Qin Haina, Lim Liangzhong, Song Jianxing
Department of Biological Sciences, Faculty of Science, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Republic of Singapore.
BMC Biophys. 2012 Jan 25;5:2. doi: 10.1186/2046-1682-5-2.
The role of dynamics in protein functions including signal transduction is just starting to be deciphered. Eph receptors with 16 members divided into A- and B- subclasses are respectively activated by 9 A- and B-ephrin ligands. EphA4 is the only receptor capable of binding to all 9 ephrins and small molecules with overlapped interfaces.
We first determined the structures of the EphA4 ligand binding domain (LBD) in two crystals of P1 space group. Noticeably, 8 EphA4 molecules were found in one asymmetric unit and consequently from two crystals we obtained 16 structures, which show significant conformational variations over the functionally critical A-C, D-E, G-H and J-K loops. The 16 new structures, together with previous 9 ones, can be categorized into two groups: closed and open forms which resemble the uncomplexed and complexed structures of the EphA4 LBD respectively. To assess whether the conformational diversity over the loops primarily results from the intrinsic dynamics, we initiated 30-ns molecular dynamics (MD) simulations for both closed and open forms. The results indicate that the loops do have much higher intrinsic dynamics, which is further unravelled by NMR H/D exchange experiments. During simulations, the open form has the RMS deviations slightly larger than those of the closed one, suggesting the open form may be less stable in the absence of external contacts. Furthermore, no obvious exchange between two forms is observed within 30 ns, implying that they are dynamically separated.
Our study provides the first experimental and computational result revealing that the intrinsic dynamics are most likely underlying the conformational diversity observed for the EphA4 LBD loops mediating the binding affinity and specificity. Interestingly, the open conformation of the EphA4 LBD is slightly unstable in the absence of it natural ligand ephrins, implying that the conformational transition from the closed to open has to be driven by the high-affinity interaction with ephrins because the weak interaction with small molecule was found to be insufficient to trigger the transition. Our results therefore highlight the key role of protein dynamics in Eph-ephrin signalling and would benefit future design of agonists/antagonists targeting Eph receptors.
动力学在包括信号转导在内的蛋白质功能中的作用才刚刚开始被破解。Eph受体有16个成员，分为A和B两个亚类，分别由9种A类和B类ephrin配体激活。EphA4是唯一能够通过重叠界面与所有9种ephrin和小分子结合的受体。
我们首先在P1空间群的两个晶体中确定了EphA4配体结合结构域（LBD）的结构。值得注意的是，在一个不对称单元中发现了8个EphA4分子，因此从两个晶体中我们获得了16种结构，这些结构在功能关键的A-C、D-E、G-H和J-K环上表现出显著的构象变化。这16种新结构与之前的9种结构可分为两组：封闭形式和开放形式，分别类似于EphA4 LBD的未结合和结合结构。为了评估环上的构象多样性是否主要源于内在动力学，我们对封闭和开放形式都进行了30纳秒的分子动力学（MD）模拟。结果表明，这些环确实具有更高的内在动力学，这通过核磁共振氢/氘交换实验得到了进一步揭示。在模拟过程中，开放形式的均方根偏差略大于封闭形式，这表明在没有外部接触的情况下，开放形式可能不太稳定。此外，在30纳秒内未观察到两种形式之间有明显的交换，这意味着它们在动力学上是分离的。
我们的研究提供了首个实验和计算结果，揭示内在动力学很可能是介导结合亲和力和特异性的EphA4 LBD环构象多样性的基础。有趣的是，在没有天然配体ephrin的情况下，EphA4 LBD的开放构象略显不稳定，这意味着从封闭到开放的构象转变必须由与ephrin的高亲和力相互作用驱动，因为发现与小分子的弱相互作用不足以触发这种转变。因此，我们的结果突出了蛋白质动力学在Eph-ephrin信号传导中的关键作用，并将有助于未来针对Eph受体的激动剂/拮抗剂的设计。
