Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i., Flemingovo nám. 2, 166 10 Prague 6, Czech Republic.
J Biol Chem. 2013 Apr 12;288(15):10230-40. doi: 10.1074/jbc.M112.448050. Epub 2013 Feb 27.
Despite the recent first structural insight into the insulin-insulin receptor complex, the role of the C terminus of the B-chain of insulin in this assembly remains unresolved. Previous studies have suggested that this part of insulin must rearrange to reveal amino acids crucial for interaction with the receptor. The role of the invariant Phe(B24), one of the key residues of the hormone, in this process remains unclear. For example, the B24 site functionally tolerates substitutions to D-amino acids but not to L-amino acids. Here, we prepared and characterized a series of B24-modified insulin analogues, also determining the structures of [D-HisB24]-insulin and [HisB24]-insulin. The inactive [HisB24]-insulin molecule is remarkably rigid due to a tight accommodation of the L-His side chain in the B24 binding pocket that results in the stronger tethering of B25-B28 residues to the protein core. In contrast, the highly active [D-HisB24]-insulin is more flexible, and the reverse chirality of the B24C(α) atom swayed the D-His(B24) side chain into the solvent. Furthermore, the pocket vacated by Phe(B24) is filled by Phe(B25), which mimics the Phe(B24) side and main chains. The B25→B24 downshift results in a subsequent downshift of Tyr(B26) into the B25 site and the departure of B26-B30 residues away from the insulin core. Our data indicate the importance of the aromatic L-amino acid at the B24 site and the structural invariance/integrity of this position for an effective binding of insulin to its receptor. Moreover, they also suggest limited, B25-B30 only, unfolding of the C terminus of the B-chain upon insulin activation.
尽管最近首次获得了胰岛素-胰岛素受体复合物的结构见解，但胰岛素 B 链 C 端在该组装中的作用仍未解决。先前的研究表明，胰岛素的这一部分必须重新排列，以揭示与受体相互作用的关键氨基酸。激素的关键残基之一不变苯丙氨酸（B24）在该过程中的作用仍不清楚。例如，B24 位点在功能上可容忍取代为 D-氨基酸，但不能取代为 L-氨基酸。在这里，我们制备并表征了一系列 B24 修饰的胰岛素类似物，还确定了[D-HisB24]-胰岛素和[HisB24]-胰岛素的结构。由于 L-组氨酸侧链在 B24 结合口袋中的紧密容纳，导致 B25-B28 残基与蛋白质核心的更强束缚，无活性的[HisB24]-胰岛素分子非常僵硬。相比之下，高度活跃的[D-HisB24]-胰岛素更灵活，B24C(α)原子的反向手性使 D-His（B24）侧链摇摆到溶剂中。此外，由苯丙氨酸（B24）空出的口袋被苯丙氨酸（B25）填充，其模拟苯丙氨酸（B24）的侧链和主链。B25→B24 的下移导致 Tyr（B26）随后下移到 B25 位点，并且 B26-B30 残基离开胰岛素核心。我们的数据表明 B24 位点芳香 L-氨基酸的重要性以及该位置的结构不变性/完整性对于胰岛素与其受体的有效结合至关重要。此外，它们还表明胰岛素激活时 B 链 C 端的 C 端仅发生有限的（B25-B30）展开。
