Polymorphism of the Signaling Molecule c-di-GMP

Zhang, Zhaoying; Kim, Se‐Ho; Gaffney, Barbara L.; Jones, R. A. C.

doi:10.1021/ja0613714

Cited by 69 publications

(133 citation statements)

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“…This may be related to the varying degree of dimerization of c-di-GMP in solution (20). Because this effect is limited to the first few injections and does not interfere with the sigmoidal part of the binding curve, we have eliminated the first three data points from the fit (Fig.…”

Section: Activation Of the Pled Diguanylate Cyclase Does Not Interfermentioning

confidence: 99%

Activation of the Diguanylate Cyclase PleD by Phosphorylation-mediated Dimerization

Paul

Abel

Wassmann

et al. 2007

Journal of Biological Chemistry

175

161

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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.

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Section: Activation Of the Pled Diguanylate Cyclase Does Not Interfermentioning

confidence: 99%

Activation of the Diguanylate Cyclase PleD by Phosphorylation-mediated Dimerization

Paul

Abel

Wassmann

et al. 2007

Journal of Biological Chemistry

175

161

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“…There is, however, no strict correlation; for example, in P. aeruginosa PA14, a mutation of the gene encoding the GGEEF protein PA3343, which is active in cyclic di-GMP synthesis, leads to hyperbiofilm formation, whereas the overexpression of PA2870 and PA3343, which both lead to increases in the cellular level of cyclic di-GMP, has no effect on biofilm formation in the wild type (35). An added complexity is that the nucleotide adopts different but related structures with different counterions and under different concentrations (65). A bimolecular intercalated structure found at low concentrations with Na ϩ and Mg 2ϩ is also the form in which cyclic di-GMP binds to PleD and may be the active form for signaling.…”

Section: The Cellular Organization Of Cyclic Di-gmp Signaling Systemsmentioning

confidence: 99%

Cyclic Di-GMP Signaling in Bacteria: Recent Advances and New Puzzles

et al. 2006

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Cyclic di-GMP [bis-(3Ј-5Ј)-cyclic di-GMP] (Fig. 1) is a novel second messenger in bacteria that was first described as an allosteric activator of cellulose synthase in Gluconacetobacter xylinus (49). It is now established that this nucleotide is almost ubiquitous in bacteria, where it regulates a range of functions including developmental transitions, aggregative behavior, adhesion, biofilm formation, and the virulence of animal and plant pathogens (for reviews, see references 15, 18, 30, 31, 47, and 48). The level of cyclic di-GMP in bacterial cells is influenced by both synthesis and degradation. The GGDEF protein domain synthesizes cyclic di-GMP, whereas the EAL and HD-GYP domains are involved in cyclic di-GMP hydrolysis (43,50,51,53,54,58). Bacterial genomes encode a number of proteins with GGDEF, EAL, and HD-GYP domains; e.g., in Pseudomonas aeruginosa, there are 40 such proteins. The majority of these proteins contain additional signal input domains. These signaling systems are presumed to use cyclic di-GMP as a second messenger to link the sensing of specific environmental cues to appropriate alterations in bacterial physiology and/or gene expression. Some details about the operation and organization of cyclic di-GMP signaling systems are emerging. Nevertheless, many questions remain to be answered, and new puzzles have arisen as a result of experimental investigations. Here, we review the recent advances and highlight the considerable gaps in our understanding of cyclic di-GMP signaling. In particular, we discuss (i) the protein domains involved in cyclic di-GMP turnover, highlighting HD-GYP, recently described as a novel cyclic di-GMP phosphodiesterase; (ii) signals that are transduced through cyclic di-GMP signaling; (iii) the role of cyclic di-GMP signaling in bacterial virulence; (iv) recent findings addressing the organization of, and interplay between, different cyclic di-GMP signaling systems; (v) the concept of localized pools of cyclic di-GMP within the cell; and (vi) the mechanisms by which cyclic di-GMP exerts its effects on diverse cellular functions.

