Aedes aegypti D7 in Complex
with Norepinephrine and Leukotriene

  Jack Crow '18 and Chris Link '17


I. Introduction

Bloodfeeding is essential for preparation, and subsequent success, of reproduction in female mosquitoes.1 Bloodfeeding is also the the primary mode by which mosquito-borne diseases are transmitted. 1 Consequently, there is a competitive balance between mosquito salivary proteins and the host inhibitory proteins that respond to the bite. Aedes aegypti saliva contains over one hundred unique proteins, with a diverse array of functions. These proteins respond to a number of different hemostatic and inflammatory host defenses. 1 Many of these proteins also play an important role in facilitating bloodfeeding. Mosquitoes must be able to penetrate the epithelial cells, and extract blood in a time-efficient manner. Inefficient bloodfeeding substantially increases the odds that the bloodfeeding will be unsuccessful, and may in turn lower disease transmission. 1 2

However, increased bloodflow also carries host defense mechanisms to the source of the bite. In this light it is not surprising that such a diverse network of interactions between salivary proteins and host defense mechanisms rapidly becomes exceedingly complex, in terms of interactions.

The mosquito D7 proteins are a family of salivary proteins distantly related to the odorant-binding superfamily.2 D7 proteins have diverse function across different mosquito genera. In Aedes spp. the D7 protein have bifunctional biogenic amine and bioactive lipid mediator binding activity. 3 The bifunctional properties in the Aedes aegypti D7 protein (AeD7) are spatially separated into two binding domains. During binding the domains undergo a conformational change that stabilizes the molecule, and may increase AeD7 affinity for a larger number of host defensive substrate. 3

This review addresses the structure, and binding properties of AeD7 elucidated by Calvo et al. in 2009. 3 We attempt to summarize the molecular mechanics involved in both biogenic amine and leukotriene binding, as well as emphasize the conformational effects binding has upon the structure of the ligand-free protein. We also seek to discuss some of the implications structural analyses have on future research on similar compounds.  


II. General Structure PDB:3DXL

AeD7 consists of two binding domains. The biogenic amine binding domain occupies the N-terminus . The leukotriene binding domain occupies the C-terminus. Each domain in ligand-free AeD7 contains seven alpha helices. The ligand-free structure is maintained by five disulfide bonds, four of which occur in other members of the arthropod OBP family. The residues that form the disulfide bond linking helix B with the C-terminus, 16 and 53, only occurs in Aedes spp. D7 proteins.

III. AeD7 Norepinephirine Complex PDB: 3DYE

AeD7 binds biogenic amines in the C-Terminus. (residues 285-301) is ordered into an alpha helix (H2). This helix corresponds to the position of the eighth helix in anopheline D7. 1 Helices A2, B2, C2, G2 and H2 interact with the biogenic amine. The binding of the ligand and the formation of helix H2 results in the rotation of residues Arg-176 and Glu-268. The rotation of these residues forms a gate, ostensibly closing the binding pocket around the ligand. The ligand is contacted by residues His 289 and Asp 265. His 189 forms two hydrogen bonds with hyrdroxyl groups on the aromatic ring. Asp 265 forms a hydrogen bond with the amine group on the aliphatic region of the biogenic amine. The electrostatic interaction between the ligand, these residues, and other surrounding residues play an important role in stabilizing ligand binding and the bound complex. For clarity, the white discs that appear in the electrostatic button represent points of electrostatic contact between the ligand and amino acid residues, they do not represent the structure of either molecule.

*For visualization of the areas of electrostatic contact without the full electrostatic map, re-click the "contacting residues" button.

