Research Journal of Recent Sciences ______ ______________________________ ______ ____ ___ ISSN 2277 - 2502 Vol. 1( 4 ), 68 - 71 , April (201 2 ) Res.J. Recent Sci. International Science Congress Association 68 Short Communication In - Silico Structure Determination of Protein Falstatin from Malaria Parasite Plasmodium Falciparum Bhatt Tarun Kumar Department of Biotechnology, Central University of Rajasthan, Kishangarh, INDIA Available online at: www.isca.in (Received 6 th March 2012 , revised 10 th March 2012 , accepted 12 th March 2012 ) Abstract Malaria is the major cause of socio - economic loss to most of the developing countries. Several drugs have been developed against the deadly malaria causing protozoan, Plasmodium falciparum. However, development of drug resistance against existing drugs has necessitated the identification of new drug targets. Several proteases have been identified from malaria parasite which is involved in various processes like haemoglobin degradation, egress of merozoite etc. But more important aspect of malaria biology is the regulation of these proteases for effective regulation of parasite life cycle. Falstatin is such a protein which binds to many cysteine proteases and regulates their activities. Therefore, Falstatin is the potential target for drug discovery. In this study, we determined the three - dimensional structure of Falstatin by molecular modelling using Swiss Modeller and Sali’s Modeller. Ramachandran plot was used for structure validation. Falstatin active site was determined using CastP. Structural analysis o f Plasmodium Falciparum Falstatin (Pf - Falstatin) could be instrumental in identifying new drug like molecules . Keywords: Falstatin, c ysteine proteases, molecular modelling, malaria, drug discovery . Introduction Malaria is one of the most devastating infectious diseases in the world. More than 100 million deaths are reported annually (WHO report, 2009). Plasmodium is the causative agent of the malaria disease. Several species of genus Plasmodium are present in the nature, out of which four species infect only human ( Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae ) . The life cycle of malaria parasites is complex, with the asexual stages occurring in mammalian hosts and the sexual st ages in female Anopheles mosquito vectors. Malaria is transmitted by the bite of a mosquito in which hundreds of sporozoites are released into the host bloodstream. The parasites eventually migrate to the liver, passing through cell types such as Kupfer ce lls and form parasitophorous vacuoles in hepatocytes. At this stage, they can either remain dormant as a hypnozoite form (P. vivax or P. ovale), or initiate development that results in the production of thousands of merozoites. The parasites then released from the infected hepatocyte and invade erythrocytes where they replicate in a cycle that may correspond to the cycle of fever and chills in malaria. Some parasites differentiate into male and female gametocytes, which are the forms taken up by the mosquit o where sexual life of parasite continues. Genome sequencing of the Plasmodium falciparum has been almost completed . There are over 30 predicted sequences of cysteine proteases 1 . Several of them have been biochemically characterized 2 . These cysteine proteases are shown to involve in various critical processes like haemoglobin degradation, erythrocyte egression and erythrocyte invasion of merozoites 3 - 9 . However, regulation of these proteases is very important in terms of proper nutrition for the paras ite inside host cell and also for the infection to be established. Pf Falstatin is one of the endogenous regulators of the cysteine proteases in the malaria parasite similar to the Chagasin, a cysteine proteases inhibitor in Trypanosoma cruzi 10 - 11 . PfFalst atin is known to inhibit many cysteine proteases and specifically inhibit PfFalcipain 2 proteases by binding to the active site of the falcipain 2 enzyme 12 . Temporal expression of this parasite protein is very critical in invasion of erythrocyte by merozoit es 13 - 15 . Thus, elucidation of three - dimensional structure of PfFalstatin will not only help in the understanding of erythrocyte invasion but also lay the foundation of identifying new effective inhibitors against the malaria parasite. Material and Methods The sequence of PfFalstatin was retrieved from PlasmoDB (PFI0580c). 