DegP蛋白酶-伴侣在革兰氏阴性细菌的周质中运作,它有助于调节蛋白质的稳态,促进毒力,对于在压力下生存至关重要。为了完成这些任务,DegP形成预组织的载脂蛋白低聚物的网络,其促进在笼状复合物的分布内捕获底物,所述笼状复合物扩展以封装各种尺寸的客户。虽然DegP笼形复合物的结构是众所周知的,对结构知之甚少,动力学,DegP笼内客户端蛋白的相互作用以及客户端结构动力学与功能之间的关系。这里,我们通过结合流体动力学测量,使用模型α-螺旋客户蛋白在整个DegP激活周期中探测600kDaDegP笼复合物中的宿主-客体相互作用。基于甲基-横向弛豫优化光谱的溶液核磁共振研究,和蛋白水解活性测定。我们发现在客户面前,DegP笼与少数中间体合作组装。我们的数据进一步表明,绑定客户端的N端一半,伸入笼子的内部,主要是展开和灵活的,以及在宽范围的时间范围内多个构象状态之间的交换。最后,我们表明,DegP的蛋白酶结构域的协同结构转变发生在客户参与时,导致激活。一起,我们的发现支持DegP模型作为一个高度合作和动态的分子机器,稳定客户的展开状态,主要通过与C末端的相互作用,产生有效的卵裂。
The DegP protease-chaperone operates within the periplasm of Gram-negative bacteria, where it assists in the regulation of protein homeostasis, promotes virulence, and is essential to survival under stress. To carry out these tasks, DegP forms a network of preorganized apo oligomers that facilitate the capture of substrates within distributions of cage-like complexes which expand to encapsulate clients of various sizes. Although the architectures of DegP cage complexes are well understood, little is known about the structures, dynamics, and interactions of client proteins within DegP cages and the relationship between client structural dynamics and function. Here, we probe host-guest interactions within a 600 kDa DegP cage complex throughout the DegP activation cycle using a model α-helical client protein through a combination of hydrodynamics measurements, methyl-transverse relaxation optimized spectroscopy-based solution nuclear magnetic resonance studies, and proteolytic activity assays. We find that in the presence of the client, DegP cages assemble cooperatively with few intermediates. Our data further show that the N-terminal half of the bound client, which projects into the interior of the cages, is predominantly unfolded and flexible, and exchanges between multiple conformational states over a wide range of time scales. Finally, we show that a concerted structural transition of the protease domains of DegP occurs upon client engagement, leading to activation. Together, our findings support a model of DegP as a highly cooperative and dynamic molecular machine that stabilizes unfolded states of clients, primarily via interactions with their C-termini, giving rise to efficient cleavage.