Structural Insight into the Mechanism of c-di-GMP Hydrolysis by EAL Domain Phosphodiesterases

Abstract

Cyclic diguanylate (c-di-GMP) is a ubiquitous second messenger regulating diverse cellular functions including motility, biofilm formation, cell cycle progression and virulence in bacteria. In the cell, degradation of c-di-GMP is catalyzed by highly specific EAL domain phosphodiesterases whose catalytic mechanism is still unclear. Here, we purified 13 EAL domain proteins from various organisms and demonstrated that their catalytic activity is associated with the presence of 10 conserved EAL domain residues. The crystal structure of the TDB1265 EAL domain was determined in a free state (1.8 Å) and in complex with c-di-GMP (2.35 Å) and unveiled the role of the conserved residues in substrate binding and catalysis. The structure revealed the presence of two metal ions directly coordinated by six conserved residues, two oxygens of the c-di-GMP phosphate, and potential catalytic water molecule. Our results support a two-metal-ion catalytic mechanism of c-di-GMP hydrolysis by EAL domain phosphodiesterases.

Inherent Regulation of EAL Domain-catalyzed Hydrolysis of Second Messenger Cyclic di-GMP

“…Correspondingly, Barends et al (12) proposed a two-metal assisted catalytic mechanism with both cations activating the water and defining the precise relative orientation of the water (W) and the proximal phosphate moiety. The same constellation was observed later for TdEAL⅐cdG⅐Mg 2ϩ (13) and can thus be predicted to represent the competent metal constellation for YahA-EAL as well.…”

“…Most probably, not this modest difference in cdG affinity, but the observed drastic change in k cat , with only the dimer being catalytically active, is crucial for the regulation of the enzyme in vivo. The inactive state of the monomer may be largely due to a compromised M2 site that prevents binding of the second Mg 2ϩ ion, which has been shown to be catalytically indispensable in RocR (10) and in TdEAL (13). Indeed, we found for YahA-EAL that the D263N mutation, which affects a residue that coordinates M2, renders the enzyme inactive, although it does not compromise substrate affinity.…”

“…The structures reported here show YahA-EAL in various ligand-dependent conformational states and qualify it as a prototypic catalytic EAL domain with dimerization propensity. Intriguingly, apart from the canonical dimeric arrangement (11)(12)(13) found for YahA-EAL⅐Mg 2ϩ , the ternary substrate complex YahA-EAL⅐cdG⅐Ca 2ϩ shows a different subunit organization, although utilizing most of the canonical interface. Whether one or both dimeric configurations can be formed in full-length YahA must await structure investigation.…”

“…Fig. 4A shows that at the C-terminal end of the central ␤-barrel a magnesium cation (M1) is coordinated by Glu-141 (of the EAL motif), Asn-200, Asp-262, and Glu-232, analogous to the situation in the binary TdEAL⅐Mg 2ϩ complex (13). Because the metal site is fully occupied and the structure was obtained at a physiological magnesium concentration (2 mM) (33), it is likely that in vivo YahA-EAL is constitutively complexed with magnesium.…”

“…Finally, based on the substrate⅐Mn 2ϩ complex structure of the active BLUF-EAL phosphodiesterase BlrP1 from Klebsiella pneumoniae, a two-metal assisted catalytic mechanism was proposed with each of the two manganese ions coordinating the hydrolytic water molecule positioned inline with the scissile phosphodiester bond (12). This was later confirmed by the TdEAL⅐Mg 2ϩ complex structure (13). The substrate complex structure of BlrP1 in the presence of calcium revealed a somewhat distinct coordination geometry with only one of the ions coordinated by the hydrolytic water (12) and, thus, rationalized the observation that calcium is inhibiting the enzyme (8).…”