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Ykul structure solves bacterial signaling puzzle

PSI-SGKB [doi:10.1038/fa_psisgkb.2009.29]
Featured Article - July 2009
Short description: The crystal structure of the phosphodiesterase YkuI reveals how it binds cyclic di-GMP and might explain its activation.J. Biol. Chem. 284, 13174-13184 (2009)

YkuI homodimer with one subunit in gray, the other in yellow (EAL domain). Magenta (PAS domain) and light-blue for the linker helix. Cyclic-diGMP substrates is shown. PDB 2BAS, 2W27

The signaling molecule cyclic di-GMP is an important second messenger found in all bacteria. It helps bacteria adapt to changing environments by assisting the switch between movement and attachment, and has an important role in increasing virulence.

Cyclic di-GMP is a cyclic dinucleotide with a macrocycle composed of the ribose and a-phosphate moieties. Its levels are highly controlled by two types of enzyme that are responsible for its synthesis and degradation. Cyclic di-GMP is synthesized by proteins with diguanylate cyclase activity and these have a GGDEF motif; it is degraded by phosphodiesterases, which typically have an EAL motif. Because of its important role in virulence, the EAL domain was a priority target for structural genomics projects.

The first crystal structure of an EAL domain as part of a full-length molecule has now been solved. Minasov et al., in collaboration with PSI MCSG, solved the structure of YkuI from Bacillus subtilis. When this protein was first targeted by the MCSG, and the coordinates deposited in the Protein Data Bank, its catalytic function was not known. But a fruitful collaboration with Tilman Schirmer and colleagues at the University of Basel, who had previously worked on the GGDEF domain, has revealed the likely catalytic mechanism.

YkuI contains an EAL domain and a carboxy-terminal PAS-like domain connected by an alpha helix. The structures of the molecule in its native form and in complex with its second messenger cyclic di-GMP in the presence of calcium were solved using X-ray crystallography.

The EAL domain has a triose-phosphate isomerise (TIM)-barrel fold but it is unusual in that it has one anti-parallel beta strand; usually all TIM-barrel beta strands are parallel. The EAL signature motif, which in YukI is EVL, is located on beta strand 2, and the active site involves residues from this motif, glutamic acid (E)33 and leucine (L)35, which form part of a shallow groove at the C-terminal end. E33 is revealed in the structure to be involved in cation coordination.

Closer examination allowed Minasov et al. to propose a phosphodiesterase mechanism in which the divalent cation magnesium and the general base glutamic acid (E)209 activate a catalytic water molecule for nucleophilic in-line attack on the phosphorus. The EAL domain catalyzes the opening of the cyclic di-GMP macrocycle by hydrolyzing one of the O-3′-P ester bonds to produce a linear dinucleotide 5′-pGpG. It appears that catalysis proceeds through a penta-coordinated transition state and eventual O-3′-P bond cleavage.

However, the EAL domain is something of a puzzle because it lacks phosphodiesterase activity and yet has a canonical binding site for cyclic di-GMP. Interestingly, the YkuI active site is coupled with the subunit interface of the dimeric enzyme. EAL activation could proceed through a signal-dependent change in the arrangement of the subunits induced by the sensory PAS domain. Experiments are underway to test this model.

Maria Hodges


  1. G. Minasov et al. Crystal structures of YkuI and its complex with second messenger cyclic di-GMP suggest catalytic mechanism of phosphodiester bond cleavage by EAL domains.
    J. Biol. Chem. 284, 13174-13184 (2009). doi:10.1074/jbc.M808221200

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