Dissection of functional residues in receptor activity-modifying proteins through phylogenetic and statistical analyses

Type I and type-II functional divergences have been stated to highlight specific residues carrying out differential functions in evolutionary-divergent protein clusters from a single common ancestor. Briefly, type I analysis is based on residue constraints reflecting a gain of function just in one cluster of an entire family of proteins; while the type-II approach is based on residue constraints showing a different chemical nature in every cluster of a protein family. This last evidence is understood as differential functionality among clusters. The Receptor Activity-Modifying Proteins constitute a family characterized by its paralogous distribution in vertebrates. They are known as G-Protein Coupled Receptor modulators. Although several studies have determined their involvement in ligand binding, specificity, and enhancement of signal transduction, the responsible residues supporting those functions are unclear. Using different bioinformatic approaches, we predicted residues involved in different RAMP functional tasks. Many residues localized in an extracellular coil of RAMP proteins were predicted to be under functional divergence suggesting a gain of function in their respective proteins. Interestingly, the transmembrane region also showed important results for residues playing relevant roles where most of them showed a biased distribution on the structure. A relevant role was conferred by the enrichment of type-II residues observed in their sequences. We show a collection of residues explaining possible gain of function and differential functionality in RAMP proteins. These residues are still experimentally unexplored with regards to functionality. Finally, an evolutionary history could be discerned. Mainly, the RAMP2 cluster has evolved in a higher manner than other RAMP clusters. However, a deacceleration in the aminoacid substitution rate of RAMP2 was observed in mammals. Such effect could be caused by the co-evolution of ligands and receptors interacting with RAMP2 through evolution and/or the specialization of this cluster in GPCR modulation.