International Journal of Scientific Research in Knowledge, 2(2), pp. 67-74, 2014 Available online at http://www.ijsrpub.

com/ijsrk ISSN: 2322-4541; 2014 IJSRPUB http://dx.doi.org/10.12983/ijsrk-2014-p0067-0074

Full Length Research Paper Bioinformatics Prediction of Interaction Silver Nanoparticles on the Disulfide Bonds of HIV-1 Gp120 Protein
Shahin Gavanji1*, Hassan Mohabatkar2*,Hojjat Baghshahi3, Ali Zarrabi2
2

Young Researchers and Elite Club, Isfahan (Khorasgan) Branch, Islamic Azad University, Isfahan, Iran Department of Biotechnology, Faculty of Advanced Sciences and Technologies, University of Isfahan, Isfahan, Iran 3 Department of Animal Sciences, College of Agriculture, Isfahan University of Technology, Isfahan, Iran *Corresponding Author: h.mohabatkar@ast.ui.ac.ir, shahin.gavanji@yahoo.com
Received 25 November 2013; Accepted 01 January 2014

Abstract. Silver nanoparticles have anti-HIV features in early stages of virus amplification. The two existing disulfide bonds in carboxyl half of the HIV-1 GP120, which cooperate in conjugation of CD4 receptor, interact with silver nanoparticles. Studies on protein disulfide bonds were done using Metal Detector Predicts V2.0 software. All Cys and His residues in amino acid sequences were identified. Protein denaturation decreases disulfide bonds when silver ions couple with sulfhydryl groups. CTRPNNNTRKRIRIQRGPGRAFVTIGKIGNMRQAHC amino acid sequence in GP 120 plays a key role. Breaking this bond can alter spatial structure of the protein and prevents this part from connecting to CD4. Ultimately, nanosilver can prevent HIV from connecting to CD4. Key words: Metal binding sites, silver, GP 120, Bioinformatics

1. INTRODUCTION Nanoparticles are defined as structures with a dimension in the range of 1100 nm. The widely technological use of nanoparticles in commercial industry will cause a great increase in price by 2015 (up to $3 trillion) (Ahamed et al., 2010). Silver is often found in form of metal silver nanoparticles. Since the particles are small in size, the exposure of their surface in solution peaks, which leads to the maximum possible effect per unit of silver. All the interacting silver nanoparticles with the bacteria were between 1 and 10 nm (Morones et al., 2005). This dependency in size probably exists because the combination of these particles can pass and react with the cell membrane and their surface-to-volume ratio is higher. The smaller is the size of the silver nanoparticles, the higher is the interaction between atoms. This explains the reason for small (110nm) silver nanoparticles interaction with the bacteria (Feng et al., 2000). The existence of silver in this minute amount doesnt have any adverse effect on human body (Berger et al., 1976). Nanoparticles have been applied in medicine in order for experts to trace, diagnose and cure different diseases. However, the biological features of these particles are yet to be

studied (Bhattacharya and Mukherjee, 2008). In the nineteenth century scientists discovered the use of silver in medicine and local antibacterial agents (Tokumaru et al., 1974). Studies run on anti-microbial potential of silver nanoparticles have shown that these nanoparticles are antibacterial agents against Gramnegative and Gram positive bacteria (Shahverdi et al., 2007). And are antiviral agents against HIV-1 (Sun et al., 2005), hepatitis B virus (Lu et al., 2008), respiratory syncytial virus (Sun et al., 2008), herpes simplex virus type 1 (Baram-Pinto et al., 2009) and monkey pox virus (Rogers et al., 2008). Many biological processes in micro organisms can be attacked by silver, namely the alteration of cell membrane structure and functions (Pal et al., 2007). This fact makes the application of silver very useful in developing many biological and pharmaceutical processes, products and devices some of which are coating materials for medical devices (Raad and Hanna, 2002), orthopedic or dental graft materials (Hotta et al., 1998), wound repair topical aids (Dowsett, 2004), water sanitization (Lin et al., 2002), textile products (Takai et al., 2002) and washing machines (Jung et al 2007). Everyday new forms of silver nanoparticles are being produced. Parts of clothing, food containers, wound dressings, ointments, implant coating ant etc. are some of these products

