How insulin binds: the B-chain alpha-helix contacts the L1 beta-helix of the insulin receptor.


Abstract

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. Study holds ProTherm entries: 17309, 17310, 17311 Extra Details: polypeptide hormone; metabolism; diabetes mellitus; non-standard mutagenesis; NMR spectroscopy

Submission Details

ID: gcAzLWDX3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:49 p.m.

Version: 1

Publication Details
Huang K;Xu B;Hu SQ;Chu YC;Hua QX;Qu Y;Li B;Wang S;Wang RY;Nakagawa SH;Theede AM;Whittaker J;De Meyts P;Katsoyannis PG;Weiss MA,J. Mol. Biol. (2004) How insulin binds: the B-chain alpha-helix contacts the L1 beta-helix of the insulin receptor. PMID:15276842
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1BZV 1998-11-04T00:00:00+0000 0 [D-ALAB26]-DES(B27-B30)-INSULIN-B26-AMIDE A SUPERPOTENT SINGLE-REPLACEMENT INSULIN ANALOGUE, NMR, MINIMIZED AVERAGE STRUCTURE
1BZV 1998-11-04T00:00:00+0000 0 [D-ALAB26]-DES(B27-B30)-INSULIN-B26-AMIDE A SUPERPOTENT SINGLE-REPLACEMENT INSULIN ANALOGUE, NMR, MINIMIZED AVERAGE STRUCTURE
3WS3 2014-02-28T00:00:00+0000 2.33 Crystal Structure of H-2D in complex with an insulin derived peptide
6CE7 2018-02-11T00:00:00+0000 7.4 Insulin Receptor ectodomain in complex with one insulin molecule
6CE9 2018-02-11T00:00:00+0000 4.3 Insulin Receptor ectodomain in complex with two insulin molecules
6CEB 2018-02-11T00:00:00+0000 4.7 Insulin Receptor ectodomain in complex with two insulin molecules - C1 symmetry
1APH 1992-10-30T00:00:00+0000 2.0 CONFORMATIONAL CHANGES IN CUBIC INSULIN CRYSTALS IN THE PH RANGE 7-11
1APH 1992-10-30T00:00:00+0000 2.0 CONFORMATIONAL CHANGES IN CUBIC INSULIN CRYSTALS IN THE PH RANGE 7-11
1BPH 1992-10-30T00:00:00+0000 2.0 CONFORMATIONAL CHANGES IN CUBIC INSULIN CRYSTALS IN THE PH RANGE 7-11
1BPH 1992-10-30T00:00:00+0000 2.0 CONFORMATIONAL CHANGES IN CUBIC INSULIN CRYSTALS IN THE PH RANGE 7-11

