Inherent regulation of EAL domain-catalyzed hydrolysis of second messenger cyclic di-GMP

Abstract

The universal second messenger cyclic di-GMP (cdG) is involved in the regulation of a diverse range of cellular processes in bacteria. The intracellular concentration of the dinucleotide is determined by the opposing actions of diguanylate cyclases and cdG-specific phosphodiesterases (PDEs). Whereas most PDEs have accessory domains that are involved in the regulation of their activity, the regulatory mechanism of this class of enzymes has remained unclear. Here, we use biophysical and functional analyses to show that the isolated EAL domain of a PDE from Escherichia coli (YahA) is in a fast thermodynamic monomer-dimer equilibrium, and that the domain is active only in its dimeric state. Furthermore, our data indicate thermodynamic coupling between substrate binding and EAL dimerization with the dimerization affinity being increased about 100-fold upon substrate binding. Crystal structures of the YahA-EAL domain determined under various conditions (apo, Mg(2+), cdG·Ca(2+) complex) confirm structural coupling between the dimer interface and the catalytic center. The built-in regulatory properties of the EAL domain probably facilitate its modular, functional combination with the diverse repertoire of accessory domains.

Keywords: Allosteric Regulation; Bacterial Signal Transduction; Chromatography; Crystal Structure; Phosphodiesterases; Second Messenger; c-di-GMP Signaling.

FIGURE 1.

Crystal structures of the YahA-EAL domain. Schematic representation with secondary structure elements and chain termini labeled. The β5-α5 loop is highlighted in magenta. A, structure of the monomer of YahA-EAL in complex with Mg²⁺ (M1). Mutation site Ser-298 is shown in full and labeled. B, structure of the monomer of YahA-EAL in complex with substrate (cdG) and Ca²⁺ (M1 and M2). C, superimposition of YahA-EAL-apo (brown) with YahA-EAL·Mg²⁺ (steel blue) and YahA-EAL·cdG·Ca²⁺ (light green). D, subunit arrangement in YahA-EAL·Mg²⁺ (canonical EAL dimer). The asymmetric unit contains two dimers, both are virtually identical. E, subunit arrangement in YahA-EAL·cdG·Ca²⁺ (closed EAL dimer). The asymmetric unit contains one dimer with virtually identical subunit structure. In panels D and E the same color code is used as in panels A and B, but with symmetry mates shown in gray.

FIGURE 2.

Sequence of the EAL domain of YahA from E. coli (UNIPROT accession name P21514) with experimentally determined secondary structural elements and HMM logo based on a non-redundant set of 62 EAL sequences. Important residues are labeled with their number. cdG binding residues are shown in red. Metal coordinating residues are represented in boxes. The anchoring glutamate is shown in blue, whereas the general base lysine is in green. Other important residues are shown in black.

FIGURE 3.

YahA-EAL dimer interfaces. A and C, canonical YahA-EAL/Mg²⁺ dimer. B and D, closed YahA-EAL·cdG·Ca²⁺ dimer. For each structure, two orthogonal views (front and top) are shown. Hydrogen bonds are represented as broken brown lines, divalent cations as spheres.

FIGURE 4.

EAL active site structures in the absence and presence of cdG substrate. Divalent cations are colored in green (Mg²⁺), yellow (Ca²⁺), or magenta (Mn²⁺). A, YahA-EAL·Mg²⁺, B, YahA-EAL·cdG·Ca²⁺, and C, BlrP1·cdG/Mn²⁺ (PBD code 3GG0) (12). Water molecules are represented as red spheres, cation coordination bonds by black broken lines, and loop β5-α5 is highlighted in magenta. In A, Asp-263 is H-bonded (orange broken lines) to surrounding carboxylic side chains. In B and C, the hydrolytic water (W) is in-line with the scissile O3′-P bond of the cdG substrate (shown in full with cyan carbons). Note that the location of the “anchoring” glutamate (Glu-235 in YahA, Glu-275 in BlrP1) is distinct in the binary and ternary complexes. D, stereoview of the superposition of YahA-EAL in complex with Mg²⁺ (ribbon and carbon atoms in gray) and cdG·Ca²⁺ (ribbon and carbons atom in light green).

FIGURE 5.

