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

Sundriyal, Amit; Massa, Claudia; Samoray, Dietrich; Zehender, F.; Sharpe, Timothy D.; Jenal, Urs; Schirmer, Tilman

doi:10.1074/jbc.m113.516195

Cited by 64 publications

(93 citation statements)

References 38 publications

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“…EAL phosphodiesterases require a divalent cation for enzymatic activity, which in most cases is a Mg 2ϩ or Mn 2ϩ ion, while Ca 2ϩ and Zn 2ϩ efficiently inhibit the enzymatic activity (39,40). Catalytically active EAL domains usually have a high substrate affinity in the physiological nanomolar range, and cyclic di-GMP binding can increase the dimerization affinity (41). Although monomers can be catalytically active, dimerization substantially enhances protein stability and catalytic activity (37).…”

Section: Functional Diversification Of the Eal Domainmentioning

confidence: 99%

Progress in Understanding the Molecular Basis Underlying Functional Diversification of Cyclic Dinucleotide Turnover Proteins

2017

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Cyclic di-GMP was the first cyclic dinucleotide second messenger described, presaging the discovery of additional cyclic dinucleotide messengers in bacteria and eukaryotes. The GGDEF diguanylate cyclase (DGC) and EAL and HD-GYP phosphodiesterase (PDE) domains conduct the turnover of cyclic di-GMP. These three unrelated domains belong to superfamilies that exhibit significant variations in function, and they include both enzymatically active and inactive members, with a subset involved in synthesis and degradation of other cyclic dinucleotides. Here, we summarize current knowledge of sequence and structural variations that underpin the functional diversification of cyclic di-GMP turnover proteins. Moreover, we highlight that superfamily diversification is not restricted to cyclic di-GMP signaling domains, as particular DHH/DHHA1 domain and HD domain proteins have been shown to act as cyclic di-AMP phosphodiesterases. We conclude with a consideration of the current limitations that such diversity of action places on bioinformatic prediction of the roles of GGDEF, EAL, and HD-GYP domain proteins.KEYWORDS cyclic dinucleotide second messenger, GGDEF domain, EAL domain, HD-GYP domain, DHH/DHHA1 domain, cyclic GAMP, cyclic di-AMP, cyclic di-GMP, second messenger T he dinucleotide cyclic di-GMP is the most abundant second messenger in bacteria. It promotes the environmental lifestyle switch between sessility and motility, as well as the host-related lifestyle switch between acute and chronic/benign infection. A hallmark of the cyclic di-GMP signaling network is an apparent redundancy of cyclic di-GMP turnover proteins encoded in one genome. However, many of these proteins have distinct N-terminal sensing and signaling domains, suggesting that their activities in cyclic di-GMP turnover respond posttranslationally to various (and different) intraand extracellular signals. In gross terms, the number of cyclic di-GMP turnover proteins is linearly correlated with genome size within the different bacterial phyla, with Thermotogae having one of the highest cyclic di-GMP-related "IQs," the density of enzymes per megabase pair, with some species harboring over 100 cyclic di-GMP turnover proteins (http://www.ncbi.nlm.nih.gov/Complete_Genomes/c-di-GMP.html). As in other domain superfamilies, extensive sequence diversity exists. Here, we review the knowledge on the translation of sequence diversity of cyclic di-GMP turnover proteins into functional diversity. We conclude by discussing whether and how a unified nomenclature for cyclic di-GMP turnover proteins can be established.

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Section: Functional Diversification Of the Eal Domainmentioning

confidence: 99%

Progress in Understanding the Molecular Basis Underlying Functional Diversification of Cyclic Dinucleotide Turnover Proteins

2017

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“…MST quantifies molecular interactions by monitoring the motion of (bio)molecules across a microscopic temperature gradient (30). It was successfully used to measure c-di-GMP binding by EAL and PilZ domain proteins (31,32). All three proteins were fused with a His tag and purified by nickel affinity chromatography (see Fig.…”

Section: Fig 3 Functional Classification Of the Ccms-identified Cyclimentioning

confidence: 99%

An Extended Cyclic Di-GMP Network in the Predatory Bacterium Bdellovibrio bacteriovorus

