Evolutionary analysis of mammalian Rem2, a member of the RGK (Rem, Rem2, Rad, and Gem/Kir) family of small GTPases, reveals the role of selection and epistasis in shaping protein functional constraints.

Rad And Gem-Like GTP-Binding Protein 2 (Rem2), is a member of the RGK family of Ras-like GTPases and has been identified in various mammalian species. Rem2 has been implicated in Huntingtons disease and Long QT Syndrome and is highly expressed in the brain and in endocrine cells. In this study, we examined the evolutionary history of Rem2 across mammals, focusing on the role of purifying selection and epistasis in shaping its sequence and structure. In our analysis of Rem2 sequences across 175 mammalian species, we found evidence for strong purifying selection in 70% of non-invariant codon sites of the protein, characteristic of essential proteins that play critical roles in biological processes and is consistent with Rem2s role in the regulation of neuronal development and function. We inferred epistatic effects in 49 pairs of coevolving codon sites in Rem2, some of which are predicted to have deleterious effects on human health. Additionally, we reconstructed the ancestral evolutionary history of mammalian Rem2 using protein structure prediction of extinct and extant sequences. This analysis revealed the dynamics of how substitutions that change the genetic distance of Rem2 can impact protein structure in variable regions while maintaining core functional mechanisms. By understanding the selective pressures, protein- and genetic-interactions that have shaped the sequence and structure of the Rem2 protein, we may gain a stronger understanding of its biological and functional constraints.