Hua Qing-Xin, Nakagawa Satoe, Hu Shi-Quan, Jia Wenhua, Wang Shuhua, Weiss Michael A
Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106, USA.
J Biol Chem. 2006 Aug 25;281(34):24900-9. doi: 10.1074/jbc.M602691200. Epub 2006 Jun 8.
How insulin binds to the insulin receptor has long been a subject of speculation. Although the structure of the free hormone has been extensively characterized, a variety of evidence suggests that a conformational change occurs upon receptor binding. Here, we employ chiral mutagenesis, comparison of corresponding d and l amino acid substitutions, to investigate a possible switch in the B-chain. To investigate the interrelation of structure, function, and stability, isomeric analogs have been synthesized in which an invariant glycine in a beta-turn (Gly(B8)) is replaced by d- or l-Ser. The d substitution enhances stability (DeltaDeltaG(u) 0.9 kcal/mol) but impairs receptor binding by 100-fold; by contrast, the l substitution markedly impairs stability (DeltaDeltaG(u) -3.0 kcal/mol) with only 2-fold reduction in receptor binding. Although the isomeric structures each retain a native-like overall fold, the l-Ser(B8) analog exhibits fewer helix-related and long range nuclear Overhauser effects than does the d-Ser(B8) analog or native monomer. Evidence for enhanced conformational fluctuations in the unstable analog is provided by its attenuated CD spectrum. The inverse relationship between stereospecific stabilization and receptor binding strongly suggests that the B7-B10 beta-turn changes conformation on receptor binding.
胰岛素如何与胰岛素受体结合长期以来一直是一个猜测的话题。尽管游离激素的结构已得到广泛表征，但各种证据表明，受体结合后会发生构象变化。在这里，我们采用手性诱变，即比较相应的d型和l型氨基酸取代，来研究B链中可能的转变。为了研究结构、功能和稳定性之间的相互关系，我们合成了异构体类似物，其中β-转角处（Gly(B8)）的一个不变甘氨酸被d-或l-丝氨酸取代。d型取代增强了稳定性（ΔΔG(u) 0.9千卡/摩尔），但使受体结合能力受损100倍；相比之下，l型取代显著损害了稳定性（ΔΔG(u) -3.0千卡/摩尔），受体结合能力仅降低2倍。尽管异构体结构各自保留了类似天然的整体折叠，但与d-Ser(B8)类似物或天然单体相比，l-Ser(B8)类似物表现出较少的与螺旋相关的和长程核Overhauser效应。不稳定类似物中构象波动增强的证据由其减弱的圆二色光谱提供。立体特异性稳定与受体结合之间的反比关系强烈表明，B7-B10β-转角在受体结合时会改变构象。
