Long-form D7 proteins are found in the salivary secretions of Phlebotomus and Lutzomyia sand flies from the new world and old world respectively (Fig. 1A). Sequence comparisons suggest that many of the residues involved in the binding of ligands in the N-terminal domain of the protein are conserved with apparent homologs in the saliva of mosquitoes including AeD7 from Ae. aegypti and AnStD7L1 from An. stephensi (Fig. 1A,B). In the genome of Phlebotomus papatasi, AGE83092 and AGE83093, a pair of related long-form D7s are separated by 6.7 kb on the chromosome indicating that they are the result of a tandem duplication event12. P. papatasi is distributed throughout the Mediterranean region and the Middle East where it acts as a major vector of Leishmania. In sub-Saharan Africa, a closely related sand fly species, P. duboscqi, serves as the major vector in this region. Although the P. duboscqi genome has not been sequenced, long-form D7 transcripts have been identified in salivary cDNA libraries from this species13. In the new world, members of a second sand fly genus, Lutzomyia, are responsible for transmission of Leishmania, and contain long-form D7 proteins in their saliva (Fig. 1A)14. Previous studies have shown that the residues involved with eicosanoid ligand binding in the putative N-terminal domain of the two-domain long-form D7 proteins from mosquitoes are conserved in the apparently homologous proteins of sand flies, but residues associated with biogenic amine binding in the C-terminal domain of the long-form D7 proteins of Aedes species are not (Fig. 1B)6,10. We produced recombinant versions of two representative forms of sand fly long-form D7 from P. papatasi (AGE83092) and P. duboscqi (ABI15936) and evaluated their structure, ligand-binding properties and physiological function using X-ray diffraction, isothermal titration calorimetry (ITC), and bioassay with human platelets.

Figure 1 Sequences of sand fly D7 proteins. (A) Comparison of long-form D7 sequences from Phlebotomus duboscqi (ABI15936), P. papatasi (AGE83092) and Lutozomyia longipalpus (LLOJ009780). Key residues contained in the N-terminal domain ligand binding pocket are highlighted in black. (B) Comparison of the N-terminal domain sequence from ABI15936 with those of AeD7 from Aedes aegypti, and AnStD7L1 from Anopheles stephensi. The highlighted residues are known to contact bound eicosanoid ligands in the crystal structures of protein ligand complexes (AeD7 PDB ID 3DZT, AnStD7L1 PDB ID 3NHT). Full size image

Ligand binding by long-form D7s from sand flies

We used ITC to examine the binding affinities of P. papatasi AGE83092 and P. duboscqi ABI15936 for the known ligands of mosquito D7 proteins, including biogenic amines, cysteinyl leukotrienes, and the thromboxane A 2 (TXA 2 ) analog U46619. The proteins were found to bind a single molecule of the cysteinyl leukotrienes LTC 4 , LTD 4 and LTE 4 (Fig. 2). The three ligands bound similarly, with LTC 4 and LTD 4 showing affinities (1/K a ) of 2–6 nM, while LTE 4 bound with a slightly lower affinity of 16–29 nM (Fig. 2). U46619 also proved to be a ligand for the D7 proteins exhibiting affinity constants of approximately 750 nM in AGE83092 and 1.3 µM in ABI15936, with apparently a single molecule binding per molecule of protein as in the mosquito homologs (Fig. 2). The proteins did not bind the biogenic amines histamine or serotonin, consistent with the lack of binding pocket conservation relative to AeD7 from Ae. aegypti or the single domain short-form D7s from An. gambiae (Fig. S1).

