|In Silico Biology 7, 0001 (2006); ©2006, Bioinformation Systems e.V.|
Centre for Research in Medical Entomology
(Indian Council of Medical Research)
4, Sarojini Street, Chinna Chokkikulam, Madurai-625002.
Tamil Nadu, India
1 Department of Bioinformatics, Bharathiar University, Coimbatore.
Tamil Nadu, India.
* Corresponding author
Edited by H. Michael; received April 21, 2006; revised November 15, 2006; accepted November 16, 2006; published January 08, 2007
Olfaction of insects is currently recognized as the major area of research for developing novel control strategies to prevent mosquito-borne infections. A 3-dimensional model (3D) was developed for the salivary gland odorant-binding protein-2 of the mosquito Culex quinquefasciatus, a major vector of human lymphatic filariasis. A homology modeling method was used for the prediction of the structure. For the modeling, two template proteins were obtained by mGenTHERADER, namely the high-resolution X-ray crystallography structure of a pheromone-binding protein (ASP1) of Apis mellifera L., [1R5R:A] and the aristolochene synthase from Penicillium roqueforti [1DI1:B]. By comparing the template protein a rough model was constructed for the target protein using MODELLER, a program for comparative modelling. The structure of OBP of the mosquito Culex quinquefasciatus resembles the structure of pheromone-binding protein ASP1 of Apis mellifera L., [1R5R:A]. From Ramachandran plot analysis it was found that the portion of residues falling into the most favoured regions was 86.0%. The predicted 3-D model may be further used in characterizing the protein in wet laboratory.
Keywords: salivary odorant-binding protein-2, homology modeling, pheromone-binding protein, aristolochene synthase, Culex quinquefasciatus, 3-D model, MODELLER, mGenTHERADER, X-ray crystallography, predictive model, Swiss-PdbViewer, PROCHECK
Olfaction plays a major role in the behavior of insects in finding food sources, selection of mates and oviposition sites . The olfactory system of terrestrial animals has an extreme sensitivity and specificity. It can detect and discriminate a large number of olfactory signals, the odorants. Olfactory perception is accomplished by specialized bipolar sensory neurons that extend their dendrites into an aqueous medium, the olfactory mucus in vertebrates and the sensillar fluid in insects.
Hence, the airborne molecules must traverse the aqueous space that separates neuronal cells from the external air and simulate the odorant receptors. These receptors are located on the dendritic membrane of the sensory neurons.
The odorant-binding proteins (OBPs) are abundant low-molecular-weight proteins that bind and solubilize hydrophobic odorants (or pheromones) in the vertebrate olfactory mucus and in the insect sensillar lymph. These small globular proteins are synthesized and secreted by some accessory cells surrounding the sensory neurons. In insects, the OBP family includes the general odorant-binding proteins (GOBPs) and the pheromone-binding proteins (PBPs), which are not homologous to vertebrate odorant-binding proteins.
Despite the low sequence similarity among different insect OBPs, most of these proteins exhibit a similar distribution of conserved hydrophobic residues with a nearly identical predicted secondary structure. Most proteins of this family contain six highly conserved cysteines located in similar positions of the protein. In Lepidoptera, these cysteines are involved in disulfide bridges in both PBPs and GOBPs. The similar distribution of cysteine residues in both groups of OBPs suggests that the disulfide-bridge pairing might be a general feature of this family of molecules in insects. Although the specific function of OBPs in olfaction is still unknown, they seem to play an important role in olfactory coding. It has been shown that several OBPs have different odorant specificities and are present in distinct subsets of antennal sensilla. Additionally, genes encoding olfactory receptors with different binding specificities are also expressed in specific areas of the olfactory organ. These observations suggest that these proteins might participate in odor detection by restricting the spectrum of odorants accessible to the underlying receptors. In addition to the established functions of OBPs as carrier molecules and in concentrating hydrophobic odorants in the aqueous medium, it has also been proposed that these proteins could participate in the deactivation of the odorant stimulus.
An OBP carries the odor molecules from antennal sensilla to odorant receptors (OR) located in the olfactory neurons . The association of different OBPs with different odorant-binding specificities in a single insect has been reported [3, 4]. Recently, olfaction has been recognized as the important area of research for developing novel control strategies by interfering with these mechanisms. To develop effective interference mechanisms, understanding the composition, chemical nature and the structure of odorant binding protein is essential.
Bancroftian filariasis is considered as the predominant infection in the continental Asia  and Culex quinquefasciatus (the Southern house mosquito) is the principal vector. Though the vector control strategies are ongoing, understanding the biology of the vector mosquitoes at the protein level is essential for developing novel control methods. The first isolated OBP was from Culex quinquefasciatus mosquito (CquiOBP), which is a hydrophobic signal peptide (24 residues). This protein was found to have highest amino acid identity (58.6%) with the OBP PBPRP-3 of Drosophila melanogaster . The salivary gland odorant-binding protein-2 of Culex quinquefasciatus mosquitoes was submitted to the sequence databank by Ribeiro et al., 2003 (http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=74795259). Studies trying to elucidate the role different proteins play in disease transmission of various mosquito vectors of public health importance have been underway.
Comparative modeling or homology modeling (HM) is becoming a useful technique in the field of bioinformatics because the knowledge of the three-dimensional structure of a protein would be an invaluable aid to understand the details of a particular protein. By using the bioinformatics tools, a three-dimensional structure model of putative salivary odorant-binding protein-2 of Culex quinquefasciatus was constructed by HM. The results are presented here.
About 29 candidate OBP of Anopheles gambiae have been characterized for similarity with Drosophila melanogaster and other insects . The three-dimensional (3D) structure details of proteins are of major importance in providing insights into their molecular functions. Further analysis of 3D structures will help in the identification of binding sites and may lead to the designing of new drugs. The protein sequence of the putative salivary odorant-binding protein-2 of Culex quinquefasciatus (Southern house mosquito) was obtained from the NCBI sequence database. Multiple alignment of the primary structure of the target protein highlights the degree of sequence conservation and high sequence similarity.
Homology modeling is only a viable technique because it produces models that can be used for further research. Homology modeling helps in predicting the 3-D structure of a macromolecule with unknown structure (target) by comparing it with a known template from another, structurally highly similar, macromolecule. The structure of the target protein is structurally similar with the template if both the target and template sequences are similar. In general, 30% sequence homology is required for generating useful models. Here, the sequence alignment score was 44 as calculated by ClustalW (http://www.ebi.ac.uk/cgi-bin/clustalw/result?tool=clustalw&jobid=clustalw-20061129-05080236&poll=yes). In our study, based on the results obtained from mGenTHREADER program, the X-ray structure of the pheromone-binding protein Asp1 of Apis mellifera L., [1R5R:A] and the aristolochene synthase from Penicillium roqueforti [1DI1:B] were selected as templates. MODELLER was used for building the model and global energy minimization. The sequence was obtained from sequence database and was submitted to blastp search. After the BLAST analysis, PROCHECK was used to validate the model.
The total energy values of the predicted 3-D model were calculated as 86.0% of Ramachandran plot (Fig. 1A) value in 30 and 40 steepest descent and conjugate gradient, respectively.
|Figure 1: (A) Predicted 3-D structure of OBP-2: Ramachandran plot analysis. Based on analysis on 118 residues of resolution of at least 20 Å and R factor no greater than 30%, a good quality model would be expected to have over 90% in the most favoured regions. The Plot statistics are: residues in most favoured regions [A,B,L] - 104 (86.0%); residues in additional allowed regions [a,b,l.p] - 14 (11.6%); residues in generously allowed regions [-a,-b,-l,-p] - 3 (2.5%); residues in disallowed regions - 0; number of non-glycine and non-proline residues- 121 (100.0%); number of end residues (excl. Gly and Pro) - 2; number of glycine residues (shown as triangles) - 9; number of proline residues - 7; total number of residues- 139. (B) Predicted 3-D structure of OBP-2 of Culex quinquefasciatus.|
The refined model was analyzed by different protein analysis programs including PROCHECK for the evaluation of the Ramachandran plot quality, and WHATIF  for the calculation of packing quality. This structure (Fig. 1B; see Supplementary material for the corresponding coordinates in pdb format) was found to be satisfactory based on the above results. The predicted 3-D model of the salivary odorant-binding protein-2 of Culex quinquefasciatus will be very useful in wet laboratory while studying the real structure of the protein.