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
1200.0 A,B,C,D,E,F,G,H,I,J,K,L Insulin P67973 INS_BALPH
1200.0 A,B,C,D,E,F,G,H,I,J,K,L Insulin P01321 INS_CANLF
1200.0 A,B,C,D,E,F,G,H,I,J,K,L Insulin P30407 INS_CHLAE
1200.0 A,B,C,D,E,F,G,H,I,J,K,L Insulin Q6YK33 INS_GORGO
1200.0 A,B,C,D,E,F,G,H,I,J,K,L Insulin P01308 INS_HUMAN
1180.2 A,B,C,D,E,F,G,H,I,J,K,L Insulin Q91XI3 INS_ICTTR
1200.0 A,B,C,D,E,F,G,H,I,J,K,L Insulin P30406 INS_MACFA
1200.0 A,B,C,D,E,F,G,H,I,J,K,L Insulin P30410 INS_PANTR
1200.0 A,B,C,D,E,F,G,H,I,J,K,L Insulin P67974 INS_PHYMC
1200.0 A,B,C,D,E,F,G,H,I,J,K,L Insulin P01315 INS_PIG
1200.0 A,B,C,D,E,F,G,H,I,J,K,L Insulin Q8HXV2 INS_PONPY
1180.2 A,B,C,D,E,F,G,H,I,J,K,L Insulin P01311 INS_RABIT
571.2 A,C,E,G,I,K Insulin P01325 INS1_MOUSE
571.2 A,C,E,G,I,K Insulin P01322 INS1_RAT
1149.6000000000001 A,B,C,D,E,F,G,H,I,J,K,L Insulin P01326 INS2_MOUSE
1149.6000000000001 A,B,C,D,E,F,G,H,I,J,K,L Insulin P01323 INS2_RAT
1131.0 A,B,C,D,E,F,G,H,I,J,K,L Insulin P01324 INS_ACOCA
1151.4000000000003 A,B,C,D,E,F,G,H,I,J,K,L Insulin P01313 INS_CRILO
1171.2 A,B,C,D,E,F,G,H,I,J,K,L Insulin P01310 INS_HORSE
1171.2 A,B,C,D,E,F,G,H,I,J,K,L Insulin Q62587 INS_PSAOB
1143.0 A,B,C,D,E,F,G,H,I,J,K,L Insulin P01316 INS_ELEMA
1143.0 A,B,C,D,E,F,G,H,I,J,K,L Insulin P01317 INS_BOVIN
1122.6 A,B,C,D,E,F,G,H,I,J,K,L Insulin P01320 INS_CAMDR
543.0 A,C,E,G,I,K Insulin P01327 INS_CHICH
1143.0 A,B,C,D,E,F,G,H,I,J,K,L Insulin P01314 INS_BALBO
1143.0 A,B,C,D,E,F,G,H,I,J,K,L Insulin P18109 INS_DIDVI
600.0 B,D,F,H,J,L Insulin F8WCM5 INSR2_HUMAN
600.0 B,D,F,H,J,L Insulin P01319 INS_CAPHI
600.0 B,D,F,H,J,L Insulin P06306 INS_FELCA
600.0 B,D,F,H,J,L Insulin P01318 INS_SHEEP
559.8 B,D,F,H,J,L Insulin P67972 INS_AOTTR
559.8 B,D,F,H,J,L Insulin P67971 INS_SAISC
578.4 B,D,F,H,J,L Insulin P01333 INS_ANAPL
578.4 B,D,F,H,J,L Insulin P68245 INS_ANSAN
578.4 B,D,F,H,J,L Insulin P68243 INS_CAIMO
578.4 B,D,F,H,J,L Insulin P51463 INS_SELRF
577.8 B,D,F,H,J,L Insulin P67970 INS_CHICK
577.8 B,D,F,H,J,L Insulin P67968 INS_MELGA
577.8 B,D,F,H,J,L Insulin P67969 INS_STRCA
577.8 B,D,F,H,J,L Insulin P69047 INS_TRADO
577.8 B,D,F,H,J,L Insulin P69048 INS_TRASC
557.4 B,D,F,H,J,L Insulin P12706 INS1_XENLA
600.0 B,D,F,H,J,L Insulin P81423 INS_ACIGU
579.6 B,D,F,H,J,L Insulin P21563 INS_RODSP
577.2 B,D,F,H,J,L Insulin C0HJI8 INS2_HUSDA
555.6 B,D,F,H,J,L Insulin P12707 INS2_XENLA
558.6 B,D,F,H,J,L Insulin P83770 INSL_VIGUN
555.6 B,D,F,H,J,L Insulin P01340 INS_KATPE
553.8 B,D,F,H,J,L Insulin P04667 INS_ONCKE
553.8 B,D,F,H,J,L Insulin P09477 INS_PLAFE
553.8 B,D,F,H,J,L Insulin Q9W7R2 INS_VERMO
553.8 B,D,F,H,J,L Insulin P09476 INS_ATRSP
553.8 B,D,F,H,J,L Insulin C0HJI3 INS2_KATPE
555.6 B,D,F,H,J,L Insulin C0HJI7 INS1_HUSDA
550.2 B,D,F,H,J,L Insulin P68988 INS_LAMFL
550.2 B,D,F,H,J,L Insulin P68987 INS_PETMA
547.8 B,D,F,H,J,L Insulin P01338 INS2_BATSP