Weiss Michael A, Wan Zhuli, Zhao Ming, Chu Ying-Chi, Nakagawa Satoe H, Burke G Thomson, Jia Wenhua, Hellmich Rosanna, Katsoyannis Panayotis G
Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.
J Mol Biol. 2002 Jan 11;315(2):103-11. doi: 10.1006/jmbi.2001.5224.
The design of insulin analogues has emphasized stabilization or destabilization of structural elements according to established principles of protein folding. To this end, solvent-exposed side-chains extrinsic to the receptor-binding surface provide convenient sites of modification. An example is provided by an unfavorable helical C-cap (Thr(A8)) whose substitution by favorable amino acids (His(A8) or Arg(A8)) has yielded analogues of improved stability. Remarkably, these analogues also exhibit enhanced activity, suggesting that activity may correlate with stability. Here, we test this hypothesis by substitution of diaminobutyric acid (Dab(A8)), like threonine an amino acid of low helical propensity. The crystal structure of Dab(A8)-insulin is similar to those of native insulin and the related analogue Lys(A8)-insulin. Although no more stable than native insulin, the non-standard analogue is twice as active. Stability and affinity can therefore be uncoupled. To investigate alternative mechanisms by which A8 substitutions enhance activity, multiple substitutions were introduced. Surprisingly, diverse aliphatic, aromatic and polar side-chains enhance receptor binding and biological activity. Because no relationship is observed between activity and helical propensity, we propose that local interactions between the A8 side-chain and an edge of the hormone-receptor interface modulate affinity. Dab(A8)-insulin illustrates the utility of non-standard amino acids in hypothesis-driven protein design.
胰岛素类似物的设计依据蛋白质折叠的既定原则，着重于结构元件的稳定化或去稳定化。为此，受体结合表面外部暴露于溶剂中的侧链提供了便利的修饰位点。一个例子是不利的螺旋C帽（苏氨酸（A8）），用有利的氨基酸（组氨酸（A8）或精氨酸（A8））对其进行取代，产生了稳定性提高的类似物。值得注意的是，这些类似物还表现出增强的活性，这表明活性可能与稳定性相关。在此，我们通过取代二氨基丁酸（Dab（A8））来检验这一假设，二氨基丁酸与苏氨酸一样，是一种螺旋倾向较低的氨基酸。Dab（A8）-胰岛素的晶体结构与天然胰岛素及相关类似物Lys（A8）-胰岛素的晶体结构相似。尽管这种非标准类似物并不比天然胰岛素更稳定，但其活性却是天然胰岛素的两倍。因此，稳定性和亲和力可以解偶联。为了研究A8取代增强活性的其他机制，引入了多个取代。令人惊讶的是，多种脂肪族、芳香族和极性侧链均增强了受体结合和生物活性。由于未观察到活性与螺旋倾向之间的关系，我们提出A8侧链与激素-受体界面边缘之间的局部相互作用调节亲和力。Dab（A8）-胰岛素说明了非标准氨基酸在假设驱动的蛋白质设计中的效用。