Figure 2 ITC analysis of eicosanoid binding. For cysteinyl leukotriene experiments, the calorimeter cell was filled with P. papatasi D7 protein AGE83092 (A–C) or P. duboscqi ABI15936 (E–G) at 2 μM in Tris-buffered saline. The syringe contained 20 μM LTC 4 (A,E), LTD 4 (B,F) and LTE 4 (C,G) in the same buffer. Injections (10 µL) were spaced at 300 s intervals. For U46619 (D,H), the protein concentration in the cell was 5 µM for AGE83092 (D) and 10 µM for ABI15936 (H). The syringe concentration was 50 µM AGE83092 and 100 µM for ABI15936. Heats were recorded on a VP-ITC MicroCalorimeter and data were fit using a single binding site model on the MicroCal software package (Origin 7) for calculation of thermodynamic parameters. Titration curves are representative of at least 3 measurements. Thermodynamic parameters: Panel A K a = 1.7 × 108 M−1, ΔH = −10.3 kcal/mol, N = 1.1 sites; Panel B K a = 5.7 × 108 M−1, ΔH = −10.2 kcal/mol, N = 1.1 sites; Panel C K a = 6.3 × 107 M−1, ΔH = −11.3 kcal/mol, N = 1.1 sites; Panel D K a = 1.33 × 106 M−1, ΔH = −8.5 kcal/mol, N = 0.6 sites; Panel E K a = 3.1 × 108 M−1, ΔH = −8.6 kcal/mol, N = 1.3 sites; Panel F K a = 2.4 × 108 M−1, ΔH = −8.9 kcal/mol, N = 1.3 sites; Panel G K a = 3.4 × 107 M−1, ΔH = −7.0 kcal/mol, N = 1.3 sites; Panel H K a = 7.8 × 105 M−1, ΔH = −11.5 kcal/mol, N = 0.25 sites. Full size image

Inhibition of platelet aggregation by sand fly D7s

On contact with collagen exposed by wounding, quiescent platelets release granules and begin secreting TXA 2 . The granule component ADP along with secreted TXA 2 potentiate the activation of platelets through specific G protein-coupled receptors, making them secondary agonists that are necessary for a robust platelet activation response. AnStD7L1, a long-form D7 from An. stephensi saliva has been shown to inhibit collagen- and U46619-mediated platelet activation indicating that it has the potential to block aggregation of platelets at the site of feeding9. A convincing case was made that this is due to direct binding and sequestration of the agonist TXA 2 . The aggregation of platelets in platelet-rich plasma was examined to determine if ABI15936 and AGE83092 exhibit similar inhibitory properties. We measured the activation and aggregation of platelet suspensions after exposure to 1.4 µg/mL collagen in the presence and absence of ABI15936 and AGE83092 (Fig. 3A). Collagen alone initiated a complete aggregation response as indicated by an increase in transmittance of dilute platelet suspensions, following a smaller decrease in transmittance due to platelet shape change (Fig. 3A). Addition of either AGE83092 or ABI15936 prior the collagen stimulus resulted in a concentration-dependent reduction in aggregation. Inhibition of aggregation was detectable at protein concentrations as low as 1.3 µM, and the inhibition was essentially complete at a concentration of 5.2 µM (Fig. 3A). At higher collagen concentrations, signaling through integrin receptors and platelet glycoprotein VI is sufficient to induce platelet aggregation without involvement of TXA 2 . We found that inhibition of platelet activation by either AGE83092 or ABI15936 was negligible at inhibitor concentrations of 5.2 µM when a collagen stimulus of 14 µg/mL was used (Fig. 3B). Inhibition of collagen-mediated aggregation at 1.4 µg/mL collagen, but not 14 µg/mL, is consistent with a mechanism of action involving TXA 2 binding.

