Sirota Fernanda L, Héry-Huynh Stephanie, Maurer-Stroh Sebastian, Wodak Shoshana J
SCMBB, Université Libre de Bruxelles, ULB, CP263, Blvd du Triomphe, 1050 Bruxelles, Belgium.
Proteins. 2008 Jul;72(1):88-104. doi: 10.1002/prot.21901.
Substitution of four amino acid residues (L5V,F30V, Y33F,A34F) in the B1 domain of the immunoglobulin G binding protein (GB1) leads to the formation of a swapped dimer, shown to be in equilibrium with a native-like monomeric state of the protein (Byeon et al., J Mol Biol 2003;333:141-152). In this study, we employ protein design calculations and molecular dynamics simulations to investigate the role of these substitutions in fostering the swapping reaction. DESIGNER, a fully automatic procedure for computing the amino acid sequences likely to stabilize a given backbone structure is used to investigate the effect of the four substitutions on the stability of the wild type native monomeric conformation. Results indicate that at least three of these substitutions (L5V,F30V, A34F) have a destabilizing effect. The L5V forms destabilizing interactions with surrounding residues, whereas F30V causes local strain due to unfavorable interactions with its own backbone. A dual role in the swapping reaction is played by A34F. It destabilizes the monomer conformation while stabilizing the swapped dimer. Our calculations find an energetically favorable conformation for the 34F side chain in the core of the monomer, but only at the expense of forcing the wild type W43 side chain into a highly strained rotamer, and forming unfavorable interactions with both W43 and V54. Although detailed calculations could not be performed on the swapped dimer, due to the lower accuracy of the model, analysis revealed that the 34F side chain from both subunits are tightly packed against each other in the dimer core, suggesting that their replacement by the smaller Ala, as in the wild type, would be highly destabilizing through the creation of a large internal cavity possibly accompanied by a substantial conformational change. Analysis of room temperature molecular dynamics (MD) simulations of the wild type and the modeled quadruple mutant structures reveals that the latter structure fluctuates more than its wild type counterpart. In addition, its C-terminal beta-hairpin, which is exchanged in the swapping reaction, undergoes a conformation change, which pushes it further away from the remainder of the protein. Simulations at higher temperature (450 K) show that the quadruple mutant unfolds earlier and more completely than the wild type following a sequence of events that is compatible with the description of the highly fluctuating monomeric state of this mutant observed by NMR. Our findings thus support the notion that domain swapping in GB1 is fostered by three main factors: a decrease in stability and increased flexibility of the monomer conformation, concomitant with stabilization of the swapped dimer conformation through new interactions that have no counterparts in the monomer.
免疫球蛋白G结合蛋白（GB1）的B1结构域中四个氨基酸残基（L5V、F30V、Y33F、A34F）的替换导致形成了一种交换二聚体，已证明其与该蛋白的天然单体状态处于平衡状态（Byeon等人，《分子生物学杂志》2003年；333:141 - 152）。在本研究中，我们采用蛋白质设计计算和分子动力学模拟来研究这些替换在促进交换反应中的作用。使用DESIGNER（一种用于计算可能稳定给定主链结构的氨基酸序列的全自动程序）来研究这四个替换对野生型天然单体构象稳定性的影响。结果表明，这些替换中至少有三个（L5V、F30V、A34F）具有去稳定作用。L5V与周围残基形成去稳定相互作用，而F30V由于与其自身主链的不利相互作用而导致局部应变。A34F在交换反应中起双重作用。它使单体构象不稳定，同时稳定交换二聚体。我们的计算发现，在单体核心中，34F侧链存在一个能量上有利的构象，但这是以迫使野生型W43侧链进入高度应变的旋转异构体并与W43和V54都形成不利相互作用为代价的。尽管由于模型精度较低，无法对交换二聚体进行详细计算，但分析表明，来自两个亚基的34F侧链在二聚体核心中彼此紧密堆积，这表明在野生型中用较小的丙氨酸取代它们可能会通过产生一个大的内部空腔并可能伴随大量构象变化而导致高度不稳定。对野生型和模拟的四重突变体结构的室温分子动力学（MD）模拟分析表明，后者结构的波动比其野生型对应物更大。此外，其在交换反应中发生交换的C端β - 发夹经历了构象变化，使其进一步远离蛋白质的其余部分。在较高温度（450 K）下的模拟表明，四重突变体比野生型更早且更完全地展开，其事件序列与通过核磁共振观察到的该突变体高度波动的单体状态描述一致。因此，我们的研究结果支持这样一种观点，即GB1中的结构域交换由三个主要因素促成：单体构象稳定性的降低和灵活性的增加，同时通过单体中不存在的新相互作用使交换二聚体构象稳定。
