Evolution-based protein engineering: functional switching between transthyretins and 5-hydroxyisourate hydrolases

Transthyretin (TTR) is a vertebrate-exclusive transport protein that plays a key role in binding and distributing thyroid hormones. However, its evolutionary origin lies in the duplication of the gene that encoding the enzyme 5-hydroxyisourate hydrolase (HIUase), which is involved in uric acid metabolism. Unlike TTR, HIUase is ubiquitous in both prokaryotes and eukaryotes, with the exception of hominids. Both HIUase and TTR subfamilies form homotetramers, possessing an internal charged cavity between the two dimer pairs. Based on their high degree of structural similarity, we hypothesized that specific <i>in silico</i> substitutions would enable the interconversion between these protein functions. Using an evolution-based approach, we engineered two putative protein sequences, where correlated locally conserved positions from one subfamily representative sequence were substituted by the other, and vice versa. Applying computational modeling techniques, the best models were refined, validated, and their cavity volumes, three-dimensional geometries, propensity to aggregation and electrostatic potentials were analyzed. Molecular dynamics simulations were performed with the reference proteins and the engineered mutants in the bound and unbound states. We demonstrate that the volumes and geometries differ from one another, due to size and physicochemical differences between their ligands. The bound state mutant complexes are stable, and the enzymatic assay demonstrated active new enzymes. Our work suggests that the evolution-based protein engineering approach used has residue-specific resolution to identify locally conserved residues in the sequence of evolutionarily related proteins, such as HIUase and TTR.

甲状腺素运载蛋白（Transthyretin, TTR）是一类仅存在于脊椎动物中的转运蛋白，在结合并转运甲状腺激素的过程中发挥关键作用。但其进化起源可追溯至编码5-羟异尿酸水解酶（5-hydroxyisourate hydrolase, HIUase）的基因复制事件，该酶参与尿酸代谢通路。与TTR不同，HIUase广泛分布于原核生物与真核生物中，仅人科动物除外。HIUase与TTR两个亚家族均形成同源四聚体，在两组二聚体之间存在内部带电空腔。基于二者高度相似的结构特征，我们提出假说：特定计算机模拟（in silico）替换可实现这两类蛋白功能间的相互转化。我们采用基于进化的研究策略，构建了两条推定的蛋白序列：将其中一个亚家族代表性序列的局部保守关联位点替换为另一亚家族的对应位点，并反之亦然。通过计算建模技术对最优模型进行精修与验证，并分析了其空腔体积、三维几何结构、聚集倾向与静电电势分布。我们对参考蛋白与工程化突变体的配体结合态与游离态分别开展了分子动力学模拟。研究结果表明，由于配体间的尺寸与理化性质差异，突变体与野生型蛋白的空腔体积及几何结构存在显著区别。结合态的突变体复合物稳定性良好，酶活实验证实其具备全新的酶催化活性。本研究证实，所采用的基于进化的蛋白质工程策略具备残基级分辨率，可在进化关联蛋白（如HIUase与TTR）的序列中精准鉴定局部保守残基。