Figure 3 Sand fly D7s inhibit collagen-induced platelet aggregation. 10 mM Tris buffer pH 7.4 (control) or different concentrations (indicated on figure) of P. dubosqi ABI15936 or P. papatasi AGE83092 in Tyrode buffer were added to a stirring platelet suspension at 37 °C and incubated for 1 min in the aggregometer. Platelet aggregation was observed as an increase in transmittance (rise of the baseline) after addition of collagen at concentrations of 1.4 μg/ mL (low concentration) (A) or 14 μg/ ml (high concentration) (B). Traces are representative of three experiments performed in duplicate. Full size image

To further establish the role of sand fly D7s as TXA 2 -binding proteins, we tested the effect of these on platelet aggregation induced by the TXA 2 analog U46619. Complete aggregation was seen after addition of 0.5 µM U46619 to a suspension of stirred platelets (Fig. 4). If either ABI15936 or AGE83092 were added and incubated with the platelet suspension prior to addition of agonist, the aggregation response was inhibited in a concentration-dependent manner (Fig. 4). Unlike the effects seen after collagen stimulation, inhibition appeared abruptly at concentrations at or above 0.5 µM protein. This is indicative of a sequestration mechanism, where the sequestering agent, in this case the D7 protein, needs to be present at a concentration equal to or higher than that of the ligand (Fig. 4). The effect is particularly pronounced when the concentration of ligand is near or above its dissociation constant. Taken together, experiments with collagen and U46619 indicate that the sand fly D7 proteins act as platelet aggregation inhibitors that function by sequestering TXA 2 . The lability of TXA 2 prevents direct measurement of its binding to the D7 proteins, but the potency of the protein in collagen-mediated aggregation assays strongly suggests that the proteins bind natural TXA 2 with similar or higher affinity than the analog U46619, as was previously seen with the An. stephensi D7 AnStD7L1.

Figure 4 Sand fly D7s inhibit U46619-induced platelet aggregation. Stirred platelets were incubated with 10 mM Tris buffer pH 7.4 (control) or buffer containing P. duboscqi ABI159356 or P. papatasi AGE83092 D7 proteins (at the concentrations indicated on the figure) for one minute. Platelet aggregation was induced by addition of U46619 at a final concentration of 0.5 μM and monitored as described in Fig. 3. Traces are representative of three experiments performed in duplicate. Full size image

The crystal structure of P. duboscqi ABI15936

The structure of ABI15936 was determined using single anomalous diffraction methods with a selenomethionine-substituted variant protein prepared in E. coli (Table 1). An initial model containing two protein monomers was built using the experimental electron density map and then positioned into a wild-type data set by molecular replacement. The model was then completed by iterative cycles of rebuilding and refinement. As suggested by sequence comparisons, the protein is made up of two linked odorant binding protein (OBP) domains with a well-ordered domain interface fixing their relative orientations (Fig. 5). The N-terminal domain contains a hydrophobic channel leading from the surface to the interior of the protein that is much like the eicosanoid binding pocket in Aedes and Anopheles long-form D7 proteins. The C-terminal domain appears generally like those of the mosquito proteins but does not contain the biogenic amine binding pocket seen in AeD7, the Ae. aegypti long D7 protein that binds both eicosanoids and biogenic amines (Fig. 5).

Table 1 Data collection and refinement statistics for the P. duboscqi ABI15936. Full size table

Figure 5 Ribbon diagram of P. duboscqi ABI15936. The models on the left and right are related by 90° rotation around the axis shown. The N-terminal domain is colored in red and the C-terminal domain in blue. Alpha helical segments are labeled α1-α13, and disulfide bonds are shown in stick representation in the right-hand figure and labeled DS1-DS5. Sulfur atoms are colored in yellow. The ligand Triton X-100 is also shown in the N-terminal binding pocket, in stick representation with carbon atoms colored light grey and oxygen colored red. Full size image

