Tishchenko Svetlana, Kostareva Olga, Gabdulkhakov Azat, Mikhaylina Alisa, Nikonova Ekaterina, Nevskaya Natalia, Sarskikh Alena, Piendl Wolfgang, Garber Maria, Nikonov Stanislav
Institute of Protein Research, Russian Academy of Sciences, Institutskaya 4, 142290 Pushchino, Moscow Region, Russian Federation.
Biocenter, Division of Medical Biochemistry, Innsbruck Medical University, Innrain 80-82, 6020 Innsbruck, Austria.
Acta Crystallogr D Biol Crystallogr. 2015 Feb;71(Pt 2):376-86. doi: 10.1107/S1399004714026248. Epub 2015 Jan 23.
Ribosomal protein L1, as part of the L1 stalk of the 50S ribosomal subunit, is implicated in directing tRNA movement through the ribosome during translocation. High-resolution crystal structures of four mutants (T217V, T217A, M218L and G219V) of the ribosomal protein L1 from Thermus thermophilus (TthL1) in complex with a specific 80 nt fragment of 23S rRNA and the structures of two of these mutants (T217V and G219V) in the RNA-unbound form are reported in this work. All mutations are located in the highly conserved triad Thr-Met-Gly, which is responsible for about 17% of all protein-RNA hydrogen bonds and 50% of solvent-inaccessible intermolecular hydrogen bonds. In the mutated proteins without bound RNA the RNA-binding regions show substantial conformational changes. On the other hand, in the complexes with RNA the structures of the RNA-binding surfaces in all studied mutants are very similar to the structure of the wild-type protein in complex with RNA. This shows that formation of the RNA complexes restores the distorted surfaces of the mutant proteins to a conformation characteristic of the wild-type protein complex. Domain I of the mutated TthL1 and helix 77 of 23S rRNA form a rigid body identical to that found in the complex of wild-type TthL1 with RNA, suggesting that the observed relative orientation is conserved and is probably important for ribosome function. Analysis of the complex structures and the kinetic data show that the number of intermolecular contacts and hydrogen bonds in the RNA-protein contact area does not correlate with the affinity of the protein for RNA and cannot be used as a measure of affinity.
核糖体蛋白L1作为50S核糖体亚基L1柄的一部分，在转位过程中参与引导tRNA通过核糖体移动。本文报道了嗜热栖热菌核糖体蛋白L1（TthL1）的四个突变体（T217V、T217A、M218L和G219V）与23S rRNA的特定80 nt片段形成复合物的高分辨率晶体结构，以及其中两个突变体（T217V和G219V）未结合RNA形式的结构。所有突变均位于高度保守的苏氨酸-甲硫氨酸-甘氨酸三联体中，该三联体负责约17%的所有蛋白质-RNA氢键和50%的溶剂不可及的分子间氢键。在没有结合RNA的突变蛋白中，RNA结合区域显示出显著的构象变化。另一方面，在与RNA形成的复合物中，所有研究突变体的RNA结合表面结构与野生型蛋白与RNA形成复合物的结构非常相似。这表明RNA复合物的形成将突变蛋白扭曲的表面恢复到野生型蛋白复合物特有的构象。突变的TthL1的结构域I和23S rRNA的螺旋77形成了一个与野生型TthL1与RNA形成的复合物中发现的刚体相同的刚体，这表明观察到的相对取向是保守的，可能对核糖体功能很重要。对复合物结构和动力学数据的分析表明，RNA-蛋白质接触区域的分子间接触和氢键数量与蛋白质对RNA的亲和力不相关，不能用作亲和力的衡量标准。
