Kranz J K, Hall K B
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, MO, 63110, USA.
J Mol Biol. 1999 Jan 8;285(1):215-31. doi: 10.1006/jmbi.1998.2296.
One of the most common structural motifs in RNA-binding proteins is the RNA-binding domain (RBD). These domains share a common alpha/beta sandwich tertiary fold, and are highly conserved, though they bind diverse RNA targets with a wide range of binding affinities. The N-terminal RNA-binding domain (RBD1) of the human U1A protein binds specifically to stem/loop II of the U1 snRNA with sub-nanomolar affinity. Solvent-exposed aromatic residues on the beta-sheet surface are highly conserved among RBD domains; in RBD1, these are Tyr13 and Phe56, with a unique Gln at position 54. Effects of substitutions at these positions were examined using energetic pairwise coupling to describe the communication between these residues in both the free and RNA-bound states of the protein. 15N NMR experiments were used to determine effects of the beta-sheet substitutions on the structural and dynamic properties of this domain. The combination of thermodynamic pairwise coupling and 15N-backbone dynamics provides direct evidence for local cooperative interactions among Y13, Q54, and F56, and a non-conserved loop that directly affect RNA-binding. The results describe how conserved and non-conserved regions of an RBD can communicate with each other to mediate recognition of the RNA.
RNA结合蛋白中最常见的结构基序之一是RNA结合结构域（RBD）。这些结构域具有共同的α/β三明治三级折叠，并且高度保守，尽管它们以广泛的结合亲和力结合多种RNA靶标。人U1A蛋白的N端RNA结合结构域（RBD1）以亚纳摩尔亲和力特异性结合U1 snRNA的茎/环II。β-折叠表面上暴露于溶剂的芳香族残基在RBD结构域中高度保守；在RBD1中，这些残基是Tyr13和Phe56，在位置54处有一个独特的Gln。使用能量成对耦合来描述蛋白质的游离态和RNA结合态中这些残基之间的通讯，研究了这些位置的取代效应。利用15N NMR实验来确定β-折叠取代对该结构域的结构和动力学性质的影响。热力学成对耦合和15N主链动力学的结合为Y13、Q54和F56以及一个直接影响RNA结合的非保守环之间的局部协同相互作用提供了直接证据。结果描述了RBD的保守区和非保守区如何相互通讯以介导对RNA的识别。
