EVOLUTION-GUIDED FUNCTIONAL INTERCONVERSION OF HIUASE INTO A TRANSTHYRETIN-LIKE PROTEIN

Protein functional diversification is a key driver of molecular evolution, allowing preexisting protein scaffolds to acquire novel biological functions. A notable example is the evolutionary relationship between 5-hydroxyisourate hydrolase (HIUase), an enzyme involved in purine catabolism, and transthyretin (TTR), a vertebrate hormone transporter responsible for thyroid hormone binding. Phylogenetic evidence suggests that TTR evolved from an ancestral HIUase through the loss of catalytic activity and the acquisition of ligand-binding properties while retaining a conserved tetrameric architecture. Although previous studies have converted TTR into an active HIUase, the reverse evolutionary transition remains unexplored. Here, we investigated whether a rationally engineered HIUase could acquire TTR-like hormone-binding features. Based on phylogenetic reconstruction and coevolution analyses, key TTR-associated residues were introduced into the HIUase scaffold from Herbaspirillum seropedicae, generating a mutant protein (HIUmut) with a redesigned central cavity. This strategy enabled the exploration of a plausible evolutionary pathway from an ancestral enzyme toward a transport-related function. A homology model of HIUmut was generated using Modeller and subjected to molecular dynamics simulations with NAMD. To evaluate its hormone-binding potential, blind docking of thyroxine (T4) into the central cavity was performed using AutoDock Vina, followed by molecular dynamics simulations of the protein–ligand complex. Cluster analysis of the trajectories using TTClust identified six predominant conformational states. Examination of the most populated binding poses revealed that T4 stabilization within the HIUmut cavity is mainly mediated by interactions involving lysine and histidine residues. In contrast, T4 stabilization in human TTR primarily involves lysine and glutamate residues, indicating that the engineered protein may accommodate the ligand through an alternative interaction network. Future analyses will focus on the residues responsible for ligand stabilization to determine whether the TTR-like cavity of HIUmut reproduces key functional features of native TTR. Together, these findings provide evidence that an ancestral enzymatic scaffold can be computationally reprogrammed toward hormone recognition, offering insights into protein evolution and advancing strategies for rational protein design.

This work was supported by Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG #APQ-01898-22).