Pin1 is an essential mitotic regulator consisting of a peptidyl-prolyl isomerase (PPIase) domain flexibly tethered to a smaller Trp-Trp (WW) binding domain. Communication between these domains is important for Pin1 in vivo activity; however, the atomic basis for this communication has remained elusive. Our previous nuclear magnetic resonance (NMR) studies of Pin1 functional dynamics suggested that weak interdomain contacts within Pin1 enable allosteric communication between the domain interface and the distal active site of the PPIase domain.1,2 A necessary condition for this hypothesis is that the intrinsic properties of the PPIase domain should be sensitive to interdomain contact. Here, we test this sensitivity by generating a Pin1 mutant, I28A, which weakens the wild-type interdomain contact while maintaining the overall folds of the two domains. Using NMR, we show that I28A leads to altered substrate binding affinity and isomerase activity. Moreover, I28A causes long-range perturbations to conformational flexibility in both domains, for both the apo and substrate-complexed states of the protein. These results show that the distribution of conformations sampled by the PPIase domain is sensitive to interdomain contact and strengthen the hypothesis that such contact supports interdomain allosteric communication in Pin1. Other modular systems may exploit interdomain interactions in a similar manner.

译文

Pin1是一种必需的有丝分裂调节剂,由肽基-脯氨酰异构酶 (PPIase) 结构域组成,灵活地束缚在较小的Trp-Trp (WW) 结合结构域上。这些结构域之间的交流对于Pin1的体内活性很重要; 但是,这种交流的原子基础仍然难以捉摸。我们先前对Pin1功能动力学进行的核磁共振 (NMR) 研究表明,Pin1内的弱域间接触使域界面与PPIase域的远端活性位点之间的变构通讯成为可能。1,2该假设的必要条件是PPIase域的固有特性应对域间接触敏感。在这里,我们通过生成Pin1突变体I28A来测试这种敏感性,该突变体削弱了野生型域间接触,同时保持了两个域的整体褶皱。使用NMR,我们显示I28A导致改变的底物结合亲和力和异构酶活性。此外,对于蛋白质的载脂蛋白和底物络合态,I28A会在两个结构域中引起构象灵活性的远距离扰动。这些结果表明,由PPIase结构域采样的构象分布对结构域间接触敏感,并加强了这种接触支持pin1中结构域间变构通讯的假设。其他模块化系统可以以类似的方式利用域间相互作用。

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