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(Arora et al., 2008; Kumar et al., 2008). Silver modes of inhibitory action to microorganisms are various(Clement and Jarrett, 1994), therefore, compared to synthetic fungicides, the use of it in controlling different forms of plant pathogens might be safer (Choi, 2006). This effect seems to be caused by those mechanisms that are the cause of bactericidal effect of silver ions. There are two groups of bactericidal effects of silver: silver ions or silver nanoparticles. Ions and particles have to be distinctively identified. Silver ions are charged atoms (Ag+) while silver nanoparticles are single crystals of nanosize dimensions. Structural changes in the cell membrane have been proven to be made by silver ions. The enzyme-containing sulfate in the membrane of the bacteria is in interaction with silver ions and. This changes the membrane morphology by pacifying the enzymes. Silver ions can easily penetrate the membrane because of the mentioned inactivation which makes the membrane vulnerable. The interaction between silver ions and sulfate groups located in the active site of the enzyme inside the cell causes the destruction of the different parts of the cell to continue. An inactivation of enzymes happens as a result of this interaction with the active site. Another interaction, which is proved to have severe effects, is the interaction between silver ions and phosphorus groups. The interaction between silver ions and DNA backbone is an example of this, which is the reason for bacterias inability in replicating itself or transcribing mRNA for new proteins. All abovementioned changes cause a decrease in the speed of bacteria growth and eventually lead to its death (Alcamo, 1997). It was shown by another study that, on the surface of cellular membrane, monovalent silver ions (Ag+) replace hydrogen cations (H+) of sulfhydryl or thiol (S-H) groups. This disables the production of proteins required to sustain the cell, which finally kills the cell (Feng et al., 2000). In addition, studies have suggested that when silver ions enter the cell they intercalate into bacterial DNA, and that prevents more pathogen proliferation. The effectiveness of silver particles as antimicrobial agents has been increased by nanotechnology in recent years. The contact between silver nanoparticles and microbes increases because of the larger area-to-volume ratio of silver nanoparticles, which results in the increase of their ability to permeate cells, and that prevents more pathogen proliferation. It is thought that silvers mode of action depends on Ag+ ions. These ions prevent bacterial growth greatly by suppression of respiratory enzymes and electron transport components and through interference with DNA functions (Li et al., 2006).

Life is believed to be sustained in bacteria through using an enzyme to metabolize oxygen. This enzyme gets crippled by silver ions therefore oxygen metabolizing gets stopped which suffocates the bacteria and kills it. Virus growth happens when they invade living cells and then produce replicas of themselves. Since cells life depends on oxygen metabolizing enzymes, silver ions kill viruses by killing the cell by suffocation (Alvarez-Puebla et al., 2004). The interactions between silver nanoparticles and the bacteria are quite similar to the interactions between silver ions and bacteria. This might mean that silver nanoparticles and silver ions are similarly able to interact with the same types of chemical groups, and therefore can damage the bacteria in the same form (Morones et al., 2005). More than any other inorganic antibacterial agent, the antimicrobial property of silver has been investigated and employed extensively (Russell et al., 1994). The slow and continual release of silver that prolonged the antimicrobial effect is supposed to be reason for this. Ionic or nanoparticle silver can potentially be used for controlling spore-producing fungal plant pathogens because of their antifungal activity. In comparison with synthetic fungicides, silver might have less toxicity for humans and animals. Antibiotic and preservative qualities of silver nanoparticles bring them close to human beings. Bacteria play a key role in human digestion, and if nanoparticles could eliminate the bacteria, it would have a fatal effect. Some recent studies have shown that silver nanoparticles have the ability to attach to HIV-1virus and avoid it from making bonds with cells (Elechiguerra et al., 2005). World Health Organization has currently listed silver sulfadiazine as an essential anti-infective topical medicine (Lara et al., 2010). Virus adsorption tests were used to prove that anti HIV activity of silver nanoparticles prevents this virus from binding or fusion to the cells. AIDS happens because of HIV-1infection (Lara et al., 2010; Allan et al., 1985). Since the binding between HIV and the target cell surface, and therefore the cellular and viral membrane fusion, is mediated by the envelope, its role is critical in infection. Because preventing HIV from entering the target cell abandons viral infectivity, replication, and the cytotoxicity caused by virus-cell interaction, fusion or entry inhibitors should be remarkably noted. Also the presence of virucidal agents is necessary for HIV/AIDS prevention because of their deactivating quality against viral particle (virion), with which the completion of viral replication cycle can be prevented (Salzwedel et al., 1999). Silver nanoparticles act like virucidal agent or viral entry inhibitor and therefore deploy anti-HIV activity at early stages of viral replication. Remarkable differences in antiviral

