Own in the 3D model. Thus, our mapping of major immunogenic
Own in the 3D model. Thus, our mapping of major immunogenic regions of EED was in good consistency with the position of accessible loops and surface exposed portions of -stands predicted by the EED 3D-model.3D structure and protein interacting regions in the EED core domain The binding site of the HIV-1 MA protein has been mapped to position 294?09 on the linear sequence of EED [15]. The newly established conformation of thisPage 5 of(page number not for citation purposes)Virology Journal 2008, 5:http://www.virologyj.com/content/5/1/Figure 3 Surface representation of the -propeller domain of EED and protein-interacting regions Surface representation of the -propeller domain of EED and protein-interacting regions. The binding residues of HIV-1 proteins are represented with the following colour code : yellow for the matrix protein (MA), red for integrase (IN). (A), Top view of the -propeller showing the MA and IN binding sites laterally oriented. Note the absence of overlapping between the MA and IN binding sites, which form a continuous binding groove. (B), Side view of the -propeller showing the MA+IN-binding groove on the lateral face, and the position of the EZH2 -helical peptide 39?8 (represented in blue), bound to the EZH2-binding pocket facing downwards.rence of ternary complex involving EED, MA and IN has previously been suggested by their colocalization observed by immuno-electron microscopy of HIV-1infected cells at early steps of the virus life cycle [16]. The EZH2 binding groove, which was oriented downwards with respect to the EED -propeller plane, was totally independent of the continuous MA-IN binding groove (Fig. 3B). Interestingly, although -helices represent privileged PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28250575 domains of protein-protein interaction, none of the newly identified helices in the EED core, 1 or PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28494239 1, represented binding domains of known cellular or viral partners of EED, e.g. EZH2 [30], MA [15], or IN [16].ConclusionThe refined structural model of the EED LY-2523355 solubility C-terminal core as a seven-bladed -propeller determined from crystallographic data provided structural support to our mapping of immunogenic epitopes recognized by our anti-EED polyclonal antibodies, and of the binding sites of HIV-1 MA and IN [15,16]. Several immunoreactive regions coincided with the MA, IN and EZH2 binding sites, confirming the accessibility of these regions at the surface of EED. According to the EED 3D-model, the domain of interaction with the HIV-1 MA protein would be localised on the lateral face of the -propeller, and be comprised of twoloops separated by the short -strand IVd (Fig. 2 and Fig. 3). The region of interaction with IN would be assigned to -strand IIId and its neighboring turn, also located on the peripheral area of the -propeller. When represented on the surface of the EED molecule, the two discrete prints of MA and IN interaction were contiguous but did not overlap, and formed a continuous protein-interacting groove running along the lateral face of the EED -propeller. This groove slightly opened towards the lower face of the propeller (Fig. 3). The absence of overlapping of the MA and IN binding sites and the possible occurrence of ternary complex involving EED, MA and IN raised the issue of the biological parameters of a simultaneous binding of EED to MA and IN, and of the role that such a ternary complex might play in the HIV-1 life cycle.MethodsPlasmids, proteins and cells Plasmids coding for GST-fused or His-tagged proteins EED, MA, IN and Nef and protein exp.