A new non-classical fold of varroa odorant-binding proteins reveals a wide open internal cavity

Abstract

Odorant-binding proteins (OBPs), as they occur in insects, form a distinct class of proteins that apparently has no closely related representatives in other animals. However, ticks, mites, spiders and millipedes contain genes encoding proteins with sequence similarity to insect OBPs. In this work, we have explored the structure and function of such non-insect OBPs in the mite Varroa destructor, a major pest of honey bee. Varroa OBPs present six cysteines paired into three disulphide bridges, but with positions in the sequence and connections different from those of their insect counterparts. VdesOBP1 structure was determined in two closely related crystal forms and appears to be a monomer. Its structure assembles five α-helices linked by three disulphide bridges, one of them exhibiting a different connection as compared to their insect counterparts. Comparison with classical OBPs reveals that the second of the six α-helices is lacking in VdesOBP1. Ligand-binding experiments revealed molecules able to bind only specific OBPs with a moderate affinity, suggesting that either optimal ligands have still to be identified, or post-translational modifications present in the native proteins may be essential for modulating binding activity, or else these OBPs might represent a failed attempt in evolution and are not used by the mites.

Figure 1

(a) Alignment of the five OBPs of V. destructor. The six conserved cysteines are highlighted. VdesOBP1 and VdesOBP2 are very similar (72% identity), as VdesOBP3 and VdesOBP4 (60%), but proteins of the two groups share only about 15% of their residues between them as well as with VdesOBP5. The six conserved cysteines are highlighted and in red font. Signal peptides are underlined. (b) Phylogenetic tree of tick and mite OBPs. Vdes: Varroa destructor; Turt: Tetranychus urticae; Hqin: Haemaphysalis qinghaiensis; Hfla: Haemaphysalis flava; Hlon: Haemaphysalis longicornis; Isca: Ixodes scapularis; Gocc: Galendromus occidentalis; Tmer: Tropilaelaps mercedesae; Rann: Rhipicephalus annulatus; Rsan: Rhipicephalus sanguineus; Ldel: Leptotrombidium delicense; Dtin: Dinothrombium tinctorium; Dpte: Dermatophagoides pteronyssinus; Ssca: Sarcoptes scabiei; Emay: Euroglyphus maynei. All sequences used to build the tree are reported in Supplementary Table S1 online. Names of ticks are in red, those of mites are in green. Highlighted clades contain VdesOBP1 and VdesOBP2 (pink), VdesOBP3 and VdesOBP4 (light blue), VdesOBP5 (light green).

Figure 2

Expression and purification of four OBPs of V. destructor. While VdesOBP1, VdesOBP2 and VdesOBP5 were easily produced in bacteria, VdesOBP3 and VdesOBP4, which are structurally related, proved difficult to express. In fact, VdesOBP4 was obtained only in very low yield, while we could not observe expression of VdesOBP3 in any of the different constructs adopted. M: Molecular weight markers; bef: crude sample before culture induction; aft: crude sample after induction with IPTG; SN: supernatant after lysis and centrifugation; P: pellet after lysis and centrifugation; OBP: sample of purified protein.

Figure 3

Fragmentation spectra of disulfide-bridged peptides identified in the tryptic-chymotryptic digest of V. destructor VdesOBP1 as revealed by nanoLC-ESI-Q-Orbitrap-MS/MS analysis. The fragments are highlighted in different colours depending on peptides present in S–S-linked species, and on the corresponding b and y ion series. Complete data on disulphide-bridged peptides are reported in Supplementary Table S2 online.

Figure 4

Crystal structure of VdesOBP1 form P2₁. (a) Ribbon view of a monomer displaying helices 1–5 and disulphide bridges. The polypeptide chain is rainbow coloured, from blue (N-terminus) to red (C-terminus). The disulphide bonds are identified by the secondary structures they belong to. (b) Ribbon view of the crystallographic dimer. One monomer is rainbow coloured, the other one is coloured brown. The serendipitously bound buffer molecule 2-(N-cyclohexylamino)-ethane sulfonic acid (NCES) is displayed as atomic spheres (C: white, N: blue and O: red). Figure made with PyMOL (version 1.5.0.2. http://www.pymol.org).

Figure 5

Comparison of crystal structures of VdesOBP1 forms P2₁ and P3₂21. (a) Ribbon view of the superposition of monomers belonging to forms P2₁ (rainbow coloured) and P3₂21 (grey). (b) Ribbon view of the crystallographic dimers of forms P2₁ and P3₂21. In form P2₁ one monomer is rainbow coloured, the other one is coloured brown. The crystallographic dimer of P3₂21 form is coloured light grey. One monomer of each form has been superimposed (left). The second monomers do not superimpose. B/same view rotated 90° around a vertical axis. Note the rotation of ~ 80° of the second monomer of form P3₂21 relative to the second monomer of form P2₁. Figure made with PyMOL (version 1.5.0.2. http://www.pymol.org).

Figure 6

The internal cavity of VdesOBP1 form P2₁. (a) Slabbed ribbon view of a monomer rainbow coloured. The cavity is represented by a grey surface and is wide open. The serendipitously bound buffer molecule 2-(N-cyclohexylamino)-ethane sulfonic acid (NCES) is displayed as atomic spheres (C: white, N: blue and O: red), and is stacked against the cavity wall. (b) Slabbed ribbon view of a crystallographic dimer rainbow coloured. The cavity is represented by a green surface originating from monomer 1 and a blue surface from monomer 2. The serendipitously bound buffer molecule NCES is displayed as atomic spheres (C: white, N: blue and O: red), and is stacked against the cavity walls from both monomers. Figure made with PyMOL (version 1.5.0.2. http://www.pymol.org).

Figure 7

Comparison of crystal structures of VdesOBP1 with the closest related OBPs from Phormia regina (PregOBP56) and Leucophera madereae (LmadPBP). (a) Ribbon view of the superposition of monomers from VdesOBP1 (rainbow coloured) and PregOBP56 (light grey) with its two fatty acid ligands (grey sticks). Note the absence of the classical OBP fold helix 2 (red squared) in VdesOBP1. (b) Ribbon view of the superposition of monomers from VdesOBP1 (rainbow coloured) and LmadPBP. Note the absence of the classical OBP fold helix 2 (red squared) in VdesOBP1 as compared to LmadPBP (red squared) and the shift of one disulphide bridge (red arrow). The 3D positions of the two other disulphide bridges are conserved (purple arrow), in the final fold, although as result of different C1-C6, C2-C3, C4-C5 (VdesOBP1) and C1-C3, C2-C5, C4-C6 (LmadPBP) connectivities. Figure made with PyMOL (version 1.5.0.2. http://www.pymol.org).

Figure 8

Ligand-binding assays. (a) Binding of the fluorescent probe N-phenylnaphthylamine (1-NPN) to VdesOBP4 and VdesOBP5. With the other two proteins expressed (VdesOBP1 and VdesOBP2), we could not observe the blue shift usually associated with binding of the fluorescent probe. (b) Structures of the ligand used in the competitive binding experiments. (c,d) Competitive binding assays. Only few chemicals were able to displace 1-NPN from the complex with VdesOBP4 and VdesOBP5. The same chemicals were also the best ligands for the other VdesOBPs (Supplementary Fig. S3 online).