The N-terminal domain of ABI15936 contains two disulfide bonds that are conserved in the mosquito proteins. These link Cys 17 with Cys 47 and Cys 45 with Cys 93 (Fig. 5). The C-terminal domain contains three disulfides linking Cys 143 with Cys 159, Cys 155 with Cys 202 and Cys 192 with Cys 211 (Fig. 5). Like the mosquito long D7 proteins the N-terminal domain is made up of seven α-helical segments (labeled α1-7, Fig. 5). Segment α5 is shortened to a single turn in the sand fly protein and is partially replaced by an extended coil region connecting α5 and α6. The segment connecting the N-terminal and C-terminal domains is essentially identical in conformation to the long-form D7 proteins from mosquitoes. The relative orientation of the C-terminal and N-terminal domains to one another is also similar in the mosquito and sand fly proteins, but the positioning of the four helical segments (α10-α13) in the C-terminal domain is somewhat different than that of the mosquito proteins. The genomic sequences of AGE83092 and AGE83093 from P. papatasi show the presence of three exons in each gene. Exon 1 encodes a signal peptide, while exons 2 and 3 encode the N- and C-terminal domains, respectively, with the exon-intron boundary located in the linker region between the two domains (Fig. 6A,D). The salivary D7 AeD7 from Ae. aegypti contains five exons with exon 1 encoding the signal peptide (Fig. 6B,D). The N- and C-terminal domains are encoded by two exons each and a third intron is present in the region encoding the domain-linking sequence (Fig. 6B). AnStD7L1 from An. stephensi shows conservation of the intron positions in the N- and C-terminal domains, but lacks the intron in the domain linker region, giving a total of four exons with one encoding the signal peptide (Fig. 6C,D).

Figure 6 Salivary long-form D7 proteins from P. duboscqi, An. stephensi and Ae. aegypti colored according to exon structure. (A) P. duboscqi ABI15936 with the region encoded by exon 2 colored magenta and encompassing the entire N-terminal domain. The region encoded by exon 3 is colored green and includes the entire C-terminal domain. (B) Ae. aegypti AeD7 (3DYE) showing the N-terminal domain split into regions encoded by exon 2 (magenta) and exon 3 (cyan). The C-terminal domain is encoded by exons 4 (light blue) and 5 (green). (C) Like the Aedes protein, AnStD7L1 from An. stephensi (3NHT) has the C-terminal domain encoded by exons 2 (magenta) and 3 (cyan) but in this case exon 3 also encodes the first part of the C-terminal domain encoded by exon 4 in AeD7. The region encoded by exon 4 (green) corresponds to exon 5 of AeD7. (D) Schematic of the exon structure of sand fly and mosquito long-form D7 proteins. Horizontal lines represent the primary amino acid sequences of the coding regions color coded as in (A–C). The vertical lines represent the intron/exon boundaries with the amino acids at the joining sites listed. Full size image

Binding pocket of the N-terminal domain

Although ABI15936 was crystallized in the absence of any potential natural ligands, electron density was observed indicative of a non-protein molecule occupying the N-terminal domain binding site. This was determined after refinement of the protein complex to be the detergent Triton X-100, which was retained through the steps of protein purification (Figs 5, 7A,C). A second sample of protein was prepared in the absence of detergent, and the binding properties of the two samples were compared using LTC 4 as a ligand. Little difference in the equilibrium constant or other thermodynamic parameters was observed, indicating that Triton-X100 binding is not of sufficient affinity to exclude the host ligand at the micromolar concentrations used in the calorimetry experiments (Fig. 7B). We were unable to produce co-crystals of either the U46619 or LTC 4 complexes under the same conditions as the ligand-free protein and when crystals were soaked with cryoprotectant solution containing LTC 4 the resulting ligand electron density was not easily interpretable, suggesting a mixture of Triton-X100 and LTC 4 complexes, with the Triton-X100 complex predominating. Perhaps elevated detergent concentrations in the crystal or steric constraints in the crystal prevented efficient ligand exchange during the soak period.

