Structural characterisation of the virulence-associated protein VapG from the horse pathogen Rhodococcus equi

Abstract

Virulence and host range in Rhodococcus equi depends on the variable pathogenicity island of their virulence plasmids. Notable gene products are a family of small secreted virulence-associated proteins (Vaps) that are critical to intramacrophagic proliferation. Equine-adapted strains, which cause severe pyogranulomatous pneumonia in foals, produce a cell-associated VapA that is necessary for virulence, alongside five other secreted homologues. In the absence of biochemical insight, attention has turned to the structures of these proteins to develop a functional hypothesis. Recent studies have described crystal structures for VapD and a truncate of the VapA orthologue of porcine-adapted strains, VapB. Here, we crystallised the full-length VapG and determined its structure by molecular replacement. Electron density corresponding to the N-terminal domain was not visible suggesting that it is disordered. The protein core adopted a compact elliptical, anti-parallel β-barrel fold with β1-β2-β3-β8-β5-β6-β7-β4 topology decorated by a single peripheral α-helix unique to this family. The high glycine content of the protein allows close packing of secondary structural elements. Topologically, the surface has no indentations that indicate a nexus for molecular interactions. The distribution of polar and apolar groups on the surface of VapG is markedly uneven. One-third of the surface is dominated by exposed apolar side-chains, with no ionisable and only four polar side-chains exposed, giving rise to an expansive flat hydrophobic surface. Other surface regions are more polar, especially on or near the α-helix and a belt around the centre of the β-barrel. Possible functional significance of these recent structures is discussed.

Keywords: Protein Structure; Rhodococcus equi; VapA; VapG; Virulence-associated protein.

Fig. 1

The virulence genes/proteins of R. equi. (A) The 21.3 kb pathogenicity island region of the equine type R. equi plasmid pVAPA1037. The genes encoding the homologous virulence associated proteins Vaps A, C, D, E, G, H are shown in blue with a one letter label. The pseudogenes vapF, vapI and vapX are shown in turquoise. The genes encoding the transcriptional regulators virR (a LysR type transcriptional regulator) and virS (an orphan two-component system response regulator) are coloured in orange. Expression of vapA appears to be controlled indirectly by VirR in response to temperatures above 30 °C and acidic pH by production of VirS (green arrow) (Russell et al., 2004), which is required for transcription of the vapAIC(orfAB)D operon (purple arrow) (Kakuda et al., 2014); the small uncharacterised ORFs (orfAB) that lie between vapC and vapD are not shown for clarity. The promoter associated with the vcgABC operon (uncharacterised vapco-expressed genes) shares homology with the vapA promoter (Miranda-CasoLuengo et al., 2011) suggesting that a similar mode of regulation may operate. Observed transcripts are related as orange arrows (Byrne et al., 2007, 2008; Kakuda et al., 2014; Miranda-CasoLuengo et al., 2011). (B) Comparison of the amino acid sequences of the Vap proteins of the R. equi horse virulence plasmid together with VapB form the porcine virulence plasmid. The sequences were aligned in CLUSTALW (Thompson et al., 1994) and displayed together with the secondary structure elements of VapG using the programme ESPRIPT (Robert and Gouet, 2014). Invariant residues in the alignment are shown in white type on a red background; conserved residues are in blue boxes (For interpretation of the colour information in this figure legend, the reader is referred to the web version of the article.).

Fig. 2

Size exclusion chromatography with multi-angle laser light scattering (SEC-MALLS) analysis of VapG. The refractive index (RI) of the eluate from a Superdex S75 column is plotted as a function of time (blue trace). Over the peak region, the molecular weight (MW) of the species (green line) in the eluate is calculated from the light scattering measurements (For interpretation of the colour information in this figure legend, the reader is referred to the web version of the article.).

Fig. 3

The chain topology in VapG. (A) and (B) Approximately orthogonal views of the VapG chain represented as a ribbon trace colour-ramped from the N-terminus (blue) to the C-terminus (red). The secondary structure elements are labelled and the potassium ion from the A chain is shown as a purple sphere. (C) and (D) The VapG molecule coloured as above viewed parallel to the long axis of the barrel. It is apparent that the barrel is closed off by the β3–β4 and β7–β8 segments when viewed from above (C) and by the α1 helix when viewed from below (D). In (C) the potassium ion has been omitted for clarity (For interpretation of the colour information in this figure legend, the reader is referred to the web version of the article.).

Fig. 4

Potassium coordination in the A molecule of VapG. (A) Electron density (2F_obs − F_calc) contoured at the 1σ and 6σ levels and displayed on the potassium ion, shown as a purple sphere, and neighbouring residues shown in ball-and-stick representation and coloured by atom type (carbon, green; nitrogen, blue; oxygen, red). (B) The bound potassium ion (K⁺) is shown as a purple sphere with residues which contribute ligands to the metal centre shown in cylinder representation and with metal–ligand bonds shown as dashed lines. The bond lengths in Å are given.

Fig. 5

Details of the structure of VapG. (A) Stereo view of the core of VapG. The protein backbone is represented as a worm tracing coloured in cyan and the side chains of residues that are significantly buried in the protein interior are shown in cylinder format and coloured by atom type (carbon, green; nitrogen, blue; oxygen, red). Two buried water molecules are shown as red spheres. Hydrogen bonding interactions between the buried side chains and these waters are shown as dashed lines. (B) The largely hydrophobic distal surface of VapG. The protein backbone is represented as a worm tracing colour ramped as in Fig. 3. Residues whose side chains are directed towards the solvent are shown in ball-and-stick representation and coloured according to their side chain type: non-polar aliphatic (Ala, Pro, Val, Leu, Ile), coral; aromatic (Trp, Tyr, Phe), light green; polar (Ser, Thr, Asn, Gln), gold; acidic (Glu, Asp) red; histidine, light blue. The Cα atoms of glycine residues are shown as grey spheres. (C) The distribution of ionisable residues in VapG. The side chains of acidic and basic residues are shown in cylinder format. (D) The distribution of the 20 glycine residues in VapG. The Cα atom of each glycine residue is shown as a grey sphere (For interpretation of the colour information in this figure legend, the reader is referred to the web version of the article.).

Fig. 6

Comparison of the crystal structures of the three R. equi virulence-associated proteins. (A) and (B) The structures of VapG molecule A (blue), VapG molecule B (light blue) VapD (red) and VapB (green) were overlaid using the SSM superpose routines in CCP4mg and the polypeptide backbones displayed as worm tracings. The superposed protein chains are viewed from the side illustrating the whole molecules (A) and from above parallel to the long axis of the barrel with the molecule clipped for clarity (B). (C) Electrostatic surface renderings of VapB, VapD and VapG with positive electrostatic potential in blue and negative electrostatic potential in red. The three molecules are viewed in two orientations which are illustrated by the ribbon tracing for VapG at the left. The extended apolar surface previously noted for VapD and VapB is evident (For interpretation of the colour information in this figure legend, the reader is referred to the web version of the article.).