National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Int J Mol Sci. 2021 Sep 22;22(19):10203. doi: 10.3390/ijms221910203.
Oculocutaneous albinism type 3 (OCA3) is an autosomal recessive disorder caused by mutations in the gene. Tyrosinase-related protein 1 (Tyrp1) is involved in eumelanin synthesis, catalyzing the oxidation of 5,6-dihydroxyindole-2-carboxylic acid oxidase (DHICA) to 5,6-indolequinone-2-carboxylic acid (IQCA). Here, for the first time, four OCA3-causing mutations of Tyrp1, C30R, H215Y, D308N, and R326H, were investigated computationally to understand Tyrp1 protein stability and catalytic activity. Using the Tyrp1 crystal structure (PDB:5M8L), global mutagenesis was conducted to evaluate mutant protein stability. Consistent with the foldability parameter, C30R and H215Y should exhibit greater instability, and two other mutants, D308N and R326H, are expected to keep a native conformation. SDS-PAGE and Western blot analysis of the purified recombinant proteins confirmed that the foldability parameter correctly predicted the effect of mutations critical for protein stability. Further, the mutant variant structures were built and simulated for 100 ns to generate free energy landscapes and perform docking experiments. Free energy landscapes formed by Y362, N378, and T391 indicate that the binding clefts of C30R and H215Y mutants are larger than the wild-type Tyrp1. In docking simulations, the hydrogen bond and salt bridge interactions that stabilize DHICA in the active site remain similar among Tyrp1, D308N, and R326H. However, the strengths of these interactions and stability of the docked ligand may decrease proportionally to mutation severity due to the larger and less well-defined natures of the binding clefts in mutants. Mutational perturbations in mutants that are not unfolded may result in allosteric alterations to the active site, reducing the stability of protein-ligand interactions.
眼皮肤白化病 3 型（OCA3）是一种常染色体隐性遗传病，由 基因突变引起。酪氨酸酶相关蛋白 1（Tyrp1）参与真黑素合成，催化 5,6-二羟基吲哚-2-羧酸氧化酶（DHICA）氧化为 5,6-吲哚醌-2-羧酸（IQCA）。在这里，首次对 Tyrp1 的四个导致 OCA3 的突变 C30R、H215Y、D308N 和 R326H 进行了计算研究，以了解 Tyrp1 蛋白的稳定性和催化活性。利用 Tyrp1 晶体结构（PDB:5M8L）进行全局突变，评估突变体蛋白的稳定性。与折叠参数一致，C30R 和 H215Y 应该表现出更大的不稳定性，而另外两个突变体 D308N 和 R326H 预计保持天然构象。纯化重组蛋白的 SDS-PAGE 和 Western blot 分析证实，折叠参数正确预测了对蛋白质稳定性至关重要的突变的影响。此外，构建并模拟了突变变体结构 100 ns，生成自由能景观并进行对接实验。Y362、N378 和 T391 的自由能景观表明，C30R 和 H215Y 突变体的结合裂缝大于野生型 Tyrp1。在对接模拟中，稳定 DHICA 在活性位点的氢键和盐桥相互作用在 Tyrp1、D308N 和 R326H 之间仍然相似。然而，由于结合裂缝在突变体中更大且不太明确，这些相互作用的强度和结合配体的稳定性可能会成比例地降低。未展开的突变体中的突变扰动可能导致活性位点的变构改变，从而降低蛋白-配体相互作用的稳定性。
