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  • Ellipticine Crystal structures from soluble E constructs Kha

    2022-05-21

    Crystal structures from soluble E2 constructs (Khan et al., 2014; Kong et al., 2013) have revealed globular protein with no regular structure in spite of having eight disulphide bonds (Kong et al., 2013). Although 62% of the protein exists in loops or disordered structures, overall it was well-defined with a central β-sheet flanked by loops, short helices and β-sheets at the front and back. Our predicted models of all four strains conform to similar structural organisation as reported in these studies (Balasco et al., 2017; Khan et al., 2014; Kong et al., 2013). Our model is also supported by E2 models as proposed by computer algorithms using class II fusion protein folds supported by FTIR spectroscopy and circular dichroism (Krey et al., 2010). Furthermore, analysis of antigenic residues as predicted to be exposed by several studies (Barone et al., 2016; Deng et al., 2014; Kong et al., 2012b; Leopold Kong et al., 2012b; Li et al., 2015; Meola et al., 2015; Pantua et al., 2013) reporting neutralizing Ellipticine against the conserved region (412–423 and 427–446) revealed that our model generated from genotype 1a also has the same residues exposed. However it was interesting to note that while the majority of these residues were also exposed in the models from the other genotypes, strain specific differences remained. This explains the lack of pan-genotypic effect of most antibodies. It is also interesting to note that although this region remains mostly conserved across genotypes, subtle structural differences do occur. The E2 binding sites for CD81 interaction have been extensively studied and have been neatly summarized in a review article by Feneant et al. (Fénéant et al., 2014). Researchers have used multiple tools to identify the E2 binding site which include the use of blocking monoclonal antibodies, E2 deletion mutants, as well as in silico modelling similar to what we have done (Callens et al., 2005; Drummer et al., 2006; Roccasecca et al., 2003; Rothwangl et al., 2008). Surprisingly all the papers report different E2 binding sites. This could be due to a predicted flexible conformation of E2 that varies during interactions with receptors (Owsianka et al., 2001). It has also been suggested that interactions and conformations may vary from cell type to cell type (Roccasecca et al., 2003). Our predicted sites have been supported by quite a few of these studies. Our prediction of the CD81 binding sites on E2 follows the same theme as suggested by Krey et al. (Krey et al., 2010) who predicted CD81 binding residues far apart in primary structure brought into close approximation in secondary structure. Similarly region spanning 407–524 suggested by Patel et al. using chimeras made from genotype 1a strains (McKeating et al., 2000) was also observed in our prediction. HVR2 residues reported to be part of the CD81 binding region using deletion mutants as well as blocking antibodies (McCaffrey et al., 2011; Roccasecca et al., 2003) support our prediction. Several residues identified from studies involving blocking antibodies such as mAb 6/41a and mAb 6/53 (Yagnik et al., 2000) have also been validated to be part of the CD81-E2 binding interface in genotype 1a and 1b. Interestingly these residues were not part of the binding interface for E2 from genotypes 3a and 3b, further highlighting strain specific differences. Although most of the published reports are based on studies on strain H77 of genotype 1a, few are based on the JFH-1 strain (genotype 2a). To the best of our knowledge, there are no reports on the CD81 binding regions in genotype 3. However several residues that we predict to be important in genotypes 3a and 3b have been reported in other studies involving genotype 1a (Drummer et al., 2006; Owsianka et al., 2001). From mutagenic studies on isolates from genotypes 1a and 1b, CD81 binding differs among strains (Roccasecca et al., 2003). While HVR1 region was speculated to inhibit CD81 interaction by steric hindrance, HVR2 (613–620) is believed to be important in CD81 interaction. While we have reported residues from HVR2, no residues of HVR1 are part of our predicted interaction region. Such was also reported in a study using HVR deletions where deletion of HVR1 had no effect whereas deletion of HVR2 abrogated CD81 interaction (McCaffrey et al., 2011). Importance of disulphide bridges between cysteines were shown to be important for E2 interaction (Petracca et al., 2000). However none of these cysteines showed up as residues in the interaction site possibly because cysteines perhaps contribute to the final structure of the CD81 but may not necessarily be part of the interacting residues.