Unravelling the Erythropoietin Heterodimeric Complex: In Silico Analysis of Complex Assembly and Binding Interface

Erythropoietin (Epo), a glycosylated protein categorized as a member of the cytokine family, is responsible for the modulation of erythrogenesis. Besides this, the nonhaematopoietic effects of Epo are associated with tissue‐protective activity via an antiapoptotic pathway. This function of Epo is linked with its heterodimeric form, i.e. EpoR/βcR belonging to the cytokine family as well. Both receptors are dissected into three domains: extracellular (also known as ligand‐binding domain), transmembrane and cytoplasmic domain. The transmembrane and cytoplasmic domains are assumed to play a paramount role in dimerization and tissue protection. However, the tertiary structure of cytoplasmic domains of both EpoR and βcR was not reported in the literature. Inspired by the potential therapeutic applications, we aimed to investigate the interaction pattern of EpoR/βcR using in silico tools. Cytoplasmic domains of EpoR and βcR were modelled, structural refinement was carried out through Rosetta, and these were then quality verified by well‐known quality matrices. The observed results were found to be structurally consistent with the amenable geometry showing stereochemical sustainability. The modelled cytoplasmic domains were then successfully merged with the remaining domains (extracellular and transmembrane domains) of the respective receptors through MOEv2019.01 and Modeller. The complete structures of EpoR and βcR were subjected to geometry optimization and energy minimization via dynamic studies. The refined structures were subjected to protein–protein docking to yield the heterocomplex. Here, we report on the optimized structure coordinates of EpoR/βcR, the plausible electrostatic interactions involving G191, V206, T214, S241, T244, E336, R337, M342, G348, T349, E362 and K373 of EpoR and D336, S337, Y338, K333, R391, M426, H494, Q495, S717, S722, Y750 and G758 of βcR. Subsequently, we performed screening utilizing the StraPep database to identify Epo mimetic agents with the potential to enhance their tissue‐protective effects. Following the virtual screening, four hits (4HQX, 4XO8, 1TPO and 3EZM) were obtained based on the binding affinity and protein–ligand interaction fingerprinting (PLIF). The stability exhibited by the four selected peptides in the binding site of the EpoR/βcR heterocomplex became visible during visual inspection. We propose that the presented model can be harnessed for future characterization studies.