PDBsum entry: 1kmf

Hormone/growth factor

PDB id: 1kmf

Contents

Protein chains

21 a.a.

30 a.a. *

* Residue conservation analysis

PDB id:

1kmf

Name:

Hormone/growth factor

Title:

Nmr structure of human insulin mutant ile-a2-allo-ile, his- b10-asp, pro-b28-lys, lys-b29-pro, 15 structures

Structure:

Insulin. Chain: a. Engineered: yes. Mutation: yes. Insulin. Chain: b. Engineered: yes. Mutation: yes

Source:

Synthetic: yes. Other_details: the peptide was chemically synthesized. The sequence of the peptide is naturally found in homo sapiens (human).. (Human).

NMR struc:

15 models

Authors:

B.Xu,Q.X.Hua,S.H.Nakagawa,W.Jia,Y.C.Chu,P.G.Katsoyannis, M.A.Weiss

Key ref:

B.Xu et al. (2002). Chiral mutagenesis of insulin's hidden receptor-binding surface: structure of an allo-isoleucine(A2) analogue. J Mol Biol, 316, 435-441. PubMed id: 11866509 DOI: 10.1006/jmbi.2001.5377

Date:

14-Dec-01 Release date: 09-Jan-02

Protein chain

P01308  (INS_HUMAN) -  Insulin

Seq: 110 a.a.

Struc: 21 a.a.*

Protein chain

P01308  (INS_HUMAN) -  Insulin

Seq: 110 a.a.

Struc: 30 a.a.*

* PDB and UniProt seqs differ at 4 residue positions (black crosses)

 Gene Ontology (GO) functional annotation 

• Cellular component: extracellular region (1 term)
• Biochemical function: hormone activity (1 term)

DOI no: 10.1006/jmbi.2001.5377

J Mol Biol 316:435-441 (2002)

PubMed id: 11866509

Chiral mutagenesis of insulin's hidden receptor-binding surface: structure of an allo-isoleucine(A2) analogue.

B.Xu, Q.X.Hua, S.H.Nakagawa, W.Jia, Y.C.Chu, P.G.Katsoyannis, M.A.Weiss.

ABSTRACT

The hydrophobic core of vertebrate insulins contains an invariant isoleucine residue at position A2. Lack of variation may reflect this side-chain's dual contribution to structure and function: Ile(A2) is proposed both to stabilize the A1-A8 alpha-helix and to contribute to a "hidden" functional surface exposed on receptor binding. Substitution of Ile(A2) by alanine results in segmental unfolding of the A1-A8 alpha-helix, lower thermodynamic stability and impaired receptor binding. Such a spectrum of perturbations, although of biophysical interest, confounds interpretation of structure-activity relationships. To investigate the specific contribution of Ile(A2) to insulin's functional surface, we have employed non-standard mutagenesis: inversion of side-chain chirality in engineered monomer allo-Ile(A2)-DKP-insulin. Although the analogue retains native structure and stability, its affinity for the insulin receptor is impaired by 50-fold. Thus, whereas insulin's core readily accommodates allo-isoleucine at A2, its activity is exquisitely sensitive to chiral inversion. We propose that the Ile(A2) side-chain inserts within a chiral pocket of the receptor as part of insulin's hidden functional surface.

Selected figure(s)

Figure 1.
Figure 1. Insulin's ``hidden'' functional-surface hypothesis and corresponding CD studies. (a) Sche- matic model proposing confor- mational change on receptor binding, exposing Ile A2 and novel non-polar surface. (b) Cylinder model of insulin T-state. 5,6 Detach- ment of C-terminal B-chain b- strand on receptor binding would expose ``hidden'' surface spanning Ile A2 and Val A3 . 2,8,23 Native or ``closed'' form of B chain is anchored by engagement of Phe B24 against hydrophobic core. (c) Far- UV CD spectra of DKP-insulin (continuous line), allo-Ile A2 -DKP- insulin (*), and Ala A2 -DKP-insulin (*) suggest attenuated helix con- tent in Ala A2 -DKP-insulin 25 but native helix content in allo-Ile A2 - DKP-insulin. (d) Thermal unfold- ing, monitored by ellipticity at 222 nm, demonstrates native stab- ility of allo-Ile A2 -DKP-insulin (*) relative to DKP-insulin (continuous line) whereas Ala A2 -DKP-insulin (*) is less stable. 25 (e) Guanidine unfolding transitions of Ala A2 -DKP- insulin (*), allo-Ile A2 -DKP-insulin (*) and DKP-insulin (continuous line) demonstrate that whereas Ala A2 -DKP variant exhibits decreased stability (deltadeltaGu 0.4-1.2 kcal/mol), 25 allo-Ile A2 -DKP-insulin has similar or greater stability (see footnote { on page 437). Two-state modeling and extracted thermodynamic parameters are provided in Supplementary Material. CD samples contained 25-50 mM insulin or analogue in 50 mM potassium phosphate (pH 7); samples were diluted to 5 mM for equilibrium denaturation studies.

Figure 4.
Figure 4. Core packing schemes. (a) Environment of Ile A2 (black) in T-state crystal structures in relation to Val A3 , Leu A16 , Tyr A19 , Leu B11 , Leu B15 , and Tyr B26 . (b) and (c) Corresponding interactions of A2 side-chain in NMR-derived ensembles of (b) DKP-insulin ensemble and (c) allo-Ile A2 -DKP-insulin. Except for chiral inversion of its b-carbon, packing interactions of allo-Ile A2 are simi- lar to those of Ile A2 in crystal and solution structures. Structures in each panel are aligned with respect to main-chain atoms of A2-A8, A13-A20 and B9-B19. Excepting A2, A-chain side-chains are shown in red and B-chain side-chains in blue.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2002, 316, 435-441) copyright 2002.

Figures were selected by an automated process.