Evolution of oligomeric state through allosteric pathways that mimic ligand binding
Evolution of oligomeric state through allosteric pathways that mimic ligand binding
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DOI：
10.1126/science.1254346
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发表时间：
2014-12-19
期刊：
影响因子：
56.9
通讯作者：
Teichmann, Sarah A.
中科院分区：
文献类型：
--
作者：
Perica, Tina;Kondo, Yasushi;Teichmann, Sarah A.
简介：蛋白质复合物的进化和设计经常通过蛋白质界面的氨基酸突变来观察，但我们之前表明，远离界面的残基也通常参与替代四级结构的进化。我们假设在这些蛋白质家族中，寡聚状态的差异是由于亚基间几何形状的变化造成的。间接突变将通过改变蛋白质构象和动力学来起作用，类似于变构小分子引入功能性构象变化的方式。我们将这些取代称为“变构突变”。 理由：在这项工作中，我们研究了变构突变对嘧啶操纵子衰减子 PyrR 家族寡聚状态的作用机制。在这个家族中，在嗜热直向同源物（BcPyrR）中形成四聚体界面的完全序列保守的螺旋转变为在嗜温直向同源物（BsPyrR）中暴露于溶剂。这导致同二聚体结构，其中两个亚基相对于它们在四聚体中的方向明显旋转。这种旋转和四级结构变化的根源是什么？为了剖析 BsPyrR 和 BcPyrR 之间 49 个取代的作用，我们使用祖先序列重建结合结构和生物物理方法来识别一组导致这种构象转变的变构突变。我们比较了鸟苷单磷酸 (GMP) 变构调节过程中突变引起的蛋白质运动构象变化。结果：我们鉴定了 11 个控制寡聚状态的关键突变，这些突变均远离界面和外部配体结合袋。我们通过设计推断的祖先 PyrR 蛋白（现有直向同源物之间的中间序列）寡聚状态的转变，证实了这些变构突变的作用。我们进一步使用推断的祖先状态及其突变体来表明变构突变是 PyrR 蛋白对较低温度的下坡适应的一部分。我们将祖先和工程化 PyrR 蛋白的 X 射线晶体结构与嗜温 BsPyrR 的游离和 GMP 结合结构进行了比较，BsPyrR 在配体结合后将其平衡从二聚体转变为四聚体。变构分子的结合引入了亚基间几何形状的变化，这相当于二聚体和四聚体同源物之间亚基间几何形状的进化差异。我们进一步发现寡聚状态的差异与二聚体内在动力学的差异相关。最后，我们使用残基-残基接触网络方法表明，当亚基间几何形状发生变化时，与变构突变相对应的残基会经历大的接触重连，进而通过 GMP 结合或引入变构突变来改变寡聚状态。结论：我们表明，进化利用该蛋白质的内在动力学以类似于小分子的方式切换构象开关。通过微妙的修饰改变不同状态的相对群体是蛋白质功能的核心过程，如此处所示，也是蛋白质进化的核心过程。这表明我们可以从进化中学习并设计具有多种构象状态的蛋白质。
INTRODUCTION: Evolution and design of protein complexes are frequently viewed through the lens of amino acid mutations at protein interfaces, but we showed previously that residues distant from interfaces are also commonly involved in the evolution of alternative quaternary structures. We hypothesized that in these protein families, the difference in oligomeric state is due to a change in intersubunit geometry. The indirect mutations would act by changing protein conformation and dynamics, similar to the way in which allosteric small molecules introduce functional conformational change. We refer to these substitutions as "allosteric mutations."RATIONALE: In this work, we investigate the mechanism of action of allosteric mutations on oligomeric state in the PyrR family of pyrimidine operon attenuators. In this family, an entirely sequence-conserved helix that forms a tetrameric interface in the thermophilic ortholog (BcPyrR) switches to being solvent-exposed in the mesophilic ortholog (BsPyrR). This results in a homodimeric structure in which the two subunits are clearly rotated relative to their orientation in the tetramer. What is the origin of this rotation and the change in quaternary structure? To dissect the role of the 49 substitutions between BsPyrR and BcPyrR, we used ancestral sequence reconstruction in combination with structural and biophysical methods to identify a set of allosteric mutations that are responsible for this shift in conformation. We compared the conformational changes introduced by the mutations to the protein motion during allosteric regulation by guanosine monophosphate (GMP).RESULTS: We identified 11 key mutations controlling oligomeric state, all distant from the interfaces and outside ligand-binding pockets. We confirmed the role of these allosteric mutations by engineering a shift in oligomeric state in an inferred ancestral PyrR protein (intermediate in sequence between the extant orthologs). We further used the inferred ancestral states and their mutants to show that the allosteric mutations are part of a downhill adaptation of the PyrR proteins to lower temperatures. We compared the x-ray crystal structures of ancestral and engineered PyrR proteins to the free and GMP-bound structure of the mesophilic BsPyrR, which shifts its equilibrium from dimer to tetramer upon ligand binding. Binding of the allosteric molecule introduces a change in intersubunit geometry that is equivalent to the evolutionary difference in intersubunit geometry between the dimeric and tetrameric homologs. We further find that the difference in oligomeric state is coupled to the difference in intrinsic dynamics of the dimers. Finally, we used the residue-residue contact network approach to show that the residues corresponding to the allosteric mutations undergo large contact rewiring when the intersubunit geometry and, in turn, oligomeric state change, either by GMP binding or by the introduction of allosteric mutations.CONCLUSION: We show that evolution employs the intrinsic dynamics of this protein to toggle a conformational switch in a manner similar to that of small molecules. Shifting the relative populations of different states by subtle modifications is a process central to protein function and, as shown here, also to protein evolution. This suggests that we can learn from evolution and design proteins with multiple conformational states.