Figure 7 Binding pocket interactions in ABI15936. (A) Stereoview of bound Triton X-100 with weighted omit electron density (Fo-Fc) contoured at 3 σ covering the ligand. Carbon atoms are colored green and oxygen atoms are colored red (B). Comparison of LTC 4 binding with ABI15936 prepared with (left, also shown in Fig. 2E) and without (right) Triton-X100 as measured by ITC using the same methods as in Fig. 2. Thermodynamic parameters without Triton X-100, K a = 2.2 × 108 M−1, ΔH = −10.0 kcal/mol, N = 1.2 sites. (C) Stereoview showing interactions of Triton X-100 with binding pocket residues of P. duboscqi ABI15936, also containing a modeled U46619 molecule positioned by superposition of the An. stephensi AnStD7L1-U46619 complex (PDB ID 3NHT). Triton X-100 atoms are colored as in panel A. In U46619, carbon is colored blue and oxygen red. In the protein, carbon is colored light grey, oxygen is colored red and nitrogen is colored blue. Potential hydrogen bonding interactions in the modeled U46619 complex are shown as dashed red lines. For modeling of the U46619 complex, only the position of the Lys 135 side chain was changed to avoid a steric clash. The refined position is shown in green and the rotated position in white. (D) Stereoview showing U46619 in the binding pocket of AnStD7L1 from An. stephensi (PDB ID 3NHT). Coloring of U46619 atoms, protein atoms and hydrogen bonds are as in panel C. Full size image

The binding pocket of ABI15936 is much like that of the mosquito long-form D7 proteins, with numerous residues important for ligand binding being conserved. The side chain of Lys 135 is oriented nearly identically to that of Lys 152 of AnStD7L1 where it is positioned to form a salt bridge with the carboxyl group of U46619 modeled into the pocket in the same conformation as in the crystal structure of the U46619-AnStD7L1 complex (Fig. 7C)9. The interior region of the pocket that accommodates the hydrophobic non-functionalized end of the eicosanoid is also highly conserved. Trp 35, Phe 14, Tyr 44 and Phe 115 are positioned much like their aromatic counterparts in AnStD7L1, and the aliphatic hydrophobic residues Leu 35, Val 48 and Leu 52 are also conserved in mosquito proteins (Fig. 7C,D). In previous studies, AeD7 from Ae. aegypti showed no affinity for U46619, while AnStD7L1 from An. stephensi bound this compound and was a potent platelet aggregation inhibitor8,9. A single phenylalanine to tyrosine change was responsible for the ability to bind TXA 2 since the tyrosine hydroxyl group forms an essential hydrogen bond with the hydroxyl group at ω-5 position of the fatty acid. Cysteinyl leukotrienes are not hydroxylated at this position and bind similarly to either protein. Both ABI15936 and AGE83092 contain tyrosine at this position, and as expected, both inhibit platelet aggregation as shown above. In the structure of ABI15936, Tyr 44 is oriented identically to the homologous residue Tyr 52 in AnStD7L1 and can be assumed to play the same role in binding (Fig. 7C,D).

Comparison of sand fly and mosquito C-terminal domains

The C-terminal domain of ABI15936 shows a clear structural relationship to mosquito long-form D7s but the protein does not contain the interior binding pocket known to accommodate biogenic amine ligands in the long-form D7 AeD7 from Ae. aegypti and the short-form D7 protein D7r4 from An. gambiae (Fig. 8). Its positional relationship with the N-terminal domain is almost identical to the mosquito long-form D7s as well as mJHBP, a hemolymph D7-like juvenile hormone (JH)-binding protein from mosquitoes, and the domain interface contains several conserved interactions15. Comparison of ABI15936 with AeD7 shows that the region connecting helices α9 and α10 is truncated relative to AeD7, eliminating the equivalent of α-helix B2 of AeD7 that makes up one side of the ligand binding pocket. Additionally, the peptide chain terminates at Tyr 230, resulting in loss of the terminal helix H2 of AeD7, which forms a second side of the binding pocket. The An. stephensi long-form D7 AnStD7L1 and Ae. aegypti mJHBP also do not bind biogenic amines, and their crystal structures show rearrangement in the binding pocket region of the C-terminal domain related to the loss of α-helix B2 and changes in the positioning of the terminal α-helix that explain the absence of this function. However, the changes are not related to those seen in ABI15936 and involve rearrangements of structural elements rather than the absence of significant stretches of peptide sequence.