendicular direction, using the same color code.

FIG 3

Structure of the PAS domain of RbdA. (A) Sequence comparisons of the PAS domains of RbdA of P. aeruginosa (accession number AAG04250.1), the two-component system sensor histidine kinase NRII of V. parahaemolyticus (accession number WP_049877818.1), the Nostoc histidine kinase NSHisKinase (accession number WP_010994604.1), the Geobacillus thermodenitrificans histidine kinase GtHiskinase (accession number WP_029761733.1), the oxygen sensor protein EcDosP of E. coli (accession number P76129.4), and FixL of Bradyrhizobium japonicum (accession number CAA40143.1). The histidine residue involved in heme chelation by FixL, which is not conserved in RbdA, is indicated with an arrow. Hydrophobic residues that form a possible ligand binding site of the PAS domain of cRbdA are indicated with stars. Secondary structure elements of the PAS domain of RbdA are displayed above the alignment. (B) Topology of the PAS domain. β-Strands are shown as purple arrows and α-helices as blue tubes, with residues at the extremities of each numbered. (C) Closeup view of the hydrophobic core housed inside the β-barrel of the PAS domain from RbdA. Hydrophobic residues forming a putative ligand binding site are depicted as white sticks and labeled.

FIG 4

Structure-based amino acid sequence alignment of cRbdA and homologous domains from other GGDEF-EAL domain-containing bacterial proteins. Secondary structure elements are based on assignments for the cRbdA protein (this work). Strictly conserved residues are highlighted in red. The catalytic motifs of the DGC domain (A site or “GGDEF” motif and I site) and the PDE domain (“ELL” motif) are highlighted in yellow boxes. Key elements of the structure are indicated, including the H-helix (hinge-helix) and S-helix (present in RbdA and LapD but not in MorA; no structure is available for the dual domains of DipA, NbdA, CC3396, and Tbd1265). Accession numbers are as follows: Tbd1265, WP_011311777.1; LapD, WP_011331847.1; MorA, WP_073670889.1; FimX, WP_033999828.1; CC3396, WP_010921225.1; and NBDA, WP_048305406.1. Blue stars indicate GTP-interacting residues (GGDEF domain) and red stars c-di-GMP binding residues (EAL domain).

FIG 5

Interaction between the GGDEF domain of cRbdA and the GTP allosteric activator of PDE activity. (A) Overall view of the cRbdA monomer, highlighting the location of the bound GTP molecule within the GGDEF domain. (B and D) Closeup views showing the atomic interactions between GTP (sticks) and residues of the A site of the GGDEF domain (blue) (B) and the Mg (green sphere) coordination shell (D). A difference electron density map with F_o-F_c Fourier coefficients, with the GTP moiety omitted from the calculation, is displayed at a level of 3σ. (C) Conformational changes in RbdA triggered by GTP binding. An overlay of the native (blue) and GTP-bound (white) cRbdA structures is shown. Residues near the phosphate tail of GTP, including in helix α_2-G, and undergoing large displacements between the GTP-free and bound states are shown as sticks and labeled. A list of atomic interactions between the GGDEF domain and GTP is given in Table S2 in the supplemental material, and contacts between the EAL domain of cRbdA and c-di-GMP are shown in Table S3.

FIG 6

Comparison of three dual domain-containing proteins, highlighting the range of relative orientations permitted by pivoting around the hinge helix. The EAL domain has the same orientation in panels A to C. (A) View of the GGDEF-EAL dual domain from RbdA, highlighting the hinge helix (magenta) that connects the EAL domain to the GGDEF domain and the S-helix (magenta) that contacts helix α_6-E′ in trans from the partner EAL′ domain (shaded). The c-di-GMP moiety bound to the EAL domain is shown as green sticks (dashed circle). A salt bridge between Asp518 (GGDEF domain) and Arg609 (EAL domain) is also represented. (B) View of LapD (PDB code 3PJT). The S-helix contacts helix α_6-E in cis from the same EAL monomer, resulting in an autoinhibited state. (C) View of MorA (PDB code 4LYK), which also possesses the H-helix, but with a flexible N-terminal segment (no visible S-helix in the available crystal structure). The Lys1076-Glu1213 salt bridge between the GGDEF and EAL domains is shown as sticks. (D) Difference electron density map with F_o-F_c Fourier coefficients, with c-di-GMP (sticks) omitted from the calculation. The inset shows a magnified view of the interactions established between the EAL domain of RbdA and c-di-GMP. Hydrogen bonds are shown as black dashed lines and van der Waals and stacking interactions as purple dashed lines.

FIG 7

Solution X-ray scattering studies of cRbdA with ligands. (A) Experimental scattering patterns (○) and calculated scattering profiles (lines) of crystal structure dimers of cRbdA alone (black) and in complex with GTP and c-di-GMP (cdG), GMP and cdG, GTP, and cdG. (Inset) Guinier plots show linearity at all concentrations used, indicating no aggregation. The scattering profiles were offset, for clarity, by applying arbitrary scale factors. (B) Overlapping of pair-distance distribution function P(r) of cRbdA and its ligand complexes. cRbdA complexes with GTP and cdG, GMP and cdG, and GTP have similar profiles; however, cRbdA with cdG has an extended tail. (C) Normalized Kratky plot of cRbdA (black) compared to its complexes and the compact globular lysozyme (gray), with a peak (gray dashed line) representing the theoretical peak and assuming an ideal Guinier region of a globular particle. The scattering pattern of cRbdA and its complexes exhibits a broad bell-shaped profile shifted toward the right with respect to standard globular proteins, indicating the presence of motion in the protein. (D) Averaged and filtered envelope (gray) from 20 independent ab initio reconstructions created by use of DAMMIF superimposed onto a cartoon representation of the CORAL model with an extended conformation for cRbdA, with the PAS domain in blue, the GGDEF domain in purple, and the EAL domain in brown. The second subunit is colored in lighter shades. The linker regions modeled between the domains are shown as green and cyan spheres for the subunits. Front (left) and side (right) views are displayed. (E) Averaged and filtered ab initio low-resolution shape of cRbdA in the presence of GTP and c-di-GMP (green) superimposed on the compact conformation generated from CORAL. (F) Fitting of the CORAL model (red lines) to the experimental scattering patterns (○) for cRbdA alone (black) and with GTP and c-di-GMP (green). The theoretical scattering curve for the mixture of 75% compact and 25% extended conformations of cRbdA dimers (black line) calculated using the OLIGOMER program fits the experimental scattering pattern of cRbdA with cdG (orange), with a χ² value of 0.8.

FIG 8

Proposed allosteric mechanism regulating the PDE activity of RbdA. The left panel schematically represents the putative autoinhibited state observed in the cRbdA crystal structure. This resting state is stabilized by interactions between helix α₆ from the EAL domain and the S′-helix immediately N-terminal to the GGDEF′ domain (and also between helix α_6-E′ and the S-helix in the other monomer, via the dyad). These interactions lock both EAL domains of the RbdA dimer in a noncanonical configuration. Upon signal detection by either the putative periplasmic sensor domain (triangle) or GTP binding to the A sites of the GGDEF domains, local conformational changes (Fig. 5D) propagate throughout the protein and lead to the release of the autoinhibitory interactions between helix α_6-E and the S′-helix. This is proposed to lead to the rearrangement of both EAL domains into a canonical dimer capable of hydrolyzing the incoming c-di-GMP substrate and to the observed higher PDE activity (Fig. 1D) conducive to biofilm dispersal (right panel).