Ahmed Rashik, Bonin Jeffrey P, Forman-Kay Julie D, Kay Lewis E
Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada.
Department of Chemistry, University of Toronto, Toronto, ON M5S 3H6, Canada.
J Am Chem Soc. 2025 Sep 10;147(36):33071-33085. doi: 10.1021/jacs.5c10918. Epub 2025 Aug 26.
Biomolecular condensates enrich specific client molecules while excluding others, often modulating conformational landscapes, and hence functions, of molecules dissolved within them. NMR-based atomic resolution studies have focused on interactions between scaffold proteins and the unfolded states of client proteins to understand the factors that influence client partitioning into condensed phases. However, characterization of interactions involving the folded client conformer is required to obtain a complete picture of how dissolution within the condensed phase affects the client energy landscape. Here, we use solution NMR spectroscopy to investigate, at atomic resolution, the interactions mediating the selective partitioning of a folded client, the FUS RNA Recognition Motif (RRM), into condensates formed by the scaffold protein CAPRIN1. At 40 °C, approximately 40% of FUS RRM remains folded in the CAPRIN1 condensed phase and high-resolution [H-N]-HSQC spectra can be recorded, enabling site-specific interactions between the folded client and scaffold to be mapped across the entire FUS RRM sequence. Using intermolecular NOE and PRE (mixed solutions), and chemical shift perturbation (demixed solution) analyses, we identify a set of interaction surfaces on the folded FUS RRM that enable multiple contacts with aromatic- and arginine-rich regions of CAPRIN1. These heterotypic interactions lead to a 30-fold enrichment of FUS RRM within the condensate and overlap with sites responsible for CAPRIN1 homotypic phase separation, suggesting a shared molecular basis for scaffold-scaffold and scaffold-client recognition. Notably, tyrosine phosphorylation of CAPRIN1 disrupts these interactions and reduces client partitioning by over 2 orders of magnitude, highlighting how post-translational modifications can profoundly regulate condensate composition.
生物分子凝聚物富集特定的客体分子，同时排除其他分子，常常调节其中溶解分子的构象格局，进而影响其功能。基于核磁共振的原子分辨率研究聚焦于支架蛋白与客体蛋白未折叠状态之间的相互作用，以了解影响客体分子分配到凝聚相的因素。然而，为了全面了解在凝聚相中溶解如何影响客体分子的能量格局，需要对涉及折叠态客体构象的相互作用进行表征。在这里，我们使用溶液核磁共振光谱，在原子分辨率下研究介导折叠态客体FUS RNA识别基序（RRM）选择性分配到由支架蛋白CAPRIN1形成的凝聚物中的相互作用。在40℃时，约40%的FUS RRM在CAPRIN1凝聚相中保持折叠状态，并且可以记录高分辨率的[H-N]-HSQC光谱，从而能够在整个FUS RRM序列上绘制折叠态客体与支架之间的位点特异性相互作用。通过分子间NOE和PRE（混合溶液）以及化学位移扰动（分离溶液）分析，我们在折叠的FUS RRM上确定了一组相互作用表面，这些表面能够与CAPRIN1富含芳香族和精氨酸的区域进行多次接触。这些异型相互作用导致凝聚物中FUS RRM富集30倍，并与负责CAPRIN1同型相分离的位点重叠，表明支架-支架和支架-客体识别具有共同的分子基础。值得注意的是，CAPRIN1的酪氨酸磷酸化破坏了这些相互作用，并使客体分配减少了2个以上数量级，突出了翻译后修饰如何深刻调节凝聚物组成。
