Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Oct 11;2(5):e00163-11.
doi: 10.1128/mBio.00163-11. Print 2011.

The structure of an unconventional HD-GYP protein from Bdellovibrio reveals the roles of conserved residues in this class of cyclic-di-GMP phosphodiesterases

Andrew L Lovering  1 , Michael J Capeness, Carey Lambert, Laura Hobley, R Elizabeth Sockett
Affiliations

The structure of an unconventional HD-GYP protein from Bdellovibrio reveals the roles of conserved residues in this class of cyclic-di-GMP phosphodiesterases

Andrew L Lovering et al. mBio. .

Abstract

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.

Importance: It is becoming apparent that many bacteria use the signaling molecule cyclic-di-GMP to regulate a variety of processes, most notably, transitions between motility and sessility. Importantly, this regulation is central to several traits implicated in chronic disease (adhesion, biofilm formation, and virulence gene expression). The mechanisms of cyclic-di-GMP synthesis via GGDEF enzymes and hydrolysis via EAL enzymes have been suggested by the analysis of several crystal structures, but no information has been available to date for the unrelated HD-GYP class of hydrolases. Here we present the multidomain structure of an unusual member of the HD-GYP family from the predatory bacterium Bdellovibrio bacteriovorus and detail the features that distinguish it from the wider structural family of general HD fold hydrolases. The structure reveals how a binuclear iron center is formed from several conserved residues and provides a basis for understanding HD-GYP family sequence requirements for c-di-GMP hydrolysis.

