Three dimensional modeling of C-terminal loop of CssA subunit in CS6 of Enterotoxigenic Escherichia coli and its interaction with the 70 KDa domain of Fibronectin.

Colonization factor CS6 of enterotoxigenic Escherichia coli (ETEC) helps to establish the adherence of CS6-expressing ETEC in the intestinal wall. CS6 is composed of two structural subunits, known as CssA and CssB. During CS6-expressing ETEC adherence in intestinal wall, 15 amino acid residues containing Cterminal region of CssA subunit, help to bind with N-terminal 70kDa domain of fibronectin (Fn). In this study, we have predicted a theoretical structural model for C-terminal domain of CssA by homology modelling using protein data bank (PDB) file, 1NTY-A as template (66.67% sequence identity) in Discovery Studio. The structural model of N-terminal region of Fn was also determined by homology modelling using PDB files 1FBR and 1E88 as templates. The structure of the model was also validated by Ramachandran plot. The energy minimization for Fn was performed in standard dynamic cascade using Steepest Descent algorithm followed by Adopted Basis NR algorithm in Discovery studio. The docking model between C-terminal domain and fibronectin were generated by using ClusPro algorithm. This docking study would be help for better understanding how CS6 interacts with fibronectin of intestinal extracellular matrix in the host during infection, and would be of great help towards subunit vaccine generation.


Background:
Enterotoxigenic Escherichia coli (ETEC), is one of the major causes of infantile diarrhoea and traveller's diarrhoea in different developing countries [1, 2, 3]. ETEC alone causes around 40-70% of the diarrhoeal incidence globally [4]. The essential step in pathogenesis is mediated by initial adherence followed by colonization in the intestine. Till date more than 25 proteinous appendages have been identified, collectively called colonization factor antigens which had been shown to be involved in adhesion in vivo and in vitro [5]. Among them, CS6 is one of the prevalent colonization factors worldwide [1,6]. Genomic studies have suggested that CS6 is mainly composed of two equally expressed subunits, CssA and CssB in a stoichiometry of 1:1 [7,8].
They are very tightly associated and cannot be separated from each other easily [7]. Functionally, CssA subunit was shown to bind to fibronectin (Fn-an extra cellular matrix protein of intestinal epithelial cells) by us (7) earlier whereas CssB has been shown to interact with glycosphingolipid [9]. Studies further showed that the C-terminal of CssA subunit of CS6 (from 112 to 126 amino acids) binds Fn in vitro [7] but residue interaction was not known. We reported purification of CS6 to a functionally active form while its organization (in terms of both subunits) is not known. No template has been reported with high sequential homology till date to explore its organizational details. Lack of any three dimensional structures for CS6 thus makes it difficult to complement the functional details of the CS6-Fn interactions with structural insights. In this study, we have reported a model for CssA and its interaction with Fn by a systematic approach using different bioinformatics softwares. The prediction will help to explore the functional aspects of CS6 in greater detail in future.

Methodology:
The amino acid sequence of the CssA subunit of CS6 was extracted from ETEC 4266 strain (GeneBank accession number EF451566   Homology modeling of N terminal region of Fn. 1FBR and 1E88 were used as template. The energy minimization for Fn was performed in standard dynamic cascade using Steepest Descent algorithm followed by Adopted Basis NR algorithm in Discovery studio. The RMSD of the derived structure was 1.67 Å with respect to the template. The arrow indicates the ramachandran plot of the structure showing more than 95% of residues are in the energetically permissible region of the Ramachandran diagram.

Result and Discussion: Homology modeling of C-terminal domain of CssA:
In order to determine the probable model for the interaction of CS6 with Fn, homology modeling for both the N-terminal region of Fn and C-terminal region of CssA were performed. For homology modelling DH/PH domain of Trio (PDB: 1NTY-A) [11] was used as a template for the C-terminal region of CssA. The theoretical prediction of secondary structure suggested that the proposed binding region of CssA (NYTSGDKEIPPGIYN) assumes a loop in between two β sheets (Figure 1). Three dimensionally it was confirmed by homology modeling (Figure 2A). The RMSD of the predicted structure of the loop was 1.27 Å from the template. More than 98% of amino acids fall in the allowed region of Ramachandran plot ( Figure 2B). Following denotes the energy content of the final structure of the C-terminal loop. Initial Potential Energy = -701.66 kcal/mol, Potential Energy of the final structure = -2517.75 kcal/mol, Van der Waals Energy = -205.03 kcal/mol, Electrostatic Energy = -2630.94 kcal/mol.

Docking of CssA and Fn in search of probable binding region:
In protein-protein docking by ClusPro [10] the probable binding region of the peptide was determined (Figure 3). The interaction energy was calculated using Discovery studio. The interaction energy of the peptide with the 5 F1 motif of 30-kDa fibrin binding domain was -71.220 (kcal/mol). Binding also occurs to the connecting region of 30 and 45 kDa domains (interaction energy = -46.106 kcal/mol) ( Table 1