Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, United Kingdom.
Proc Natl Acad Sci U S A. 2011 Apr 5;108(14):5596-601. doi: 10.1073/pnas.1101752108. Epub 2011 Mar 21.
Members of the homeodomain superfamily are three-helix bundle proteins whose second and third helices form a helix-turn-helix motif (HTH). Their folding mechanism slides from the ultrafast, three-state framework mechanism for the engrailed homeodomain (EnHD), in which the HTH motif is independently stable, to an apparent two-state nucleation-condensation model for family members with an unstable HTH motif. The folding intermediate of EnHD has nearly native HTH structure, but it is not docked with helix1. The determinant of whether two- or three-state folding was hypothesized to be the stability of the HTH substructure. Here, we describe a detailed Φ-value analysis of the folding of the Pit1 homeodomain, which has similar ultrafast kinetics to that of EnHD. Formation of helix1 was strongly coupled with formation of HTH, which was initially surprising because they are uncoupled in the EnHD folding intermediate. However, we found a key difference between Pit1 and EnHD: The isolated peptide corresponding to the HTH motif in Pit1 was not folded in the absence of H1. Independent molecular dynamics simulations of Pit1 unfolding found an intermediate with H1 misfolded onto the HTH motif. The Pit1 folding pathway is the connection between that of EnHD and the slower folding homeodomains and provides a link in the transition of mechanisms from two- to three-state folding in this superfamily. The malleability of folding intermediates can lead to unstable substructures being stabilized by a variety of nonnative interactions, adding to the continuum of folding mechanisms.
同源域超家族成员是三螺旋束蛋白，其第二和第三螺旋形成螺旋-转角-螺旋基序（HTH）。它们的折叠机制从超快的、三态框架机制（EnHD）滑动，其中 HTH 基序独立稳定，到具有不稳定 HTH 基序的家族成员的明显两态成核-凝聚模型。EnHD 的折叠中间体具有几乎天然的 HTH 结构，但未与 helix1 对接。假设二态或三态折叠的决定因素是 HTH 亚结构的稳定性。在这里，我们描述了对 Pit1 同源域折叠的详细Φ值分析，其折叠动力学与 EnHD 相似。helix1 的形成与 HTH 的形成强烈耦合，这最初令人惊讶，因为它们在 EnHD 折叠中间体中是解耦的。然而，我们发现了 Pit1 和 EnHD 之间的一个关键区别：在没有 H1 的情况下，对应于 Pit1 中 HTH 基序的分离肽没有折叠。对 Pit1 展开的独立分子动力学模拟发现，存在 H1 错误折叠到 HTH 基序上的中间体。Pit1 折叠途径是 EnHD 和较慢折叠同源域之间的连接，并为该超家族中从两态到三态折叠机制的转变提供了联系。折叠中间体的可变性可以导致不稳定的亚结构通过各种非天然相互作用稳定化，增加了折叠机制的连续性。
