Activation of the Diguanylate Cyclase PleD by Phosphorylation-mediated Dimerization

Ralf Paul,Paul Wassmann,Sören Abel,Urs Jenal,Heiko Heerklotz,Andreas Beck

doi:10.1074/jbc.m704702200

Abstract

Diguanylate cyclases (DGCs) are key enzymes of second messenger signaling in bacteria. Their activity is responsible for the condensation of two GTP molecules into the signaling compound cyclic di-GMP. Despite their importance and abundance in bacteria, catalytic and regulatory mechanisms of this class of enzymes are poorly understood. In particular, it is not clear if oligomerization is required for catalysis and if it represents a level for activity control. To address this question we perform in vitro and in vivo analysis of the Caulobacter crescentus diguanylate cyclase PleD. PleD is a member of the response regulator family with two N-terminal receiver domains and a C-terminal diguanylate cyclase output domain. PleD is activated by phosphorylation but the structural changes inflicted upon activation of PleD are unknown. We show that PleD can be specifically activated by beryllium fluoride in vitro, resulting in dimerization and c-di-GMP synthesis. Cross-linking and fractionation experiments demonstrated that the DGC activity of PleD is contained entirely within the dimer fraction, confirming that the dimer represents the enzymatically active state of PleD. In contrast to the catalytic activity, allosteric feedback regulation of PleD is not affected by the activation status of the protein, indicating that activation by dimerization and product inhibition represent independent layers of DGC control. Finally, we present evidence that dimerization also serves to sequester activated PleD to the differentiating Caulobacter cell pole, implicating protein oligomerization in spatial control and providing a molecular explanation for the coupling of PleD activation and subcellular localization.

Highlights

Lytic domains and the integrity of the catalytic site
Because stimulation of PleD diguanylate cyclases (DGCs) activity by BeF3 required the phosphoryl acceptor side Asp-53, the changes in structure and activity observed most likely reflect the activation mechanism normally evoked by phosphorylation
Because the constitutive active form PleD* still showed ϳ10-fold higher DGC activity and formed more stable dimers than BeF3-modified PleD, it is possible that PleD is only partially activated by BeF3 in the non-toxic concentration range used (Fig. 2)

Summary

Introduction

Lytic domains and the integrity of the catalytic site. Much less is known about catalysis and regulation mechanisms of the recently discovered family of diguanylate cyclases (DGCs). DGCs are responsible for the synthesis of cyclic diGMP, a ubiquitous second messenger involved in bacterial biofilm formation and persistence [2]. A simple model proposes that dimerization mediates an antiparallel arrangement of two DGC domains, each of which is loaded with one GTP substrate molecule. A simple mechanistic model for the activation of PleD proposes that phosphorylation at the conserved Asp-53 of Rec induces repacking of the Rec1/Rec interface This in turn would mediate dimer formation by isologous Rec1-Rec contacts across the interface and thereby facilitate reorientation and assembly of two C-terminal DGC domains [5]. Controlled dimerization modulates DGC activity but is employed to couple PleD activity to its subcellular sequestration This is the first demonstration that GGDEF protein dimers represent the active conformation of diguanylate cyclases and confirms that oligomerization can be used to regulate the activity of this abundant class of signaling proteins

Methods

Results

Conclusion