The Structure of an Unconventional HD-GYP Protein from Bdellovibrio Reveals the Roles of Conserved Residues in this Class of Cyclic-di-GMP Phosphodiesterases

School of Biosciences, University of Birmingham, Birmingham, United Kingdom.
mBio (Impact Factor: 6.79). 08/2011; 2(5). DOI: 10.1128/mBio.00163-11
Source: PubMed


Cyclic-di-GMP is a near-ubiquitous bacterial second messenger that is important in localized signal transmission during the control of various processes, including virulence and switching between planktonic and biofilm-based lifestyles. Cyclic-di-GMP is synthesized by GGDEF diguanylate cyclases and hydrolyzed by EAL or HD-GYP phosphodiesterases, with each functional domain often appended to distinct sensory modules. HD-GYP domain proteins have resisted structural analysis, but here we present the first structural representative of this family (1.28 Å), obtained using the unusual Bd1817 HD-GYP protein from the predatory bacterium Bdellovibrio bacteriovorus. Bd1817 lacks the active-site tyrosine present in most HD-GYP family members yet remains an excellent model of their features, sharing 48% sequence similarity with the archetype RpfG. The protein structure is highly modular and thus provides a basis for delineating domain boundaries in other stimulus-dependent homologues. Conserved residues in the HD-GYP family cluster around a binuclear metal center, which is observed complexed to a molecule of phosphate, providing information on the mode of hydroxide ion attack on substrate. The fold and active site of the HD-GYP domain are different from those of EAL proteins, and restricted access to the active-site cleft is indicative of a different mode of activity regulation. The region encompassing the GYP motif has a novel conformation and is surface exposed and available for complexation with binding partners, including GGDEF proteins.

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    • "Finally, a single HD-GYP-domain protein was identified that carried a non-canonical HT-GYP motif and a predicted N-terminal PilZ domain. The only two structures for this family of proteins have carried either an HD-GYP or HD- G.P domain, as such it is unclear what the effect of a noncanonical HT-GYP motif may be on the PDE activity of this protein (Bellini et al., 2014; Lovering et al., 2011). PilZ domains have been demonstrated to bind c-di-GMP and act as receptor domains on several proteins (Pratt et al., 2007; Ryjenkov et al., 2006; Zorraquino et al., 2013; Guzzo et al., 2013). "
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    ABSTRACT: Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is a ubiquitous bacterial signalling molecule produced by diguanylate cyclases of the GGDEF-domain family. Elevated c-di-GMP levels or increased GGDEF protein expression is frequently associated with the onset of sessility and biofilm formation in numerous bacterial species. Conversely, phosphodiesterase-dependent diminution of c-di-GMP levels by EAL- and HD-GYP-domain proteins is often accompanied by increased motility and virulence. In this study we individually overexpress 23 predicted GGDEF, EAL or HD-GYP-domain proteins encoded by the phytopathogen Pectobacterium atrosepticum strain SCRI1043 (Pba). Mass spectrometry-based detection of c-di-GMP and 5'-phosphoguanylyl-(3'-5')-guanosine (pGpG) in these strains revealed that overexpression of most genes promoted modest 1-10-fold changes in cellular levels c-di-GMP, with the exception of the GGDEF-domain proteins ECA0659 and ECA3374, which induced 1290 and 7660-fold increases respectively. Overexpression of most EAL domain proteins increased motility while overexpression of most GGDEF domain proteins reduced motility and increased poly-N-acetyl-β-(1-6)-glucosamine-dependent flocculation. In contrast to domain-based predictions, overexpression of the EAL protein ECA3549 or the HD-GYP protein ECA3548 increased c-di-GMP concentrations and reduced motility. Most overexpression constructs altered the levels of secreted cellulases, pectinases and proteases, confirming c-di-GMP regulation of virulence in Pba. However there was no apparent correlation between virulence factor induction and the domain class expressed or cellular c-di-GMP levels, suggesting that regulation was in response to specific effectors within the network, rather than total c-di-GMP concentration. Finally we demonstrate that the cellular localisation patterns vary considerably for GGDEF/EAL/HD-GYP proteins indicating it is a likely factor restricting specific interactions within the c-di-GMP network.
    Microbiology 04/2014; 160(Pt 7). DOI:10.1099/mic.0.076828-0 · 2.56 Impact Factor
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    • "Site-directed mutagenesis showed that alanine substitutions of the eight metal-ligating residues either abolished or significantly reduced the catalytic activity, indicating that all three metal ions contribute to catalysis, although a more structural role in co-ordination of the active site cannot be ruled out (Bellini et al., 2013). Consistent with the crystal structure of the HD-G_P protein Bd1817 reported earlier (Lovering et al., 2011), the conserved His 221 and Asp 222 that constitute the HD-GYP motif were shown to be directly involved in metal binding. Thus, the authors further clarify the role for several of the metal co-ordination residues, which have previously been shown to be essential for PDE activity and are highly conserved within the HD-GYP family (Ryan, 2013). "
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    ABSTRACT: The major sessility-motility life style change and additional fundamental aspects of bacterial physiology, behavior and morphology are regulated by the secondary messenger cyclic di-GMP (c-di-GMP). Although the c-di-GMP metabolizing enzymes and many receptors have been readily characterized upon discovery, the HD-GYP domain c-di-GMP phosphodiesterase family remained underinvestigated. In this issue of Molecular Microbiology, Bellini et al., (2013) provide an important step towards functional and structural characterization of the previously neglected HD-GYP domain family by resolving the crystal structure of PmGH, a catalytically active family member from the thermophilic bacterium Persephonella marina. The crystal structure revealed a novel tri-nuclear catalytic iron center involved in c-di-GMP binding and catalysis and provides the structural basis to subsequently characterize in detail the catalytic mechanism of hydrolysis of c-di-GMP to GMP by HD-GYP domains.
    Molecular Microbiology 11/2013; 91(1). DOI:10.1111/mmi.12463 · 4.42 Impact Factor
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    • "HnoD, the second cognate RR linked to c-di-GMP metabolism, is identified as a HD-GYP domain, but active site residues responsible for coordination of crucial catalytic metals are missing in HnoD (Lovering et al., 2011) rendering the protein inactive for c-di-GMP hydrolysis. Compared to EAL domains, HD-GYP domains remain poorly characterized with only few reports of direct PDE activity (Hammer and Bassler, 2009; Ryan et al., 2006). "
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    ABSTRACT: Nitric oxide (NO) signaling in vertebrates is well characterized and involves the heme-nitric oxide/oxygen-binding (H-NOX) domain of soluble guanylate cyclase as a selective NO sensor. In contrast, little is known about the biological role or signaling output of bacterial H-NOX proteins. Here, we describe a molecular pathway for H-NOX signaling in Shewanella oneidensis. NO stimulates biofilm formation by controlling the levels of the bacterial secondary messenger cyclic diguanosine monophosphate (c-di-GMP). Phosphotransfer profiling was used to map the connectivity of a multicomponent signaling network that involves integration from two histidine kinases and branching to three response regulators. A feed-forward loop between response regulators with phosphodiesterase domains and phosphorylation-mediated activation intricately regulated c-di-GMP levels. Phenotypic characterization established a link between NO signaling and biofilm formation. Cellular adhesion may provide a protection mechanism for bacteria against reactive and damaging NO. These results are broadly applicable to H-NOX-mediated NO signaling in bacteria.
    Molecular cell 04/2012; 46(4):449-60. DOI:10.1016/j.molcel.2012.03.023 · 14.02 Impact Factor
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