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activities of silver nanoparticles against different drug-resistant strains were not found; therefore changes made in antiretroviral HIV strains that confer resistance do not have any influence on the efficacy of silver nanoparticles (Al-Jabri and Alenzi, 2009). Furthermore, an interaction between silver nanoparticles and the two disulfide bonds located in the carboxyl half of the HIV-1 gp120 glycoprotein might happen. This mentioned area has been shown in binding to the CD4 receptor (Lekutis et al., 1992). The binding between sulfhydryl groups causes the protein denaturation through reducing disulfide bonds. Because silver nanoparticles have antiviral activity, they are able to prevent HIV-1 infection from binding to gp120. This inhabitation prevents CD4-dependant viron binding, fusion and infectivity and inhibits HIV1 cell-free and cell-associated infection. This happens because they act as virucidal agents (Borkow and Lapidot, 2005). It is concluded that, through stopping the activity of HIV particles at an early stage of viral replication, silver nanoparticles act as effective virucides by inactivating HIV particles in a short period of time. The aim of the study was to investigate the bioinformatics prediction of interaction silver

nanoparticles on the disulfide bonds of HIV-1 Gp120 protein 2. MATERIALS AND METHODS 2.1. Preparing 3 dimensional structure GP120 protein In the first step, amino acid sequences of GP120 protein with an accession number of p03378.1 were taken from NCBI website (www.ncbi.nlm.nih.gov). The GP120 protein consists of 478 amino acids (Figure 1) and its molecular weight is 23.469Da .Then the GP120 viral protein with the number of 1GC1 linked to CD4 protein was taken from Protein Data Bank website. The CD4 and additional ligands were separated from that GP120 viral protein through Molegro software and this protein was prepared for research, without having any other protein or ligand linked to it (Figure 2) (www.rcsb.com). In the next step, and silver with Ag formula (number 22394) were provided from ChemSpider website (www.chemispider.com).

Fig. 1: Amino acid sequence of GP120 protein

Fig. 2: Structure of GP120 protein

2.2. Studying disulphide bonds in proteins 2.2.1. Metal Detector Web Server The web server Metal Detector classifies histidine residues in proteins into one of two states (free or metal bond) and cysteines into one of three states (free, metal bond or disulfide bridged). It is freely

available at (http://metaldetector.dsi.unifi.it/v2.0). This web server takes the protein sequence as input and outputs predictions of transition-metal binding for cysteine and histidine residues; for cysteines it also predicts disulfide bonding bridges. Residues predicted to coordinate the same ion will share the same identifier. Every identifier is an integer ranging from 1 to 4. Its value has no special biochemical semantics

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but lower values correspond to a higher level of confidence for the predictor. 2.2.2. Studying disulphide bonds

Studies on disulfide bonds were performed using Metal Detector Predicts v2.0 software. All Cys and His residues in amino acid sequences were identified. (http://metaldetector.dsi.unifi.it).

