Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsin Chu, Taiwan and Graduate Institute of Biochemistry and Molecular Biology, National Taiwan University, Taipei 10048, Taiwan.
Nucleic Acids Res. 2014 Apr;42(7):4712-22. doi: 10.1093/nar/gkt1407. Epub 2014 Jan 23.
TDP-43 is an important pathological protein that aggregates in the diseased neuronal cells and is linked to various neurodegenerative disorders. In normal cells, TDP-43 is primarily an RNA-binding protein; however, how the dimeric TDP-43 binds RNA via its two RNA recognition motifs, RRM1 and RRM2, is not clear. Here we report the crystal structure of human TDP-43 RRM1 in complex with a single-stranded DNA showing that RRM1 binds the nucleic acid extensively not only by the conserved β-sheet residues but also by the loop residues. Mutational and biochemical assays further reveal that both RRMs in TDP-43 dimers participate in binding of UG-rich RNA or TG-rich DNA with RRM1 playing a dominant role and RRM2 playing a supporting role. Moreover, RRM1 of the amyotrophic lateral sclerosis-linked mutant D169G binds DNA as efficiently as the wild type; nevertheless, it is more resistant to thermal denaturation, suggesting that the resistance to degradation is likely linked to TDP-43 proteinopathies. Taken together all the data, we suggest a model showing that the two RRMs in each protomer of TDP-43 homodimer work together in RNA binding and thus the dimeric TDP-43 recognizes long clusters of UG-rich RNA to achieve high affinity and specificity.
TDP-43 是一种重要的病理蛋白，在病变神经元细胞中聚集，并与各种神经退行性疾病有关。在正常细胞中，TDP-43 主要是一种 RNA 结合蛋白；然而，二聚体 TDP-43 如何通过其两个 RNA 识别基序（RRM1 和 RRM2）结合 RNA 尚不清楚。在这里，我们报告了人 TDP-43 RRM1 与单链 DNA 复合物的晶体结构，表明 RRM1 通过保守的β-折叠残基以及环残基广泛地结合核酸。突变和生化分析进一步表明，TDP-43 二聚体中的两个 RRMs 都参与了富含 UG 的 RNA 或富含 TG 的 DNA 的结合，其中 RRM1 起主导作用，RRM2 起辅助作用。此外，与肌萎缩性侧索硬化症相关的突变 D169G 的 RRM1 与野生型一样有效地结合 DNA；然而，它对热变性更具抗性，这表明降解的抗性可能与 TDP-43 蛋白病有关。综上所述，我们提出了一个模型，表明 TDP-43 同源二聚体中每个单体的两个 RRMs 共同参与 RNA 结合，因此二聚体 TDP-43 识别富含 UG 的长簇 RNA 以实现高亲和力和特异性。