3PNR and 2C34 pdb structures were used as a template for homology modeling. Identification of template structures was carried out using NCBI BlastP. Sali’s Modeller 16 and Swiss Model Server were used to build the in - silico structure of PfFalstatin. Structure validation was performed with ramachandran plot using online server rampage 17 . Modelled structure of PfFalstatin was submitted to CASTp for active site prediction 18 . Figures and images were developed using chimera 19 . Results and Discussion The modelled structure of PfFalstatin is shown in f ig ure 1. Panel A represent the ribbon diagram of structure while panel B Research Journal of Recent Sciences ______ _ _ _______________________________ ______________ _ ____ __ __ ISSN 2277 - 2502 Vol. 1( 4 ), 68 - 71 , April (201 2 ) Res. J. Recent Sci. International Science Congress Association 69 shows the surface topology of PfFalstatin . Helices, sheets are present along with loops in the structure. An extended loop is also present with short helix in it. Surface topology diagram shows that most of the surface is positively charges with intermittent negative charged patches. When structu re of PfFalstatin was compared with its homologous structure of Plasmodium berghei , several distinct features were observed in spite of similar fold of the proteins ( f ig ure 2). Two helices were missing in the P. berghei falstatin structures compared to PfF alstatin, where both the helices were nicely build and modelled. In addition, structure validation was done by using ramachandran plot which shows that most of the residues are in favoured and allowed region ( f ig ure 3). Further, prediction of active site o f PfFalstatin was carried out with CASTp ( f ig ure 4). Computed a tlas of s urface t opography o f p roteins (CASTp) gave the prediction of actve site and the number of amino acid involved as best active site with volume of 223.5 and area of 203.7. P anel A of the fig ure 4 shows the probable active site in one - dimension protein sequence whereas lower panels depict the active site residues in three - dimensional space (B and C). Conclusion Very little is known about the three - dimensional structure of protein Falst atin from Plasmodium falciparum and also it is very difficult to determine structure of proteins experimentally. This lack of information clearly blocks the possibility of transferring available facts of cysteine proteases regulation for development of new anti - malarial drugs. Thus, an in - silico approach is the most efficient way of structural characterization of proteins. Molecular modeling of the PfFalstatin provided us the 3D structures of the protein. Three - di mension structure of the parasite protein could act as a staring material for the in - silico drug screening. Not only that, but the prediction of the active site might also be useful in understanding the enzymatic activity of the protein which is crucial in deciphering the regulation of cysteine proteases. In addition, comparison of modelled PfFalstatin with the structure of cysteine proteases inhibitor from P. berghei, revealed major structural differences. These discrepancies could also fasten the process of drug development against malaria parasites. [A] [B] Figure 1 Modelled structure of PfFalstatin. A) Ribbon diagram; B) Surface topology Figure 2 Figure 3 Structural comparison of PfFalstatin with Ramachandran plot of PfFalstatin using Rampage cysteine protease inhibitor of Plasmodium berghei Research Journal of Recent Sciences ______ _ _ _______________________________ ______________ _ ____ __ __ ISSN 2277 - 2502 Vol. 1( 4 ), 68 - 71 , April (201 2 ) Res. J. Recent Sci. International Science Congress Association 70 253 - D Q I I K L G D I I N S V N E K I I S I N S T V N N V L C I N L D S V N G N G F V W T L L G V H K K 303 - K P L I D P S N F P T K R V T Q S Y V S P D I S V T N P V P I P K N S N T N K D D S I N N K Q D G S 353 - Q N N T T T N H F P K P R E Q L V G G S S M L I S K I K P H K P G K Y F I V Y S Y Y R P F D P T R D 403 - T N T R I V E L N V Q A B C Figure 4 Prediction of active site of PfFalstatin using CASTp A) Active si te residues shown in green color B) and C) Position of active site residues in three - dimensional space Research Journal of Recent Sciences ______ _ _ _______________________________ ______________ _ ____ __ __ ISSN 2277 - 2502 Vol. 1( 4 ), 68 - 71 , April (201 2 ) Res. J. Recent Sci. International Science Congress Association 71 Acknowledgement I would like to thank Central University of Rajasthan, Department of Biotechnology for providing resources to conduct these studies. References 1. 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