Structure of Rhodococcus equi virulence-associated protein B (VapB) reveals an eight-stranded antiparallel β-barrel consisting of two Greek-key motifs

Abstract

Members of the virulence-associated protein (Vap) family from the pathogen Rhodococcus equi regulate virulence in an unknown manner. They do not share recognizable sequence homology with any protein of known structure. VapB and VapA are normally associated with isolates from pigs and horses, respectively. To contribute to a molecular understanding of Vap function, the crystal structure of a protease-resistant VapB fragment was determined at 1.4 Å resolution. The structure was solved by SAD phasing employing the anomalous signal of one endogenous S atom and two bound Co ions with low occupancy. VapB is an eight-stranded antiparallel β-barrel with a single helix. Structural similarity to avidins suggests a potential binding function. Unlike other eight- or ten-stranded β-barrels found in avidins, bacterial outer membrane proteins, fatty-acid-binding proteins and lysozyme inhibitors, Vaps do not have a next-neighbour arrangement but consist of two Greek-key motifs with strand order 41238567, suggesting an unusual or even unique topology.

Keywords: Greek-key motif; Rhodococcus equi; Vap protein family; antiparallel β-barrel; single-wavelength anomalous dispersion.

Figure 1

(a) Cartoon representation of VapB coloured from dark blue to red from the N-terminus to the C-terminus. The two views are rotated 180° around a vertical axis. (b) Electrostatic potential of VapB oriented as in (a) and coloured on a scale from −15 (red) to +15 (blue) kT/e. (c) Distribution of hydrophilic (left) and hydrophobic (right) residues in VapB oriented as in the left of (a).

Figure 2

(a) View from the top down the axis of the VapB β-barrel. Side chains of residues forming the hydrophobic core are represented as sticks. (b) Cartoon representation of streptavidin (PDB entry 1stp; Weber et al., 1989 ▶) coloured from dark blue to red from the N-terminus to the C-terminus. Biotin is shown as spheres. (c) VapB coloured by B factor (blue, low; red, high) and oriented as streptavidin with respect to strand β1 (note that the structural alignment generated by DALI is different). The loops at the top exhibit high B factors and double backbone conformations for residues that are represented as sticks.

Figure 3

Topology diagrams of eight-stranded antiparallel β-barrels (a) with next-neighbour connections as found in outer membrane proteins, avidins and lysozyme inhibitors; (b) two Greek-key motifs as present in VapB; (c) two Greek-key motifs as described for RPB8 (adapted from Krapp et al., 1998 ▶).