Fig. 4: disulfide bonds and metal binding site in GP120 protein

3. RESULTS AND DISCUSSIONS 3.1. Protein Structure Analysis The GP120 protein consists of 478 amino acids, and its molecular weight is 23.469 kD. This location is in extracellular form, and the location of CD4-binding loop is from amino acids 366 to 376 which contain 11 amino acids. According to the results taken from Metal Detector Predicts v2.0 online server, the disulfide bonds in GP120 protein amino acid sequence are sited between cysteine amino acids in locations 186, 173, 164, 125, 99, 94, 87, 42, 22, 413, 386, 353, 346, 299, 264, 215, 207 and 196 as they are shown in

figure 4. The 346 location is the most important site of this sequence, which is the site for metal group bonds. Therefore, the sequence of CTRPNNNTRKRIRIQRGPGRAFVTIGKIGNMRQA HC in GP120 protein plays a key role (Figure 5). This sequence is in the first and most important part of this protein to bind GP120 and CD4 proteins. In Molegro software the basis of the three dimensional structure of this protein is positioned between 264 to 299 amino acids where sulfide bonds are located. Breaking this binding can change the spatial structure of this protein, therefore prevents this part from binding to CD4.

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Fig. 5: The structure of sequence amino acid in GP120 protein

Penetration and distribution inhibitors are considered remarkable since inhibiting HIV from penetrating the target cell will in turn lead to prevention from viral infections, duplication, and cytotoxicity caused by virus-cell interaction (Borkow and Lapidot, 2005). The antivirus activity of nanosilver particles let us inhibit HIV1 infections regardless of virus types or resistant profiles against GP120 binding. This inhibition is done so that viralparticle-dependant CD4 cannot bind and there will not be any infection and propagation, and it prevents HIV-1 free cells and infected cells. Thus, nanosilver particles are effective antivirus particles since they are short term HIV deactivators. These particles act at the beginning stages of viral propagation (penetration or spread) and at pre-penetration stages (Lara et al., 2010). The binding of silver ions to sulfhydryl groups causes protein denaturation through reducing disulfide bonds (McDonnell, 2007). Results of our research show that the first and most important part of this protein binds GP120 and CD4 proteins. In Molegro software the basis of the three dimensional structure of this protein is positioned between 264 to 299 amino acids where sulfide bonds are located. Breaking this binding can change the spatial structure of this protein, therefore prevents this part from binding with CD4.

4. CONCLUSION At the first stages of HIV proliferation, silver nano particles can act as antivirus agent for deactivation of the virus in a short period of time. This process is done through interaction of nano silver with 2 disulfide bonds located in carboxyl )HIV-1 gp120) so that silver ions, through interaction with thiol group, decrease disulfide bonds leading to protein denaturation. Considering other similar scientific investigations, one can conclude that the mentioned process is fulfilled through substitution of single valence Ag+ with H+ existed in thiol group. Bioinformatic results using software and servers show that silver ions make interaction with thiol group through which the disulfide bonds decrease and the virus will be destroyed. REFERENCES Achal V, Kumari D, Pan X (2011). Bioremediation of Chromium Contaminated Soil by a Brown-rot Fungus, Gloeophyllum sepiarium. Research Journal of Microbiology, 6: 166-171. APHA (1998). Standard Methods for Examination of Water and Wastewater, 20th ed. American Public Health Association, Washington, DC, USA.

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Shahin Gavanji graduated in Biotechnology at MSc at the Department of Biotechnology, Faculty of Advanced Sciences and Technologies, University of Isfahan, Isfahan, Iran. He has over 10 international medals in invention. Shahin Gavanji's research has focused on Pharmacy and Pharmacology, Nano Biotechnology, Bioinformatics, Biotechnology - Medical Biotechnology. He is editor in chief of International Journal of Scientific Research in Inventions and New Ideas.

Dr. Hassan Mohabatkar is a faculty member at Department of Biotechnology, Faculty of Advanced Sciences and Technologies, University of Isfahan, Isfahan, Iran. His research has focused on Bioinformatics. h_mohabatkar@yahoo.com

Hojjat Baghshahi graduated in Animal Science at MSc at the Department of Animal Sciences, College of Agriculture, Isfahan University of Technology (IUT), Isfahan, IRAN.

Dr. Ali Zarrabi, Assistant Professor of Nano-biotechnology, Department of Biotechnology, Faculty of Advanced Sciences and Technologies, University of Isfahan, Isfahan, Iran.

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