Wang Ziyi, Zhang Yun Jing, Zhang Qian-Yi, Bilsborrow Kate, Leslie Matthew, Suhandynata Raymond T, Zhou Huilin
Biomedical Science graduate program, School of Medicine, University of California at San Diego.
Department of Cellular and Molecular Medicine, School of Medicine, University of California at San Diego.
bioRxiv. 2024 Nov 14:2024.11.14.623588. doi: 10.1101/2024.11.14.623588.
Proteins with nuclear localization sequences (NLSs) are directed into the cell nucleus through interactions between the NLS and importin proteins. NLSs are generally short motifs rich in basic amino acids; however, identifying NLSs can be challenging due to the lack of a universally conserved sequence. In this study, we characterized the sequence specificity of an essential and conserved NLS in Mcm3, a subunit of the replicative DNA helicase. Through mutagenesis and AlphaFold 3 (AF3) modeling, we demonstrate that the precise positioning of basic residues within the NLS is critical for nuclear transport of Mcm3 through optimal interactions with importin. Disrupting these interactions impairs the nuclear import of Mcm3, resulting in defective chromatin loading of MCM and poor cell growth. Our results provide a structure-guided framework for predicting and analyzing monopartite NLSs, which, despite lacking a single consensus sequence, retain key characteristics shared between the NLSs of Mcm3 and the SV40 large T antigen.
带有核定位序列（NLSs）的蛋白质通过NLS与输入蛋白之间的相互作用被导向细胞核。NLSs通常是富含碱性氨基酸的短基序；然而，由于缺乏普遍保守的序列，鉴定NLSs可能具有挑战性。在本研究中，我们表征了复制性DNA解旋酶的一个亚基Mcm3中一个必需且保守的NLS的序列特异性。通过诱变和AlphaFold 3（AF3）建模，我们证明NLS内碱性残基的精确位置对于Mcm3通过与输入蛋白的最佳相互作用进行核转运至关重要。破坏这些相互作用会损害Mcm3的核输入，导致MCM的染色质加载缺陷和细胞生长不良。我们的结果为预测和分析单部分NLSs提供了一个结构导向框架，这些NLSs尽管缺乏单一的共有序列，但保留了Mcm3和SV40大T抗原的NLSs之间共有的关键特征。
