TY - JOUR
T1 - How insulin binds
T2 - The B-chain α-helix contacts the L1 β-helix of the insulin receptor
AU - Huang, Kun
AU - Xu, Bin
AU - Hu, Shi Quan
AU - Chu, Ying Chi
AU - Hua, Qing Xin
AU - Qu, Yan
AU - Li, Biaoru
AU - Wang, Shuhua
AU - Wang, Run Ying
AU - Nakagawa, Satoe H.
AU - Theede, Anne Mette
AU - Whittaker, Jonathan
AU - De Meyts, Pierre
AU - Katsoyannis, Panayotis G.
AU - Weiss, Michael A.
N1 - Funding Information:
We thank D. F. Steiner for kindly providing the P3-A cell line overexpressing the human insulin receptor; C. Yip and G. D. Smith for the gift of isolated receptor ectodomain; M. DeFellipis and B. H. Frank (Eli Lilly and Co) for generously providing human insulin (to M.A.W. and P.G.K.); Wenhua Jia for assistance with CD measurements; and R. M. Shymko for computer curve-fitting of the B12 receptor-binding data. S.H.N. was supported, in part, by an NIH R21 grant to M.A.W. (R21 DK61651) and by the Diabetes Research & Training Center of the University of Chicago. J.W. was supported, in part, by a grant from the Juvenile Diabetes Research Foundation (1-2000-198. 2). The Receptor Biology Laboratory and the Hagedorn Research Institute are independent basic research conmponents of Novo Nordisk A/S. A.M.J. and P.D.M. were supported by a grant from the Danish Research Councils through the Danish Growth and Regeneration Center. This work was supported, in part, by grants from the National Institutes of Health to M.A.W. (DK40949) and P.G.K. (DK56673) and by a grant from the Danish Research Council (P.D.M.). This is a contribution from the Cleveland Center for Structural Biology.
PY - 2004/8/6
Y1 - 2004/8/6
N2 - Binding of insulin to the insulin receptor plays a central role in the hormonal control of metabolism. Here, we investigate possible contact sites between the receptor and the conserved non-polar surface of the B-chain. Evidence is presented that two contiguous sites in an α-helix, Val B12 and TyrB16, contact the receptor. Chemical synthesis is exploited to obtain non-standard substitutions in an engineered monomer (DKP-insulin). Substitution of TyrB16 by an isosteric photo-activatable derivative (para-azido-phenylalanine) enables efficient cross-linking to the receptor. Such cross-linking is specific and maps to the L1 β-helix of the α-subunit. Because substitution of ValB12 by larger side-chains markedly impairs receptor binding, cross-linking studies at B12 were not undertaken. Structure-function relationships are instead probed by side-chains of similar or smaller volume: respective substitution of Val B12 by alanine, threonine, and α-aminobutyric acid leads to activities of 1(±0.1)%, 13(±6)%, and 14(±5)% (relative to DKP-insulin) without disproportionate changes in negative cooperativity. NMR structures are essentially identical with native insulin. The absence of transmitted structural changes suggests that the low activities of B12 analogues reflect local perturbation of a "high-affinity" hormone-receptor contact. By contrast, because position B16 tolerates alanine substitution (relative activity 34(±10)%), the contribution of this neighboring interaction is smaller. Together, our results support a model in which the B-chain α-helix, functioning as an essential recognition element, docks against the L1 β-helix of the insulin receptor.
AB - Binding of insulin to the insulin receptor plays a central role in the hormonal control of metabolism. Here, we investigate possible contact sites between the receptor and the conserved non-polar surface of the B-chain. Evidence is presented that two contiguous sites in an α-helix, Val B12 and TyrB16, contact the receptor. Chemical synthesis is exploited to obtain non-standard substitutions in an engineered monomer (DKP-insulin). Substitution of TyrB16 by an isosteric photo-activatable derivative (para-azido-phenylalanine) enables efficient cross-linking to the receptor. Such cross-linking is specific and maps to the L1 β-helix of the α-subunit. Because substitution of ValB12 by larger side-chains markedly impairs receptor binding, cross-linking studies at B12 were not undertaken. Structure-function relationships are instead probed by side-chains of similar or smaller volume: respective substitution of Val B12 by alanine, threonine, and α-aminobutyric acid leads to activities of 1(±0.1)%, 13(±6)%, and 14(±5)% (relative to DKP-insulin) without disproportionate changes in negative cooperativity. NMR structures are essentially identical with native insulin. The absence of transmitted structural changes suggests that the low activities of B12 analogues reflect local perturbation of a "high-affinity" hormone-receptor contact. By contrast, because position B16 tolerates alanine substitution (relative activity 34(±10)%), the contribution of this neighboring interaction is smaller. Together, our results support a model in which the B-chain α-helix, functioning as an essential recognition element, docks against the L1 β-helix of the insulin receptor.
KW - Aba, α-aminobutyric acid
KW - CD, circular dichroism
KW - CHO, Chinese hamster ovary
KW - CR, cysteine-rich domain of receptor α-subunit
KW - DG, distance geometry
KW - NMR spectroscopy
KW - diabetes mellitus
KW - metabolism
KW - non-standard mutagenesis
KW - polypeptide hormone
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U2 - 10.1016/j.jmb.2004.05.023
DO - 10.1016/j.jmb.2004.05.023
M3 - Article
C2 - 15276842
AN - SCOPUS:3342960841
SN - 0022-2836
VL - 341
SP - 529
EP - 550
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 2
ER -
