Crabtree Michael D, Borcherds Wade, Poosapati Anusha, Shammas Sarah L, Daughdrill Gary W, Clarke Jane
Department of Chemistry, University of Cambridge , Cambridge CB2 1EW, U.K.
Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida , Tampa, Florida 33620, United States.
Biochemistry. 2017 May 9;56(18):2379-2384. doi: 10.1021/acs.biochem.7b00179. Epub 2017 Apr 26.
Appropriate integration of cellular signals requires a delicate balance of ligand-target binding affinities. Increasing the level of residual structure in intrinsically disordered proteins (IDPs), which are overrepresented in these cellular processes, has been shown previously to enhance binding affinities and alter cellular function. Conserved proline residues are commonly found flanking regions of IDPs that become helical upon interacting with a partner protein. Here, we mutate these helix-flanking prolines in p53 and MLL and find opposite effects on binding affinity upon an increase in free IDP helicity. In both cases, changes in affinity were due to alterations in dissociation, not association, rate constants, which is inconsistent with conformational selection mechanisms. We conclude that, contrary to previous suggestions, helix-flanking prolines do not regulate affinity by modulating the rate of complex formation. Instead, they influence binding affinities by controlling the lifetime of the bound complex.
细胞信号的适当整合需要配体 - 靶点结合亲和力的微妙平衡。在这些细胞过程中大量存在的内在无序蛋白（IDP）中，增加其残余结构水平，此前已证明可增强结合亲和力并改变细胞功能。保守的脯氨酸残基通常位于IDP与伴侣蛋白相互作用时会形成螺旋的区域两侧。在这里，我们对p53和MLL中这些螺旋侧翼脯氨酸进行突变，发现随着游离IDP螺旋度增加，对结合亲和力产生相反的影响。在这两种情况下，亲和力的变化是由于解离速率常数的改变，而非缔合速率常数的改变，这与构象选择机制不一致。我们得出结论，与之前的观点相反，螺旋侧翼脯氨酸并非通过调节复合物形成速率来调节亲和力。相反，它们通过控制结合复合物的寿命来影响结合亲和力。