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“…BcsA's C-terminal PilZ domain binds an intercalated c-di-GMP dimer (Zhang et al, 2006) (Fig. 24 and Fig.…”

Section: Bcsa Binds a C-di-gmp Dimer On The β-Barrel Surfacementioning

confidence: 99%

“…C-di-GMP monomers and dimers (Zhang et al, 2006;Gentner et al, 2012) are both recognized by effector proteins via PilZ domains, first identified as regulatory components of cell motility (Christen et al, 2007), which comprise an "RxxxR" motif in a flexible linker region followed by a β-sheet or β-barrel that contains a "DxSxxG" motif (Amikam and Galperin, 2006). Both sequence motifs have been shown to interact with cdi-GMP in structures of isolated PilZ domains (Benach et al, 2007;Ko et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

“…In many cases, biofilm formation occurs in response to an elevated cytosolic C-di-GMP activates the synthesis of bacterial cellulose (Ross et al, 1987;Römling et al, 2013), an extracellular polysaccharide often found in biofilms (Zogaj et al, 2003). C-di-GMP monomers and dimers (Zhang et al, 2006;Gentner et al, 2012) are both recognized by effector proteins via PilZ domains, first identified as regulatory components of cell motility (Christen et al, 2007), which comprise an "RxxxR" motif in a flexible linker region followed by a β-sheet or β-barrel that contains a "DxSxxG" motif 56 (Amikam and Galperin, 2006). Both sequence motifs have been shown to interact with cdi-GMP in structures of isolated PilZ domains (Benach et al, 2007;Ko et al, 2010).…”

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confidence: 99%

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Structural Studies of Biopolymer Membrane Transport

Morgan¹

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The selective transport of specific substrates across the membrane is an essential part of biology.Organisms have evolved a basic mechanism for the transport of ions and small molecule such as sugars across the membrane. This mechanism is termed 'alternating access' and involves the alternating exposure of a substrate-binding site to either side of the membrane. While the molecular details of 'alternating access' have been revealed for a number of different transporters, this scheme seems infeasible for the transport of long biopolymers such as nucleic acids, proteins, and polysaccharides because 'alternating access' requires that the transporter forms a binding site that is large enough to bind the entire substrate. Here, I present novel data addressing the transport mechanism for two bacterial biopolymer transporter proteins: Bacterial Cellulose Synthase which synthesizes and secretes the polysaccharide, cellulose; and PrtD, an ABC transporter which is involved in the secretion of an extracellular protease.Cellulose is a linear polymer of glucose units that forms a major component of plant cell walls as well as many bacterial biofilms. In bacteria, the components required for cellulose biosynthesis are encoded in a single operon minimally made up of the genes bcsA, bcsB, bcsC, and bcsZ. BcsA is an integral inner-membrane (IM) glycosyltransferase enzyme that is responsible for coupling the synthesis of cellulose with its transport across the IM. BcsB is a periplasmic protein with Cterminal IM anchor, and BcsB forms a complex with BcsA (BcsA-B) that is sufficient for in vitro cellulose synthesis. BcsZ is a periplasmic cellulase enzyme, and BcsC is an outer-membrane β-barrel that presumably forms a pore for the cellulose polymer to cross the outer membrane.iii BcsA-B activity is stimulated by the bacterial signaling molecule, cyclic-di-GMP (c-di-GMP), which is a key regulator of biofilm formation. I present crystal structures of the c-di-GMP-bound BcsA-B complex in the presence and absence of UDP, a competitive inhibitor and substrate mimic. The structures reveal that c-di-GMP releases an auto-inhibited state of the enzyme. A salt bridge stabilizes one of the signature c-di-GMP-binding Arg residues in a position to tether a conserved 'gating loop' in front of the active site. The binding of c-di-GMP releases the tether and allows substrate to access the active site. Additionally, the UDP-bound structure reveals an additional role for the 'gating loop' in coordinating substrate at the active site. Functional experiments confirm the structural interpretation by revealing that disruption of the auto-inhibitory salt bridge by mutagenesis generates a constitutively-active cellulose synthase. The mechanistic insights presented here represent the first examples of how c-di-GMP allosterically modulates enzymatic functions.To address the mechanism by which BcsA-B transports cellulose across the IM, I use in crystallo enzymology with the c-di-GMP-bound BcsA-B crystals. Because crystallized BcsA-B is catalytically a...

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Polymorphism of the Signaling Molecule c-di-GMP

Cited by 69 publications

References 53 publications

Activation of the Diguanylate Cyclase PleD by Phosphorylation-mediated Dimerization

Activation of the Diguanylate Cyclase PleD by Phosphorylation-mediated Dimerization

Cyclic Di-GMP Signaling in Bacteria: Recent Advances and New Puzzles

Structural Studies of Biopolymer Membrane Transport

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