Larsen-Ledet Sven, Lindemose Søren, Panfilova Aleksandra, Gersing Sarah, Suhr Caroline H, Genzor Aitana Victoria, Lanters Heleen, Nielsen Sofie V, Lindorff-Larsen Kresten, Winther Jakob R, Stein Amelie, Hartmann-Petersen Rasmus
Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen, Denmark.
Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen, Denmark.
Structure. 2025 Feb 6;33(2):262-273.e6. doi: 10.1016/j.str.2024.11.017. Epub 2024 Dec 19.
Gene variants resulting in insertions or deletions of amino acid residues (indels) have important consequences for evolution and are often linked to disease, yet, compared to missense variants, the effects of indels are poorly understood and predicted. We developed a sensitive protein folding sensor based on the complementation of uracil auxotrophy in yeast by circular permutated orotate phosphoribosyltransferase (CPOP). The sensor reports on the folding of disease-linked missense variants and de-novo-designed proteins. Applying the folding sensor to a saturated library of single-residue indels in human dihydrofolate reductase (DHFR) revealed that most regions that tolerate indels are confined to internal loops, the termini, and a central α helix. Several indels are temperature sensitive, and folding is rescued upon binding to methotrexate. Rosetta and AlphaFold2 predictions correlate with the observed effects, suggesting that most indels destabilize the native fold and that these computational tools are useful for the classification of indels observed in population sequencing.
导致氨基酸残基插入或缺失（插入缺失）的基因变异对进化具有重要影响，且常常与疾病相关联。然而，与错义变异相比，插入缺失的影响却鲜为人知且难以预测。我们基于环状排列的乳清酸磷酸核糖转移酶（CPOP）对酵母尿嘧啶营养缺陷型的互补作用，开发了一种灵敏的蛋白质折叠传感器。该传感器可报告与疾病相关的错义变异和从头设计蛋白质的折叠情况。将折叠传感器应用于人类二氢叶酸还原酶（DHFR）单残基插入缺失的饱和文库，结果表明，大多数耐受插入缺失的区域局限于内部环、末端和一个中央α螺旋。有几个插入缺失对温度敏感，与甲氨蝶呤结合后可恢复折叠。Rosetta和AlphaFold2的预测结果与观察到的效应相关，这表明大多数插入缺失会破坏天然折叠结构，并且这些计算工具对于在群体测序中观察到的插入缺失的分类很有用。
