Fletcher Jordan M, Bartlett Gail J, Boyle Aimee L, Danon Jonathan J, Rush Laura E, Lupas Andrei N, Woolfson Derek N
School of Chemistry, University of Bristol , Cantock's Close, Bristol BS8 1TS, United Kingdom.
Department of Protein Evolution, Max Planck Institute for Developmental Biology , 72076 Tübingen, Germany.
ACS Chem Biol. 2017 Feb 17;12(2):528-538. doi: 10.1021/acschembio.6b00935. Epub 2017 Jan 9.
The α-helical coiled coil is one of the best-studied protein-protein interaction motifs. As a result, sequence-to-structure relationships are available for the prediction of natural coiled-coil sequences and the de novo design of new ones. However, coiled coils adopt a wide range of oligomeric states and topologies, and our understanding of the specification of these and the discrimination between them remains incomplete. Gaps in our knowledge assume more importance as coiled coils are used increasingly to construct biomimetic systems of higher complexity; for this, coiled-coil components need to be robust, orthogonal, and transferable between contexts. Here, we explore how the polar side chain asparagine (Asn, N) is tolerated within otherwise hydrophobic helix-helix interfaces of coiled coils. The long-held view is that Asn placed at certain sites of the coiled-coil sequence repeat selects one oligomer state over others, which is rationalized by the ability of the side chain to make hydrogen bonds, or interactions with chelated ions within the coiled-coil interior of the favored state. We test this with experiments on de novo peptide sequences traditionally considered as directing parallel dimers and trimers, and more widely through bioinformatics analysis of natural coiled-coil sequences and structures. We find that when located centrally, rather than near the termini of such coiled-coil sequences, Asn does exert the anticipated oligomer-specifying influence. However, outside of these bounds, Asn is observed less frequently in the natural sequences, and the synthetic peptides are hyperthermostable and lose oligomer-state specificity. These findings highlight that not all regions of coiled-coil repeat sequences are equivalent, and that care is needed when designing coiled-coil interfaces.
α-螺旋卷曲螺旋是研究最为深入的蛋白质-蛋白质相互作用基序之一。因此，序列与结构之间的关系可用于预测天然卷曲螺旋序列以及从头设计新的序列。然而，卷曲螺旋具有多种寡聚状态和拓扑结构，我们对这些状态的确定及其之间的区分的理解仍不完整。随着卷曲螺旋越来越多地用于构建更高复杂性的仿生系统，我们知识上的空白变得更加重要；为此，卷曲螺旋组件需要强大、正交且可在不同环境之间转移。在这里，我们探讨了极性侧链天冬酰胺（Asn，N）在卷曲螺旋原本疏水的螺旋-螺旋界面中是如何被容忍的。长期以来的观点是，位于卷曲螺旋序列重复特定位置的Asn会选择一种寡聚状态而非其他状态，这可以通过侧链形成氢键的能力或与偏好状态的卷曲螺旋内部螯合离子的相互作用来解释。我们通过对传统上被认为导向平行二聚体和三聚体的从头肽序列进行实验，并更广泛地通过对天然卷曲螺旋序列和结构的生物信息学分析来验证这一点。我们发现，当Asn位于此类卷曲螺旋序列的中心而非末端附近时，它确实会发挥预期的寡聚体确定影响。然而，在这些范围之外，Asn在天然序列中出现的频率较低，并且合成肽具有高热稳定性且失去了寡聚体状态特异性。这些发现突出表明，并非卷曲螺旋重复序列的所有区域都是等效的，在设计卷曲螺旋界面时需要谨慎。