et al. 2016

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Over the course of the last 3 decades the role of the second messenger cyclic di-GMP (c-di-GMP) as a master regulator of bacterial physiology was determined. Although the control over c-di-GMP levels via synthesis and breakdown and the allosteric regulation of c-di-GMP over receptor proteins (effectors) and riboswitches have been extensively studied, relatively few effectors have been identified and most are of unknown functions. The obligate predatory bacterium Bdellovibrio bacteriovorus has a peculiar dimorphic life cycle, in which a phenotypic transition from a free-living attack phase (AP) to a sessile, intracellular predatory growth phase (GP) is tightly regulated by specific c-di-GMP diguanylate cyclases. B. bacteriovorus also bears one of the largest complement of defined effectors, almost none of known functions, suggesting that additional proteins may be involved in c-di-GMP signaling. In order to uncover novel c-di-GMP effectors, a c-di-GMP capture-compound mass-spectroscopy experiment was performed on wild-type AP and host-independent (HI) mutant cultures, the latter serving as a proxy for wild-type GP cells. Eightyfour proteins were identified as candidate c-di-GMP binders. Of these proteins, 65 did not include any recognized c-di-GMP binding site, and 3 carried known unorthodox binding sites. Putative functions could be assigned to 59 proteins. These proteins are included in metabolic pathways, regulatory circuits, cell transport, and motility, thereby creating a potentially large c-di-GMP network. False candidate effectors may include members of protein complexes, as well as proteins binding nucleotides or other cofactors that were, respectively, carried over or unspecifically interacted with the capture compound during the pulldown. Of the 84 candidates, 62 were found to specifically bind the c-di-GMP capture compound in AP or in HI cultures, suggesting c-di-GMP control over the whole-cell cycle of the bacterium. High affinity and specificity to c-di-GMP binding were confirmed using microscale thermophoresis with a hypothetical protein bearing a PilZ domain, an acyl coenzyme A dehydrogenase, and a two-component system response regulator, indicating that additional c-di-GMP binding candidates may be bona fide novel effectors. IMPORTANCEIn this study, 84 putative c-di-GMP binding proteins were identified in B. bacteriovorus, an obligate predatory bacterium whose lifestyle and reproduction are dependent on c-di-GMP signaling, using a c-di-GMP capture compound precipitation approach. This predicted complement covers metabolic, energy, transport, motility and regulatory pathways, and most of it is phase specific, i.e., 62 candidates bind the capture compound at defined modes of B. bacteriovorus lifestyle. Three of the putative binders further demonstrated specificity and high affinity to c-di-GMP via microscale thermophoresis, lending support for the presence of additional bona fide c-di-GMP effectors among the pulled-down protein repertoire.

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“…In this case, one would expect that most or possibly all enzymes are expressed at any given time but that the majority of them are not active due to the absence of an input signal. In the past few years, the amount of information about biochemical and structural characteristics of DGCs and PDEs has increased rapidly (13)(14)(15)(16)(17)(18). Despite such rapid progress, in vivo results often remain controversial.…”

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confidence: 99%

Expression and Genetic Activation of Cyclic Di-GMP-Specific Phosphodiesterases in Escherichia coli

et al. 2016

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Intracellular levels of the bacterial second messenger cyclic di-GMP (c-di-GMP) are controlled by antagonistic activities of diguanylate cyclases and phosphodiesterases. The phosphodiesterase PdeH was identified as a key regulator of motility in Escherichia coli, while deletions of any of the other 12 genes encoding potential phosphodiesterases did not interfere with motility. To analyze the roles of E. coli phosphodiesterases, we demonstrated that most of these proteins are expressed under laboratory conditions. We next isolated suppressor mutations in six phosphodiesterase genes, which reinstate motility in the absence of PdeH by reducing cellular levels of c-di-GMP. Expression of all mutant alleles also led to a reduction of biofilm formation. Thus, all of these proteins are bona fide phosphodiesterases that are capable of interfering with different c-di-GMP-responsive output systems by affecting the global c-di-GMP pool. This argues that E. coli possesses several phosphodiesterases that are inactive under laboratory conditions because they lack appropriate input signals. Finally, one of these phosphodiesterases, PdeL, was studied in more detail. We demonstrated that this protein acts as a transcription factor to control its own expression. Motile suppressor alleles led to a strong increase of PdeL activity and elevated pdeL transcription, suggesting that enzymatic activity and transcriptional control are coupled. In agreement with this, we showed that overall cellular levels of c-di-GMP control pdeL transcription and that this control depends on PdeL itself. We thus propose that PdeL acts both as an enzyme and as a c-di-GMP sensor to couple transcriptional activity to the c-di-GMP status of the cell. IMPORTANCEMost bacteria possess multiple diguanylate cyclases and phosphodiesterases. Genetic studies have proposed that these enzymes show signaling specificity by contributing to distinct cellular processes without much cross talk. Thus, spatial separation of individual c-di-GMP signaling units was postulated. However, since most cyclases and phosphodiesterases harbor N-terminal signal input domains, it is equally possible that most of these enzymes lack their activating signals under laboratory conditions, thereby simulating signaling specificity on a genetic level. We demonstrate that a subset of E. coli phosphodiesterases can be activated genetically to affect the global c-di-GMP pool and thus influence different c-di-GMP-dependent processes. Although this does not exclude spatial confinement of individual phosphodiesterases, this study emphasizes the importance of environmental signals for activation of phosphodiesterases.

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

Cited by 64 publications

References 38 publications

Progress in Understanding the Molecular Basis Underlying Functional Diversification of Cyclic Dinucleotide Turnover Proteins

Progress in Understanding the Molecular Basis Underlying Functional Diversification of Cyclic Dinucleotide Turnover Proteins

An Extended Cyclic Di-GMP Network in the Predatory Bacterium Bdellovibrio bacteriovorus

Expression and Genetic Activation of Cyclic Di-GMP-Specific Phosphodiesterases in Escherichia coli

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