Atsrim Ernest S, Eichhorn Catherine D
Department of Chemistry, University of Nebraska, Lincoln, Nebraska, USA.
Nebraska Center for Integrated Biomolecular Communication, Lincoln, Nebraska, USA.
Protein Sci. 2025 May;34(5):e70127. doi: 10.1002/pro.70127.
The heterogeneous nuclear ribonucleoprotein (hnRNP) R-like family is a class of RNA binding proteins in the hnRNP superfamily with diverse functions in RNA processing. Here, we present the 1.90 Å X-ray crystal structure and solution NMR studies of the first RNA recognition motif (RRM) of human hnRNPR. We find that this domain adopts an extended RRM (eRRM1) featuring a canonical RRM with a structured N-terminal extension (N) motif that docks against the RRM and extends the β-sheet surface. The adjoining loop is structured and forms a tryptophan cage motif to position the N motif for docking to the RRM. Combining mutagenesis, solution NMR spectroscopy, and thermal denaturation studies, we evaluate the importance of residues in the N-RRM interface and adjoining loop on eRRM folding and conformational dynamics. We find that these sites are essential for protein solubility, conformational ordering, and thermal stability. Consistent with their importance, mutations in the N-RRM interface and loop are associated with several cancers in a survey of somatic mutations in cancer studies. Sequence and structure comparison of the human hnRNPR eRRM1 to experimentally verified and predicted hnRNPR-like proteins reveals conserved features in the eRRM.
不均一核核糖核蛋白（hnRNP）R样家族是hnRNP超家族中的一类RNA结合蛋白，在RNA加工过程中具有多种功能。在此，我们展示了人hnRNPR首个RNA识别基序（RRM）的1.90Å X射线晶体结构及溶液核磁共振研究结果。我们发现该结构域采用了一种扩展的RRM（eRRM1），其具有一个典型的RRM以及一个结构化的N端延伸（N）基序，该基序与RRM对接并扩展了β折叠表面。相邻的环是结构化的，并形成一个色氨酸笼基序，用于定位N基序以与RRM对接。结合诱变、溶液核磁共振光谱和热变性研究，我们评估了N-RRM界面和相邻环中残基对eRRM折叠和构象动力学的重要性。我们发现这些位点对于蛋白质的溶解性、构象有序性和热稳定性至关重要。与它们的重要性一致，在癌症研究的体细胞突变调查中，N-RRM界面和环中的突变与多种癌症相关。将人hnRNPR eRRM1与经过实验验证和预测的hnRNPR样蛋白进行序列和结构比较，揭示了eRRM中的保守特征。