Loop β4-α4, β5-α5, and loop 290 conformations of apo YahA-EAL (A), YahA-EAL·Mg²⁺ (B), and YahA-EAL·cdG·Ca²⁺ (C). The distinct interaction patterns between the anchoring glutamate (Glu-235) and loop β5-α5 are represented by broken lines in brown.

FIGURE 6.

EAL/substrate and EAL/EAL affinity. Concentration of divalent cations was 2 mm, where applicable. A, titration of cdG to YahA-EAL wild-type (squares) and S298W mutant (diamonds) resulting in displacement of fl-cdG (60 nm) from the protein as measured by microscale thermophoresis. Data have been acquired in the absence (black) or presence of Ca²⁺ (red). The data were fitted to a ligand competition model (solid lines) (28) yielding the cdG dissociation constants given in Table 2. For the wild-type, fit curves for K_d = 0.1 (solid line) and 1.0 nm (broken line) are shown. B, SEC-MALS chromatograms (loading concentration 180 μm) for Yaha-EAL wild-type (top) and the S298W mutant (bottom). The proteins were analyzed in the absence of divalent cations (black), in the presence of Ca²⁺ (orange), and in the presence of Ca²⁺ and 540 μm cdG (red). Continuous lines represent the dRI signal (left axis), broken lines the MALS derived apparent mass values (right axis). C, compilation of MALS data (weight-average molecular mass) acquired at various loading concentrations for wild-type YahA-EAL. Data shown are for the apo protein (black), for the binary complexes with Mg²⁺ (light green) or Ca²⁺ (orange), and for the ternary complexes (molar cdG:protein ratio, 3:1) with cdG/Mg²⁺ (green) or cdG·Ca²⁺ (red). The data were fitted with a dynamic monomer-dimer model in fast exchange with the values of the pure species set to their nominal values (horizontal lines). The derived K_d values are listed in Table 3.

FIGURE 7.

AUC-SE data for YahA-EAL in the presence of 2 mm CaCl₂. Protein and substrate concentrations are indicated. For each sample, data are shown for three speeds (9,700, 16,500, and 28,000 rpm). All six runs were fitted globally to a dynamic monomer-dimer self-association model with the monomer mass set to 31 kDa. The fit yielded a dimerization K_d of 0.5 μm (Table 3).

FIGURE 8.

Catalytic activity of the YahA-EAL domain. A, FPLC chromatograms showing time-dependent conversion of cdG to pGpG. CdG (200 μm) was incubated for the indicated time spans with 1 μm YahA-EAL. B, progress curves of pGpG production with the initial cdG substrate concentrations indicated. Data were fitted to a simple Michaelis-Menten kinetics model. C, specific activity v_init/[YahA-EAL] as a function of [YahA-EAL] concentration, acquired at saturating substrate concentration. For the wild-type protein (green), data points are shown from two separate experiments. The data were fitted (continuous line) to a simple monomer-dimer equilibrium model and indicate that the enzyme is inactive as monomer. The dimer interface mutant S298W (blue) is virtually inactive.

FIGURE 9.

A, thermodynamic scheme showing five YahA-EAL states that are in fast thermodynamic equilibrium: monomeric YahA-EAL in the uncomplexed (0) and cdG (ring symbol) complexed (1) state as well as dimeric YahA-EAL in the uncomplexed (00) and singly (10) or doubly (11) occupied dimeric state. Magnesium ions in sites M1 and M2 are indicated. The second-order ligand (K_s1, K_s10, and K_s11) and dimer (K₀₀, K₁₁) association constants and turnover numbers (k_cat,10 and k_cat,11) are indicated. Assuming no cooperativity, K_s10 = K_s11 and k_cat,10 = k_cat,11. Monomeric YahA-EAL is inactive, due to the postulated absence of a divalent cation in site M2. Dimeric YahA-EAL hydrolyzes cdG to yield the linear pGpG dinucleotide (open ring symbol). B, generic regulatory mechanism for a full-length EAL phosphodiesterase with associated regulatory domain (R). Wavey lines indicate interfaces that undergo structural changes. The protein is monomeric (top), but dimerizes via the R domains upon signal perception (bottom left). This promotes dimerization of the EAL domains, due to the increase of their local concentration (bottom left). Finally, structural changes in the EAL/EAL interface induced by dimerization are coupled to structural changes in the active site that would affect substrate affinity and/or catalytic activity.