Molecular effects of mutated amino acids involved in Transmembrane and Domain regions on the BCR oncogene protein using In silico techniques

BCR gene is expressed in patients with Philadelphia-positive Leukemias, known as chronic myeloid leukaemia (CML). Here, we focus on how the intramolecular domains and transmembrane segments are involved in the mutated sites of BCR. In this researchwork, we thoroughly analysed the transmembrane segments and the functional domains and predicted the 3D structure. We applied two kinds of techniques in our work. One is sequencebased, where we proved that the transmembrane segments in the functional domains contain the mutated sites. The second technique is structure-based, where we predicted the 3D structure of BCR gene-coded protein and visualised the transmembrane segments, which included the mutated sites. By using advanced molecular visualisation tools, the molecular structural properties of the respective transmembrane regions of amino acids will be determined. Both the techniques involved the use of advanced insilico tools and database. Our results elucidated that both the sequence and structure-based outcomes represented the identi ied transmembrane segments in the functional domains, which are potential candidates for drug docking studies. Hence, we inally concluded that this research work would play a vital role in clinical oncology and structure-based drug designing. Our research work is the irst attempt to prove that potential drug binding sites are present in BCR oncogene-protein using insilico techniques. The results of this research investigation form a basic foundation for structure-based drug designing.


INTRODUCTION
In recent years, leukaemia is a major challenge in the ield of medicine. In our research, we focus on the BCR gene. Heisterkamp and his colleagues estab-lished the structural organisation of the BCR gene, which consists of 23 exons and is positioned in a region of about 135 kb on chromosome 22 (Heisterkamp et al., 1985). The irst exon contains a unique serine/threonine kinase activity and no less than 2 SH2 binding sites. Stam and his co-workers showed that the BCR gene is positioned with its 5prime end toward the centromere of chromosome 22 (Stam et al., 1987).
It was ascertained that the BCR gene consists of 23 BCR exons with putative alternative BCR irst and second exons (Chissoe et al., 1995). It was shown that BCR, when puri ied, contains autophosphorylation activity and transphosphorylation activity for several protein substrates (Maru and Witte, 1991). It was found out that for this new phosphotransferase activity, a region encoded by the irst exon was essential.
Zhao and his researchers reported that impairment of hematopoietic stem cell renewal and reduced induction of chronic myelogenous leukaemia (CML; 608232), by the BCR-ABL1 oncoprotein were due to the loss of Smoothened (Smo;601500) which is an essential component of the hedgehog pathway (600725) (Zhao et al., 2009). Depletion of CML stem cells was caused by the loss of Smo, which propagated leukaemia, whereas constitutively CML stem cell number was increased, and active Smo accelerated disease.
Recently, with the ef icient demonstration of generic strategies which stabilise and crystallise unstable, eukaryotic membrane proteins, we have probably entered the third phase of membrane protein structure analysis. For crystallisation, such unstable membrane proteins should be stabilised either by prompt reinsertion into a lipid bilayer, such as with 2D membrane crystals (Kühlbrandt, 1992) or with lipidic phase, detergent-free crystallisation methods (Nollert et al., 1999), or by the inclusion of speci ic active-site ligands or inhibitors (Pebay-Peyroula et al., 2003;Vedadi et al., 2006;Toyoshima, 2008), or by systematic mutagenesis to create a protein which has increased intrinsic stability Serrano-Vega et al., 2008). This third post-genomic phase should enable the determination of the structure of any membrane protein or complex of interest.

MATERIALS AND METHODS
We, irst, retrieved the gene-coded protein sequence of BCR based on various clinical literature studies (OMIM, ID, Uniprot) (). Next, the retrieved sequence was applied into TMRPRED (Pasquier et al., 1999) tool to identify the potential transmembrane regions present in the BCR. Finally, the 3D structure of the retrieved sequence was studied using the CPH model 3.0 server (Nielsen et al., 2010;      Lund et al., 2002). CPHmodels-3.0 is a webserver which predicts the 3D structure of the query protein (BCR (Breakpoint cluster region protein) in our case) by using single template homology modelling. The modelled protein structure was visualised using a molecular visualisation tool called Discovery Studio Software.

RESULTS AND DISCUSSION
The length of the amino acid sequence of human BCR is 1271 amino acids, and its various details were retrieved, as shown in Table 1. The FASTA format of BCR (Breakpoint cluster region protein) was given in Figure 1.
The analysis of Transmembrane segments by PRED-TMR in the BCR human protein sequence revealed the start position as 1167 and end position as 1183.
The domain length of potential domain regions of BCR was reported to be 1054-1248 (Chuang et al., 1995). Chuang and his co-workers also explained that the region involved in binding to ABL1-SH2domain was rich in serine residues, and prior to SH2 binding, it needs to be Ser/Thr phosphorylated. This region plays a vital role in the activation of the ABL1 tyrosine kinase and transforming potential of the chimeric BCR-ABL oncogene.
The domain length of human BCR was shown in Table 2 and Figure 2. The DH domain was involved in an interaction with CCPG1. The amino terminus consists of intrinsic kinase activity. The central Dbl homology (DH) domain acts as guanine nucleotide exchange factor (GEF) which modulates the GTPases CDC42, RHOA and RAC1, and promotes the conversion of CDC42, RHOA and RAC1 from the GDPbound to the GTP-bound form. The C-terminus is a Rho-GAP domain which promotes GTP hydrolysis by RAC1, RAC2 and CDC42. The protein has a special structure having two opposing regulatory activities towards small GTP-binding proteins. Table 3 showed that the identi ied Transmembrane segments fall within the domain regions of BCR (Chuang et al., 1995). The research proved that the identi ied segments were potentially involved in the various mutations of BCR. The identi ied SNPs were found to be potential targets for drugs. These SNPs are the over-expression of the BCR gene (Diekmann et al., 1991).

3D Structure Prediction
The three dimensional structure of the protein that was predicted using CPH model server was shown in the Figure 4.
The Figure 4 showed the secondary structure view of BCR in which red colour indicates alpha-helix, blue indicates beta sheets, green indicates turns and white indicates coils.
In the above depicted Figure 5, we can visualize the transmembrane segments of BCR protein representedin space-ill model.
The breakpoints in CML to sub-bands 22q11.21 and 9q34.1 were located by Prakash and Yunis (1984). Even if the breakpoint's position in chromosome 9 is quite variable, the breakpoint in chromosome 22 is grouped in an area called BCR for breakpoint cluster region. Shtivelman and his group referred to BCR as a gene and declared that the ABL oncogene is transferred into the BCR gene on chromosome 22. They found that an 8-kb RNA which was speci ic to CML is a fused transcript of the two genes. It is probably the fused protein which is involved in the malignant process (Shtivelman et al., 1985).
Our results clearly showed that transmembrane segments are present within the functional domain of Oncogene BCR-Breakpoint cluster region protein.
We also proved it with the help of 3D structures and sequences. The identi ied regions could act as potential drug binding sites for the upcoming drugs.

CONCLUSION
In this research, we found out that Transmembrane amino acid segments act as potential candidates for drug docking studies. These segments are directly involved in various mutations. The predicted 3D structure clearly showed the target drugbinding sites (BCR-Breakpoint cluster region protein). Hence, we inally conclude that our results play a vital role in structure-based drug designing and clinical oncology studies.
ACKNOWLEDGEMENT I want to thank Dr Balaji Munivelan (bioinfobal-aji@gmail.com) for his assistance towards Insilico works in this paper.