How insulin binds: the B-chain alpha-helix contacts the L1 beta-helix of the insulin receptor.
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 alpha-helix, Val(B12) and Tyr(B16), contact the receptor. Chemical synthesis is exploited to obtain non-standard substitutions in an engineered monomer (DKP-insulin). Substitution of Tyr(B16) 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 beta-helix of the alpha-subunit. Because substitution of Val(B12) 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 alpha-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 alpha-helix, functioning as an essential recognition element, docks against the L1 beta-helix of the insulin receptor.
Department of Biochemistry, Case Western Reserve School of Medicine, Cleveland OH 44106-4935, USA.
Kun
K
Huang
Bin
B
Xu
Shi-Quan
SQ
Hu
Ying-Chi
YC
Chu
Qing-Xin
QX
Hua
Yan
Y
Qu
Biaoru
B
Li
Shuhua
S
Wang
Run-Ying
RY
Wang
Satoe H
SH
Nakagawa
Anne Mette
AM
Theede
Jonathan
J
Whittaker
Pierre
P
De Meyts
Panayotis G
PG
Katsoyannis
Michael A
MA
Weiss
D000409:Alanine; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D001665:Binding Sites; D002942:Circular Dichroism; D006801:Humans; D007328:Insulin / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D011972:Receptor, Insulin / Q000737:chemistry* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014633:Valine
D007328:Insulin; D010446:Peptide Fragments; D011972:Receptor, Insulin; D014633:Valine; D000409:Alanine