Eberini Ivano, Guerini Rocco Alessandro, Ientile Anna Rita, Baptista António M, Gianazza Elisabetta, Tomaselli Simona, Molinari Henriette, Ragona Laura
Gruppo di Studio per la Proteomica e la Struttura delle Proteine, Dipartimento di Scienze Farmacologiche, Università degli Studi di Milano, Italy.
Proteins. 2008 Jun;71(4):1889-98. doi: 10.1002/prot.21875.
The correlation between protein motions and function is a central problem in protein science. Several studies have demonstrated that ligand binding and protein dynamics are strongly correlated in intracellular lipid binding proteins (iLBPs), in which the high degree of flexibility, principally occurring at the level of helix-II, CD, and EF loops (the so-called portal area), is significantly reduced upon ligand binding. We have recently investigated by NMR the dynamic properties of a member of the iLBP family, chicken liver bile acid binding protein (cL-BABP), in its apo and holo form, as a complex with two bile salts molecules. Binding was found to be regulated by a dynamic process and a conformational rearrangement was associated with this event. We report here the results of molecular dynamics (MD) simulations performed on apo and holo cL-BABP with the aim of further characterizing the protein regions involved in motion propagation and of evaluating the main molecular interactions stabilizing bound ligands. Upon binding, the root mean square fluctuation values substantially decrease for CD and EF loops while increase for the helix-loop-helix region, thus indicating that the portal area is the region mostly affected by complex formation. These results nicely correlate with backbone dynamics data derived from NMR experiments. Essential dynamics analysis of the MD trajectories indicates that the major concerted motions involve the three contiguous structural elements of the portal area, which however are dynamically coupled in different ways whether in the presence or in the absence of the ligands. Motions of the EF loop and of the helical region are part of the essential space of both apo and holo-BABP and sample a much wider conformational space in the apo form. Together with NMR results, these data support the view that, in the apo protein, the flexible EF loop visits many conformational states including those typical of the holo state and that the ligand acts stabilizing one of these pre-existing conformations. The present results, in agreement with data reported for other iLBPs, sharpen our knowledge on the binding mechanism for this protein family.
蛋白质运动与功能之间的相关性是蛋白质科学中的核心问题。多项研究表明，在细胞内脂质结合蛋白（iLBP）中，配体结合与蛋白质动力学密切相关，其中高度的灵活性主要出现在螺旋II、CD和EF环（所谓的门户区域）水平，在配体结合后显著降低。我们最近通过核磁共振研究了iLBP家族成员鸡肝胆汁酸结合蛋白（cL-BABP）的无配体形式和全配体形式（与两个胆汁盐分子形成的复合物）的动态特性。发现结合受动态过程调控，并且构象重排与该事件相关。我们在此报告对无配体和全配体cL-BABP进行分子动力学（MD）模拟的结果，目的是进一步表征参与运动传播的蛋白质区域，并评估稳定结合配体的主要分子相互作用。结合后，CD和EF环的均方根波动值大幅下降，而螺旋-环-螺旋区域的均方根波动值增加，这表明门户区域是受复合物形成影响最大的区域。这些结果与核磁共振实验得出的主链动力学数据很好地相关。对MD轨迹的主成分动力学分析表明，主要的协同运动涉及门户区域的三个相邻结构元件，然而，无论有无配体，它们以不同方式动态耦合。EF环和螺旋区域的运动是无配体和全配体BABP基本空间的一部分，并且在无配体形式中采样更广泛的构象空间。与核磁共振结果一起，这些数据支持这样的观点，即在无配体蛋白质中，灵活的EF环会访问许多构象状态，包括全配体状态的典型构象，并且配体起到稳定这些预先存在的构象之一的作用。目前的结果与其他iLBP报道的数据一致，加深了我们对该蛋白质家族结合机制的了解。
