Preliminary studies have shown BRCA1 (170–1600) residues to be intrinsically disordered with unknown structural details. However, thousands of clinically reported variants have been identified in this central region of BRCA1. Therefore, we aimed to characterize h-BRCA1(260–553) to assess the structural basis for pathogenicity of two rare missense variants Ser282Leu, Gln356Arg identified from the Indian and Russian populations respectively. Small-angle X-ray scattering analysis revealed WT scores R_g−32 Å, D_max−93 Å, and Rflex-51% which are partially disordered, whereas Ser282Leu variant displayed a higher degree of disorderedness and Gln356Arg was observed to be aggregated. WT protein also possesses an inherent propensity to undergo a disorder-to-order transition in the presence of cruciform DNA and 2,2,2-Trifluoroethanol (TFE). An increased alpha-helical pattern was observed with increasing concentration of TFE for the Gln356Arg mutant whereas Ser282Leu mutant showed significant differences only at the highest TFE concentration. Furthermore, higher thermal shift was observed for WT-DNA complex compared to the Gln356Arg and Ser282Leu protein-DNA complex. Moreover, mature amyloid-like fibrils were observed with 30 μM thioflavin T (ThT) at 37°C for Ser282Leu and Gln356Arg proteins while the WT protein exists in a protofibril state as observed by TEM. Gln356Arg formed higher-order aggregates with amyloidogenesis over time as monitored by ThT fluorescence. In addition, computational analyses confirmed larger conformational fluctuations for Ser282Leu and Gln356Arg mutants than for the WT. The global structural alterations caused by these variants provide a mechanistic approach for further classification of the variants of uncertain clinical significance in BRCA1 into amyloidogenic variants which may have a significant role in disease pathogenesis.

中文翻译：

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h-BRCA1 中鉴定出的淀粉样蛋白罕见变体 Ser282Leu 和 Gln356Arg 的结构意义

初步研究表明 BRCA1 (170-1600) 残基本质上是无序的，结构细节未知。然而，在 BRCA1 的这个中心区域已发现了数千种临床报告的变异。因此，我们旨在表征 h-BRCA1(260–553)，以评估分别从印度和俄罗斯人群中鉴定出的两种罕见错义变异 Ser282Leu、Gln356Arg 的致病性结构基础。小角X射线散射分析显示，WT分数R _g -32 Å、D _max -93 Å和Rflex-51%部分无序，而Ser282Leu变体显示出更高程度的无序性，并且观察到Gln356Arg聚集。WT 蛋白还具有在十字形 DNA 和 2,2,2-三氟乙醇 (TFE) 存在下经历无序到有序转变的固有倾向。随着 Gln356Arg 突变体 TFE 浓度的增加，观察到 α 螺旋模式增加，而 Ser282Leu 突变体仅在最高 TFE 浓度下表现出显着差异。此外，与 Gln356Arg 和 Ser282Leu 蛋白质-DNA 复合物相比，WT-DNA 复合物观察到更高的热位移。此外，在 37°C 下，用 30 μM 硫代黄素 T (ThT) 观察到 Ser282Leu 和 Gln356Arg 蛋白的成熟淀粉样蛋白样原纤维，而 TEM 观察到 WT 蛋白以原原纤维状态存在。通过 ThT 荧光监测，Gln356Arg 随着时间的推移形成了具有淀粉样蛋白生成的高阶聚集体。此外，计算分析证实 Ser282Leu 和 Gln356Arg 突变体比 WT 具有更大的构象波动。这些变异引起的整体结构改变为进一步将 BRCA1 中临床意义不确定的变异分类为淀粉样变体提供了一种机制方法，淀粉样变体可能在疾病发病机制中发挥重要作用。