Reconstructing Protein Evolution: Converting HIUase into a Transthyretin-like Transport Protein

Protein functional diversification is a key driver of molecular evolution, often resulting in novel biochemical and physiological roles. A representative case is the evolutionary relationship between 5-hydroxyisourate hydrolase (HIUase), an enzyme broadly distributed among prokaryotes and eukaryotes, and transthyretin (TTR), a tetrameric protein restricted to vertebrates that is responsible for the transport of thyroid hormones and retinoic acid. Phylogenetic studies indicate that TTR emerged from the duplication of an ancestral HIUase gene, followed by loss of enzymatic activity and the acquisition of a distribution function. Despite their functional divergence, HIUase and TTR share high structural similarity, assembling as homotetramers with central charged cavities. Our previous studies have successfully demonstrated the conversion of TTR into an active HIUase through evolutionarily guided substitutions; however, the reverse evolutionary path – transforming HIUase into a protein with TTR-like properties – remains unexplored.

This project aims to provide a computational proof of concept by engineering HIUase into a protein with structural and functional traits of TTR, with particular focus on thyroxine (T4) binding. Phylogenetic and covariance analyses will be performed to identify conserved and coevolving residues relevant to functional divergence. These data will guide the rational design of HIUase mutants through targeted substitutions. Structural models will be generated and evaluated for stereochemical quality, stability, cavity geometry, and electrostatic properties. Molecular dynamics simulations will further assess tetrameric stability and conformational flexibility under physiological conditions. Finally, docking analyses with T4 will be conducted to investigate binding affinity, orientation, and intermolecular interactions, with comparison to the binding mode of Homo sapiens wild-type TTR.

By reconstructing an evolutionary trajectory in silico, this work seeks to demonstrate that an ancestral enzyme can be reprogrammed into a transport protein, elucidating the mechanisms underlying functional innovation in protein evolution. Beyond its theoretical relevance, this study also advances methodological frameworks for protein design, with implications for biotechnology and medicine.