Patel Sunita, Vierling Elizabeth, Tama Florence
Department of Chemistry and Biochemistry, University of Arizona, Tucson, Arizona.
Department of Biochemistry & Molecular Biology, University of Massachusetts, Amherst, Massachusetts.
Biophys J. 2014 Jun 17;106(12):2644-55. doi: 10.1016/j.bpj.2014.04.048.
The small heat shock proteins (sHSPs) are a virtually ubiquitous and diverse group of molecular chaperones that can bind and protect unfolding proteins from irreversible aggregation. It has been suggested that intrinsic disorder of the N-terminal arm (NTA) of sHSPs is important for substrate recognition. To investigate conformations of the NTA that could recognize substrates we performed replica exchange molecular dynamics simulations. Behavior at normal and stress temperatures of the dimeric building blocks of dodecameric HSPs from wheat (Ta16.9) and pea (Ps18.1) were compared because they display high sequence similarity, but Ps18.1 is more efficient in binding specific substrates. In our simulations, the NTAs of the dimer are flexible and dynamic; however, rather than exhibiting highly extended conformations they retain considerable α-helical character and contacts with the conserved α-crystallin domain (ACD). Network analysis and clustering methods reveal that there are two major conformational forms designated either "open" or "closed" based on the relative position of the two NTAs and their hydrophobic solvent accessible surface area. The equilibrium constant for the closed to open transition is significantly different for Ta16.9 and Ps18.1, with the latter showing more open conformations at elevated temperature correlated with its more effective chaperone activity. In addition, the Ps18.1 NTAs have more hydrophobic solvent accessible surface than those of Ta16.9. NTA hydrophobic patches are comparable in size to the area buried in many protein-protein interactions, which would enable sHSPs to bind early unfolding intermediates. Reduced interactions of the Ps18.1 NTAs with each other and with the ACD contribute to the differences in dynamics and hydrophobic surface area of the two sHSPs. These data support a major role for the conformational equilibrium of the NTA in substrate binding and indicate features of the NTA that contribute to sHSP chaperone efficiency.
小热休克蛋白（sHSPs）是一类几乎普遍存在且多样的分子伴侣，能够结合并保护正在解折叠的蛋白质，使其免于不可逆聚集。有人提出，sHSPs的N端臂（NTA）的内在无序性对于底物识别很重要。为了研究能够识别底物的NTA的构象，我们进行了复制交换分子动力学模拟。比较了来自小麦（Ta16.9）和豌豆（Ps18.1）的十二聚体HSPs的二聚体构建块在正常温度和应激温度下的行为，因为它们具有高度的序列相似性，但Ps18.1在结合特定底物方面更有效。在我们的模拟中，二聚体的NTA是灵活且动态的；然而，它们并非呈现高度伸展的构象，而是保留了相当多的α-螺旋特征，并与保守的α-晶状体蛋白结构域（ACD）有接触。网络分析和聚类方法表明，基于两个NTA的相对位置及其疏水溶剂可及表面积，存在两种主要的构象形式，分别称为“开放”或“封闭”。Ta16.9和Ps18.1从封闭到开放转变的平衡常数显著不同，后者在升高温度时显示出更多的开放构象，这与其更有效的伴侣活性相关。此外，Ps18.1的NTA比Ta16.9的具有更多的疏水溶剂可及表面。NTA疏水斑块的大小与许多蛋白质-蛋白质相互作用中埋藏的面积相当，这将使sHSPs能够结合早期解折叠中间体。Ps18.1的NTA彼此之间以及与ACD的相互作用减少，导致了这两种sHSPs在动力学和疏水表面积上的差异。这些数据支持了NTA的构象平衡在底物结合中的主要作用，并表明了NTA有助于sHSP伴侣效率的特征。