IV. AeD7 Leukotriene Complex PDB: 3DZT

AeD7 binds host bioactive lipid mediators involved in the inflammatory response, such as leukotrienes, in the N-terminal domain. The ligand-binding pocket is bounded by helices A–C, F and G. When bound, the lipid chain of the leukotriene, or other host lipid inflammatory compound, is inserted into the binding channel. Positional isomers that contained gluthionine conjagated at C14 of the lipid chain and a hydroxyl group at C15 (14,15-LTC4) showed no detectable binding. 3 It is this unconjugated lipid chain in leukotriene that forms hydrogen bonds with Lys-149 and Thr-135. At C5 of leukotriene the hydroxyl group forms hydrogen bonds with Trp-37 and Gly-130. Unlike the changes that accompany norepinephrine binding, leukotriene binding induces little conformational change to the C-terminus. Again, ligand binding is heavily mediated by electrostatic interactions with the surrounding AeD7 residues. As in the norepinephrine complex, the white discs that appear in the electrostatic button represent points of electrostatic contact between the ligand and amino acid residues.

*For visualization of the areas of electrostatic contact without the full electrostatic map, re-click the "contacting residues" button.

V. Conclusions and Implications

The bifunctional properties in the Aedes aegypti D7 protein (AeD7) are spatially separated into two domains. As previously shown, during norepinephrine binding the domains undergo a conformational change, stabilizing the molecule. It has been suggested that this conformational change increases AeD7 affinity for a wider diversity of host defensive substrate. This assumption would be in accordance with the relatively rapid evolutionary race between host defense compounds, and mosquito bloodfeeding proteins.

As previously discussed, mosquito salivary proteins often interact with each other, as well as with host defense compounds. These interactions form a complex network of pathways to combat host defense mechanisms. However, certain salivary proteins function relatively independently of other salivary proteins. It is difficult to determine the extent of such interactions from structural studies alone. Therefore, the path to understanding how these proteins function is also tied to binding assays, containing multiple proteins and substrate, to assess characteristics of cooperation in salivary proteins. These assays are further complicated by salivary proteins that exhibit varying structural states depending on the substrate present, such as the conformational change in AeD7 in the presence of norepinephrine.

In this review we discussed how norepinephrine binding in the C-terminus causes a conformational change in domains beyond the binding pocket. Conformational changes do not necessarily implicate functional variance upon ligand binding. However, it should be noted that conformational changes can induce functional change. Therefore, such models may provide an additional level of support, in developing assays for determining protein function, and functional changes in the presence of different substrate and different physiological environments.

As previously discussed, elucidation of salivary protein function provides a pathway for inhibition of bloodfeeding; exploring salivary protein function may also reveal proteins that play a limited role in bloodfeeding success, but are essential for disease transmission. Such a protein would be the ideal target for genetic manipulation that decreases vector capability, without decreasing host fitness.

Interestingly, reconstituted AeD7, among other bloodfeeding related compounds, has been shown to be present in fractionation samples that exhibit inhibitory effects on the dengue virus.4 Furthermore, binding assays have revealed direct interactions between AeD7 and dengue virus. 1 While interaction does not constitute binding, these studies suggest AeD7 may suppress dengue virus transmission. It is astounding that AeD7, a salivary protein that targets host inflammatory response elements, may also interact with a completely different type of substrate. These findings emphasize the striking complexity of mosquito salivary proteins, and the breadth of unelucidated information regarding their function.    

VI. References

1. Ribeiro JM and Francischetti IM. 2003. Role of arthropod saliva in blood feeding: Sialome and post-sialome perspectives*. Annu Rev Entomol 48(1):73-88.

2. Calvo E, Mans BJ, Andersen JF, Ribeiro JMC. 2006. Function and evolution of a mosquito salivary protein family. Journal of Biological Chemistry 281(4):1935-42.

3. Calvo E, Mans BJ, Ribeiro JM, Andersen JF. 2009. Multifunctionality and mechanism of ligand binding in a mosquito antiinflammatory protein. Proc Natl Acad Sci U S A 106(10):3728-33.

4. Conway MJ, Londono-Renteria B, Troupin A, Watson AM, Klimstra WB, Fikrig E, Colpitts TM. 2016. Aedes aegypti D7 saliva protein inhibits dengue virus infection. PLoS Negl Trop Dis 10(9):e0004941.

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