Structural insights into the regulatory mechanism of the response regulator RocR from Pseudomonas aeruginosa in cyclic Di-GMP signaling

Abstract

The nucleotide messenger cyclic di-GMP (c-di-GMP) plays a central role in the regulation of motility, virulence, and biofilm formation in many pathogenic bacteria. EAL domain-containing phosphodiesterases are the major signaling proteins responsible for the degradation of c-di-GMP and maintenance of its cellular level. We determined the crystal structure of a single mutant (R286W) of the response regulator RocR from Pseudomonas aeruginosa to show that RocR exhibits a highly unusual tetrameric structure arranged around a single dyad, with the four subunits adopting two distinctly different conformations. Subunits A and B adopt a conformation with the REC domain located above the c-di-GMP binding pocket, whereas subunits C and D adopt an open conformation with the REC domain swung to the side of the EAL domain. Remarkably, the access to the substrate-binding pockets of the EAL domains of the open subunits C and D are blocked in trans by the REC domains of subunits A and B, indicating that only two of the four active sites are engaged in the degradation of c-di-GMP. In conjunction with biochemical and biophysical data, we propose that the structural changes within the REC domains triggered by the phosphorylation are transmitted to the EAL domain active sites through a pathway that traverses the dimerization interfaces composed of a conserved regulatory loop and the neighboring motifs. This exquisite mechanism reinforces the crucial role of the regulatory loop and suggests that similar regulatory mechanisms may be operational in many EAL domain proteins, considering the preservation of the dimerization interface and the spatial arrangement of the regulatory domains.

Fig 1

Structure of the RocR R286W mutant protein. (A) Domain organization of RocR. (B) The RocR tetramer is shown from three angles separated by 90°. Its subunits are colored green (subunit A), cyan (subunit B), magenta (subunit C), and yellow (subunit D). The position of the single dyad that runs through the crystallographic tetramer is indicated in each panel. The region involved in forming intermolecular EAL-EAL contacts is colored in white, and a magnified view of this interaction is shown in panel C. (C) Schematic depiction of the RocR tetramer highlighting the open conformation of its C and D subunits, which form a saddle-like structure into which the REC domains of the closed subunits A and B are inserted, forming the core of the tetramer. The EAL active sites are represented as three black triangles. EAL active sites of subunits C and D are concealed (crossed circle) in trans by the REC domains located at the center of the structure. The interdomain linker is depicted as a line (dashed line if it is partially disordered in the crystal structure). (D) Enzymatic activity of the wild-type and R286W mutant RocR. (Left panel) HPLC analysis of c-di-GMP degradation in the presence of no enzyme and R286W mutant at 25 and 60°C. (Center and right panels) HPLC analysis of c-di-GMP degradation for wild type and R286W mutant across temperatures. The mutant exhibits diminished activity that is recovered at elevated temperatures. Reaction conditions are described in the experimental section.

Fig 2

(A and B) Closed (subunits A and B, in cyan, in panel A) and open conformations (subunits C and D, in magenta, in panel B) that constitute the RocR tetramer. Residues from the REC and EAL active sites are represented as yellow sticks. (C) Superposition of the two conformers based on their EAL domains. The residual rotation (angle needed to bring their respective REC domains into coincidence) is indicated.

Fig 3

SAXS analysis of the conformation of RocR in solution. (A) Experimental scattering from the wild-type protein (1, black dots), calculated scattering from the closed tetramer observed in the crystal (2, blue line); calculated scattering from the putative half-open and open RocR tetramers (3 and 4, red and green dashed lines). The logarithm of the scattering intensity is plotted as a function of momentum transfer s = 4π sin(θ)/λ, where θ is the scattering angle and λ is the X-ray wavelength. (B) ab initio low-resolution shape reconstructed from the wild-type RocR data (gray mesh) superimposed on the structure of the crystallographic tetramer using SUPCOMB (21).

Fig 4

Observed conformations in the individual domains of RocR. (A) Superposition of REC_B (cyan, side chains shown as white sticks) with REC_C (yellow, side chains shown as black sticks). Phe¹⁰⁵, the “switch residue” located on strand β5′, adopts the same rotamer in a buried conformation. The β3′-α3′ and β4′-α4′ loop regions display different conformations between the open (subunit C) and closed conformers (subunit B). (B) Close-up view of the EAL domain of RocR using a diagram representation (helices shown as cyan ribbons and β-strands as magenta arrows). Secondary structure elements of the additional lobe that protrudes over the TIM barrel are labeled. EAL active-site residues and loop 6 are represented as sticks, and the Mg²⁺ ion is represented as a green sphere. A surface representation of the EAL domain is overlaid. (C) Magnified view of the EAL active-site region with electron density displayed at a level of 3 σ calculated using Fourier coefficients Fo-Fc, where the metal and one bound water were omitted from the phase calculation. (D) Close-up view of the entrance to EAL active sites in the open (yellow) and closed (cyan) conformers. The catalytically important Loop 6 displays two distinct conformations, with Glu²⁹⁶ in two rotameric conformations and Tyr³⁰¹ flipped toward opposite directions.

Fig 5

Inhibition of c-di-GMP binding in trans by REC domains. (A) In the observed crystal structure, binding of c-di-GMP to the open RocR conformer (subunit C, magenta) is prevented due to physical occlusion by the REC domain of a closed conformer (subunit B, blue). (B) The in trans REC-EAL interaction is mediated mainly through the interactions between hydrophobic residues preceding REC helix α4′ and an EAL hydrophobic pocket (C) and interactions between Y195 of the EAL lobe and residues projecting from helix α5′ (D).

Fig 6

Proposed regulatory mechanism. Only the relevant domains from one half of the symmetric protein are shown for clarity. Phosphorylation of Asp⁵⁶ of the REC_C/D domain induces local conformational changes in the REC domains (see the text). These structural changes are transmitted to the adjacent EAL domain through the direct contact that exist between the terminal residues of helix α5′ (Ile⁸⁹ and Leu⁹⁰, depicted as blue spheres) and residues Phe³¹⁰ and Pro³¹¹ (depicted as yellow spheres) situated immediately downstream of loop 6 of EAL_D/C. The signal is further transmitted down to the active site of EAL_B/A through loop 6 that constitute EAL_D/C-EAL_B/A dimer interface. Based on the H/D exchange-coupled mass spectrometry results reported previously (31), the peptides of RocR that exhibit significant conformational changes upon D56N mutation are colored in blue (REC) and red (EAL), respectively. W286 (green spheres) is located at the REC-EAL interface and may reduce the R286W mutant's activity by disrupting the signal propagation. See also Fig. S6 in the supplemental material. (Inset) Schematic view of the signal transmission pathway across the RocR tetramer. The same color code is adopted.