Protein modeling of apical membrane antigen-1 ( AMA-1 ) of Plasmodium cynomolgi

Apical membrane Antigen-1(AMA-1), an asexual blood stage antigen of Plasmodium cynomolgi, is an important candidate for testing as a component of malarial vaccine. The degree of conservation of AMA-1 sequences implies a conserved function for this molecule across different species of Plasmodium. Since the AMA-1 of plasmodium cyanomolgi is yet to be structured, the authors have generated a homology model of AMA-1 by using the Swiss-PDB server. The protein’s conservity has been verified by performing multiple alignments using Bioedit and conserved domain database. The model was further checked for its correctness by predicting 2D and 3D structures, which validates the structure.


INTRODUCTION
Most of the activities in living organisms are regulated by proteins.All proteins start on a ribosome as a linear sequence of amino acids, this linear sequence must fold during and after the synthesis, so that the protein takes up its native conformation.The native conformation of protein is a stable three dimensional structure that strongly determines a protein's biological function.The knowledge of detailed structural organization is crucial in understanding the role of protein in the cell and the related molecular mechanisms.Insight of three dimensional (3D) structure of a protein are of great assistance when planning experiments aimed at the understanding of protein function and during the drug design process.The experimental elucidation of the 3D structure of the proteins is however often hampered by difficulties to obtain sufficient protein, diffracting crystals and many other technical aspects.The number of solved 3D structures is small, when compared to the rate of sequencing of genes, and no structural information is available for the vast majority of the protein sequences.
At this juncture, prediction methods have gained much *Corresponding author.E-mail: drdmmamatha@gmail.cominterest (Schwede et al. 2003).Genome sequencing projects continuously detect new protein sequences, this provides new information for the application of computational methods, representing a good alternative to the relatively slow experimental processes for predicting protein structure (Rodriguez et al., 1998, West Head et al., 1998).This study focuses on building molecular model of AMA-1 protein of Plasmodium cynomolgi using molecular modeling techniques using SWISS MODEL-SERVER (Guex and Peltsch, 1997) as follows: To build a theoretical model for AMA-1 protein by homology modeling and to study the fundamental characteristics of AMA-1 protein by comparative analysis with the parent/template.The model was generated using SWISS-MODEL v 3.7.an automated protein modeling server available at www.expasy.org.

MATERIALS AND METHOD
Homology/comparative modeling is carried out, if the native sequence similarity with the similar crystal/NMR structure is greater than 35%.The process of building a comparative model is conceptually straight forward.First, an alignment is performed between the sequences for which the structure has been determined by experimental methods (the parent/template) with the sequence to be modeled (the target, in our case AMA-1 of P. cynomolgi) The comparative model of AMA-1 was built using a web-based homology-modeling server, SWISS-MODEL.It predicts the model in 5 steps.Firstly, it searches for suitable template for the submitted protein to be modeled in the EXNRL-3D database using BLAST P2 program.In the second, step it selects the suitable templates with sequence identity above 25%.Furthermore, this step also detect domains, which can be modeled based on unrelated templates .Thirdly, jobs are created for PROMOD2 module of SWISS MODEL server, which generates all the probable models of the submitted protein sequence in the fourth step.Fifth step minimizes the energy of all the predicted models with GROMOS96 module of SWISS MODEL server (Van Gunsteren et al., 1996) (GROningen Molecular Simulation).Finally, the best 3D model having minimum energy is sent to the submitter.
The sequence identity of known structure was checked with AMA-1 protein using SIM program that selected all templates with sequence identities above 25%.Furthermore, this step also detected the AMA-1 domains which were further cross checked with CDD (conserved domain database) Garnier et al., (1978).The energy minimization of the generated model was done with GROMOS96 (GROningen, Molecular simulation) (Peitsch, 1995;Peitsch et al., 2000).

Secondary and tertiary structure prediction
The multiple alignments of AMA-1 protein from Plasmodium sp. were generated using Bioedit and conserved domain database.The secondary structure of the model was predicted by using NPS@GOR4, predict protein server and SWISS-MODEL server.The model was analyzed for its correctness using PROCHECK.

RESULTS AND DISCUSSION
The multiple alignments of protein sequences (Dutta et.al., 1995) of the AMA-1 of P. cynomolgi highlights the degree of sequence similarity (Cheng and Saul, 1994) and high degree of sequence conservation with the templates selected, which implies the conserved function for this molecule across the different species of Plasmodium Figure 1, http://www.ncbi.nlm.nih.gov/BLAST/,http://www2.ebi.ac.uk/clustalw/.Pair-wise comparison (Bairoch and Rolf, 1997;Bairoch et al., 2004) between all the known amino acid sequences of AMA-1 homologs indicate greater than 50% identity, with 16 cysteine residues conserved in all sequences.All these cysteins are found in the ectodomain of the molecule, which are stabilized by 8 intramolecular disulphide bonds (Figure 2).The ability of this molecule to induce a protective immune response has been shown to be dependent up on a conformation stabilized by disulphide bonds (Hodder et al., 1996).The secondary structure predicted by protein server (Tables 1  and 2) matched almost exactly with the secondary structure of AMA-1 homology model (Figure 3).The over all summary (Guex and Peltsch, 1997) of AMA-1 protein was classified as 24 β-sheets, 7 helices and 27 turns.The results from SWISS MODEL and predict protein almost coincided exactly and also shown a high degree of conservity (Marshall et al., 1989) to the parent /template used.
Energy minimization/computations were done with GROMOS 96 implementation of SWISS-PDB SERVER (Van Gussteren et al., 1996) (Table 3).Further, tertiary structure validation done on the Procheck z-score shown 84.2% resides in the most favored regions, 13.4% in the additional allowed regions, 1.2% in the generously allowed regions and 1.2% in the disallowed regions for   the generated homology model of AMA-1, to the normal Z-score of parent /template shown 83.9% residues in the most favoured regions, 13.3% in the additional allowed regions, 1.2% in the generously allowed regions and1.2% in the disallowed regions (Table 4).The Z-score results of the generated model almost coincides to the Zscore results of the parent/template used, thus proving the model to be almost correct.

Conclusion
Using protein modeling tools, building a homology model for this protein has been accomplished and visualized.In the absence of 3D structure of AMA-1 protein of P. cynomolgi, the generated protein model of AMA-1 may be used to understand the antigenic sites and regulatory function by visualizing its domains and structural conformation (Rajesh and Saravanan, 2005;Yogesh et al. 2005).The degree of conservation of AMA-1 sequence and structure implies a conserved function for this molecule across different species of Plasmodium, Mary et al., (1996).Apical membrane Antigen (AMA-1), which is an asexual blood-stage antigen of Plasmodium, is an important candidate for testing as a component of a malarial vaccine (Crewther et al., 1990;1996).However, before using the model for further work, it should be checked for its geometrical, stereo chemical and conformational accuracy before taking up for rational drug design, to avoid later complications.

Figure 3 .
Figure 3.The structure of the protein.Secondary structure validation of AMA-1 protein.

Table 3 .
Energy computations of AMA-1 protein done with the GROMOS96 implementation of SWISS-PDB viewer.