PubMed Disclaimer

Figures

FIG 1
FIG 1
Sequence alignment of B. bacteriovorus Bd1817 and other HD-GYP proteins. Bacterial strains and associated UNIPROT accession codes are as follows: Bd1817 (B. bacteriovorus, Q6MM30, aa 131 to 308), RpfG (Xanthomonas campestris, Q4UU85, aa 180 to 349), PA4108 (Pseudomonas aeruginosa, Q9HWS0, aa 141 to 308), PA4781 (P. aeruginosa, Q9HV27, aa 161 to 344), VCA_0681 (Vibrio cholerae, Q9KLR1, aa 237 to 405), 3,508 bp (Bordetella pertussis, Q7VTL7, aa 14 to 183), KPK_3322 (Klebsiella pneumoniae, B5XSE7, aa 204 to 372), SCO5218 (Streptomyces coelicolor, Q9K4A3, aa 226 to 393), BAS0914 (Bacillus anthracis, Q81UA6, aa 187 to 355). Conserved residues are boxed in white font on a red background, and partially conserved residues are boxed in red font on a white background. Metal-liganding and phosphate-liganded residues are indicated by blue triangles and magenta ovals, respectively. Secondary structure elements for Bd1817 are given above the alignment, corresponding to the HD-GYP domain only. Residue numbering refers to Bd1817, which terminates at L308; the other sequences have been cropped to match the region from A131 to the common DP element preceding α7. This alignment was prepared using T-Coffee (33) and ESPript (34).
FIG 2
FIG 2
Comparative transcriptional profiling of Bd1817 expression. Lanes AP to 4h contain total RNA taken from predatory B. bacteriovorus HD100 attacking E. coli prey cells, i.e., wild-type attack phase cells outside prey (AP) and B. bacteriovorus HD100 wild-type cells that invaded and were replicating inside E. coli prey cells for 15, 30, or 45 min or 1, 2, 3, 4 h. Lanes 1 to 4 contain Bd1817 RT-PCR products showing expression levels from matched 10-ng total RNA samples from attack phase Bdellovibrio (lane 1) and axenically grown, independently isolated wild-type HI strains of B. bacteriovorus HD100. Lane 2, strain HID2; lane 3, strain HID13; lane 4, strain HID26. Control lanes are as follows: S17-1, E. coli S17-1 RNA alone; –, no-template negative control; +, B. bacteriovorus genomic DNA positive control; L, 100-bp DNA ladder. This figure shows that Bd1817 is most highly expressed in the growth and septation phases of the Bdellovibrio axenic and predatory life cycles.
FIG 3
FIG 3
Electron density of chain A P21A crystal form, 1.28-Å resolution data, final refined map at 1.2σ. (a) Binuclear metal-binding site, with bridging hydroxide ion (W1) and tetrahedral ion, modeled as phosphate. (b) Region of GYP motif (glycine 245 and proline 246, “missing” the conserved tyrosine), electron density of aa 237 to 249 demonstrating the ordered nature of the fold.
FIG 4
FIG 4
Modular nature of the Bd1817 HD-GYP protein and active-site coordination of metal ions. Selected side chains and bound phosphate are shown in stick form, and metal ions (tan, M1 leftmost and M2 rightmost) and bound hydroxide (red, W1) are shown in sphere form. (a) Ribbon diagram of Bd1817 with individual domains colored separately as follows: NTD (aa 1 to 78), blue; linker helices (aa 79 to 146), yellow; HD-GYP domain (aa 147 to 308), white; lid region subdomain (aa 188 to 211), green; GYP motif subdomain (aa 239 to 255), orange. (b) Detail of binuclear metal active site (HD-GYP domain). Fe-protein interactions, purple dashed lines; hydroxide-protein interaction, green dashed line. (c) Schematic of binuclear metal site with coordination distances shown (taken from chain A of the 1.28-Å P21A crystal form).
FIG 5
FIG 5
Features of the NTD. (a) The Bd1817 NTD (blue) possesses weak structural homology with the Ral-binding domain of Exo84 (cyan; PDB code 1ZC3 [11]), ending at the point where the Bd1817 linker region (yellow) starts. In the P21B crystal form, residues −11 to 3 of chain B (the polyhistidine tag with the thrombin site left uncleaved [red]) are highly ordered and bind into a cleft formed between the NTD and linker region of chain A. This interaction occurs in a region identical to that of the Exo84 binding partner RalA (magenta), albeit running in different chain directions. (b) Detail of affinity tag-NTD interaction. Selected side chains are shown in stick form.
FIG 6
FIG 6
Comparison of the HD-GYP fold with a representative of the wider HD family. Bd1817 (yellow; GYP motif orange) and the HD protein YfbR (PDB code 2PAQ [13]; blue) are shown superimposed, with the HD residues of Bd1817 in stick form and the active-site binuclear metal center in sphere form. It is apparent that all of the α-helices of the HD-GYP fold have counterparts in the generalized HD fold, although α6 sits at an angle different from that of its counterpart in YfbR. The lid region of Bd1817 corresponds to a flexible loop of YfbR (lower left-hand area of image, left unmodeled). Comparatively, the GYP motif of Bd1817 replaces the short interhelical loop of HD proteins (between α4 and α5 equivalents, the 20 residues between Bd1817 aa 236 and 255 contrasting with the 5 residues between YfbR aa 122 and 126).
FIG 7
FIG 7
Structure of the GYP region of the fold and possible site for c-di-GMP binding. (a) Detail of the GYP motif (residues 239 to 255; orange), illustrating fold stabilization by the conserved E239 side chain and tightly bound solvent molecule W2 (hydrogen bond interactions represented by dashed green line, backbone amide and carbonyl groups represented by blue and red spheres, respectively). Proline 246 of the GYP motif (G-P for Bd1817, this structure) sits at the edge of the second of two perpendicular U-shaped turns (with glycine 245, N atom shown as a blue sphere, preceding it). The GYP motif is located in a region close to that of the binuclear metal active site (liganding residues are yellow). (b) Possible binding site modeled for a c-di-GMP substrate in HD-GYP proteins. The bound phosphate in the Bd1817 structure was used as a guide to place c-di-GMP (stick form, C atoms magenta), with the 3′ end located proximal to H187 (postulated to protonate this group upon bond cleavage; see text). One of the guanine bases is positioned toward the GYP motif and a region formed by the S262 and H293 residues (consensus A and D, respectively), while the other base clashes with the lid region (green). Regardless of how c-di-GMP is positioned next to the binuclear center, the restricted space available in this region suggests that a small conformational rearrangement of the lid region would be required to accommodate the substrate if this structure of Bd1817 is representative of other HD-GYP active-site geometry. (c) Detail of several nonconsensus residues of Bd1817 (single-letter code, C atoms blue, HD-GYP family consensus in parentheses; derived from Fig. 1), illustrating the position around the predicted c-di-GMP-binding site (in surface representation, transparent to show side chain detail, metal sites and bound phosphate ion). In particular, the consensus arginine at Bd1817 E274 would be well placed to contact the second c-di-GMP phosphate group (oriented toward the top in this view). The nonconsensus GYP motif at aa 245 and 246 has been left unnumbered for clarity.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Römling U, Gomelsky M, Galperin MY. 2005. C-di-GMP: the dawning of a novel bacterial signalling system. Mol. Microbiol. 57:629–639 - PubMed
    1. Schirmer T, Jenal U. 2009. Structural and mechanistic determinants of c-di-GMP signalling. Nat. Rev. Microbiol. 7:724–735 - PubMed
    1. Ross P, et al. 1987. Regulation of cellulose synthesis in Acetobacter xylinum by cyclic diguanylic acid. Nature 325:279–281 - PubMed
    1. Amikam D, Galperin MY. 2006. PilZ domain is part of the bacterial c-di-GMP binding protein. Bioinformatics 22:3–6 - PubMed
    1. Ryan RP, et al. 2006. Cell-cell signaling in Xanthomonas campestris involves an HD-GYP domain protein that functions in cyclic di-GMP turnover. Proc. Natl. Acad. Sci. U. S. A. 103:6712–6717 - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources