In Silico Biology 7, 0001 (2006); ©2006, Bioinformation Systems e.V.  


Prediction of 3-dimensional structure of salivary odorant-binding protein-2 of the mosquito Culex quinquefasciatus, the vector of human lymphatic filariasis


Rajaiah Paramasivan*, Ramamoorthy Sivaperumal1, Kutty Jegadeeswaran Dhananjeyan, Velayutham Thenmozhi and Brij Kishore Tyagi




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
   Email: rpsivan2000@yahoo.co.in





Edited by H. Michael; received April 21, 2006; revised November 15, 2006; accepted November 16, 2006; published January 08, 2007



Abstract

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



Introduction

Olfaction plays a major role in the behavior of insects in finding food sources, selection of mates and oviposition sites [1]. 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 [2]. 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 [5] 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 [6]. 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.



Methods

Retrieval of target sequence:
The amino acid sequence of the putative salivary odorant binding protein-2 of Culex quinquefasciatus was obtained from the sequence database of NCBI (www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=74795259) (ID: Q6TRY1). It was ascertained that the three-dimensional structure of the protein was not available in Protein Data Bank (http://www.rcsb.org/pdb/home/home.do), hence the present exercise of developing the 3D model of the putative salivary odorant-binding protein-2 of Culex quinquefasciatus was undertaken. The protein is 139 amino acids in length with a molecular weight of 15,426 Da.
Template searching:
An attempt was made to find a suitable template protein for the modeling of the target protein. The template protein was searched through mGenTHREADER [7], which is an online tool for searching similar sequences, based on sequence and structure-wise similarity. From the homology searching, two templates were selected. High-resolution X-ray crystallography structure of the pheromone-binding protein ASP1 of Apis mellifera L., [1R5R:A] [8] and the aristolochene synthase from Penicillium roqueforti [1DI1:B] [9] were selected as template proteins. From the query protein, the first 17 residues MKCSPITLLALASIITL were modeled by comparing with 1DI1.pdb as the template. Remaining residues were modeled using 1R5R.pdb, since the first 15 residues of the target protein were not aligned with 1R5R.pdb.
Sequence Alignment:
Amino acid sequence alignment of target and template proteins was derived using the Swiss-PdbViewer package (http://www.expasy.ch/spdbv/). Default parameters were applied and the aligned sequences were inspected and adjusted manually to minimize the number of gaps and insertions.
Rough Model:
A rough 3-D model was constructed from the sequence alignment between OBP and the template proteins using MODELLER 8v0 [10] (http://salilab.org/modeller/) with parameters of energy minimization value.
Refinement:
The rough model constructed was solvated and subjected to constraint energy minimization with a harmonic constraint of 100 kJ/mol/Å2, applied for all protein atoms, using the steepest descent and conjugate gradient technique to eliminate bad contacts between protein atoms and structural water molecules. Computations were carried out in vacuo with the GROMOS96 43B1 parameters set, implementation of Swiss-PdbViewer.
Evaluation of Refined Model:
In the last step of homology modeling the refined structure of the model was subjected to a series of tests for testing its internal consistency and reliability. Backbone conformation was evaluated by the inspection of the Psi/Phi Ramachandran plot obtained from PROCHECK (http://biotech.ebi.ac.uk:8400/cgi-bin/sendquery) [11] analysis. The Swiss-PdbViewer energy minimization test was applied to check for energy criteria in comparison with the potential of mean force derived from a large set of known protein structures. Packing quality of the refined structure was investigated by the calculation of PROCHECK Quality Control value.



Results and discussion

About 29 candidate OBP of Anopheles gambiae have been characterized for similarity with Drosophila melanogaster and other insects [12]. 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 [13] 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.




References


  1. Hekmat-Scafe, D. S., Scafe, C. R., McKinney, A. J. and Tanouye, M. A. (2002). Genome-wide analysis of the odorant-binding protein gene family in Drosophila melanogaster. Genome Res. 12, 1357-1369.

  2. Justice, R. W., Dimitratos, S., Walter, M. F., Woods, D. F. and Biessmann, H. (2003). Sexual dimorphic expression of Putative antennal carrier protein genes in the malaria vector Anopheles gambiae. Insect Mol. Biol. 12, 581-594.

  3. Du, G., Ng, C. S. and Prestwich, G. D. (1994). Odorant binding by a pheromone binding protein: active site mapping by photoaffinity labeling. Biochemistry 33, 4812-4819.

  4. Plettner, E., Lazar, J., Prestwich, E. G. and Prestwich, G. D. (2000). Discrimination of pheromone enantiomers by two pheromone binding proteins from the gypsy moth Lymantria dispar. Biochemistry 39, 8953-8962.

  5. Gyapong, J. O., Kumaraswami, V., Biswas, G. and Ottesen, E. A. (2005). Treatment strategies underpinning the global programme to eliminate lymphatic filariasis. Expert Opin. Pharmacother. 6, 179-200.

  6. Ishida, Y., Cornel, A. J. and Leal, W. S. (2002). Identification and cloning of a female antenna-specific odorant- binding protein in the mosquito Culex quinquefasciatus. J. Chem. Ecol. 28, 867-871.

  7. McGuffin, L. J. and Jones, D. T. (2003). Improvement of the GenTHREADER method for genomic fold recognition. Bioinformatics 19, 874-881.

  8. Lartigue, A., Gruez, A., Briand, L., Blon, F., Bezirad, V., Walsh, M., Pernollet, J. C., Tegoni, M. and Cambillau, C. (2004). Sulfur single-wavelength anomalous diffraction crystal structure of a pheromone-binding protein from the honeybee Apis mellifera L. J. Biol. Chem. 279, 4459-4464.

  9. Caruthers, J. M., Kang, L., Rynkiewicz, M. J., Cane, D. E. and Christianson, D. W. (2000). Crystal structure determination of aristolochene synthase from the blue cheese mold, Penicillium roqueforti. J. Biol. Chem. 275, 25533-25539.

  10. Marti-Renom, M. A., Stuart, A. C., Fiser, A., Sanchez, R., Melo, F. and Sali, A. (2000). Comparative protein structure modeling of genes and genomics. Annu. Rev. Biophys. Biomol. Struct. 29, 291-325.

  11. Laskowski, R. A., MacArthur, M. W., Moss, D. S. and Thornton, J. M. (1993). PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Cryst. 26, 283-291.

  12. Vogt, R. G. (2002). Odorant binding protein homologues of the malaria Anopheles gambiae; possible orthlogues of the OS-E and OS-F OBPs of Drosophila melanogaster. J. Chem. Ecol. 28, 2371-2376.

  13. Vriend, G. (1990). WHAT IF: A molecular modeling and drug design program. J. Mol. Graph. 8, 52-56.