Insight into the Structural and Biological Relevance of the T/R Transition of the N-Terminus of the B-Chain in Human Insulin
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- 作者：Lucie Kosinov谩 ; V谩clav Veverka ; Pavl铆na Novotn谩 ; Michaela Collinsov谩 ; Marie Urbanov谩 ; Nicholas R. Moody ; Johan P. Turkenburg ; Ji艡铆 Jir谩膷ek ; Andrzej M. Brzozowski ; Lenka 沤谩kov谩
- 刊名：Biochemistry
- 出版年：2014
- 出版时间：June 3, 2014
- 年：2014
- 卷：53
- 期：21
- 页码：3392-3402
- 全文大小：370K
- 年卷期：v.53,no.21(June 3, 2014)
- ISSN：1520-4995
文摘
The N-terminus of the B-chain of insulin may adopt two alternative conformations designated as the T- and R-states. Despite the recent structural insight into insulin鈥搃nsulin receptor (IR) complexes, the physiological relevance of the T/R transition is still unclear. Hence, this study focused on the rational design, synthesis, and characterization of human insulin analogues structurally locked in expected R- or T-states. Sites B3, B5, and B8, capable of affecting the conformation of the N-terminus of the B-chain, were subjects of rational substitutions with amino acids with specific allowed and disallowed dihedral 蠁 and 蠄 main-chain angles. 伪-Aminoisobutyric acid was systematically incorporated into positions B3, B5, and B8 for stabilization of the R-state, and N-methylalanine and mallcaps">d-proline amino acids were introduced at position B8 for stabilization of the T-state. IR affinities of the analogues were compared and correlated with their T/R transition ability and analyzed against their crystal and nuclear magnetic resonance structures. Our data revealed that (i) the T-like state is indeed important for the folding efficiency of (pro)insulin, (ii) the R-state is most probably incompatible with an active form of insulin, (iii) the R-state cannot be induced or stabilized by a single substitution at a specific site, and (iv) the B1鈥揃8 segment is capable of folding into a variety of low-affinity T-like states. Therefore, we conclude that the active conformation of the N-terminus of the B-chain must be different from the 鈥渃lassical鈥?T-state and that a substantial flexibility of the B1鈥揃8 segment, where GlyB8 plays a key role, is a crucial prerequisite for an efficient insulin鈥揑R interaction.