PDBsum entry: 2jzq

Hormone

PDB id: 2jzq

Contents

Protein chain

57 a.a. *

* Residue conservation analysis

PDB id:

2jzq

Name:

Hormone

Title:

Design of an active ultra-stable single-chain insulin analog 20 structures

Structure:

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

Source:

Synthetic: yes

NMR struc:

20 models

Authors:

Q.X.Hua,S.Nakarawa,W.H.Jia,K.Huang,N.F.Philips,S.Q.Hu, M.A.Weiss

Key ref:

Q.X.Hua et al. (2008). Design of an active ultrastable single-chain insulin analog: synthesis, structure, and therapeutic implications. J Biol Chem, 283, 14703-14716. PubMed id: 18332129 DOI: 10.1074/jbc.M800313200

Date:

11-Jan-08 Release date: 26-Feb-08

Protein chain

No UniProt id for this chain

DOI no: 10.1074/jbc.M800313200

J Biol Chem 283:14703-14716 (2008)

PubMed id: 18332129

Design of an active ultrastable single-chain insulin analog: synthesis, structure, and therapeutic implications.

Q.X.Hua, S.H.Nakagawa, W.Jia, K.Huang, N.B.Phillips, S.Q.Hu, M.A.Weiss.

ABSTRACT

Single-chain insulin (SCI) analogs provide insight into the inter-relation of hormone structure, function, and dynamics. Although compatible with wild-type structure, short connecting segments (<3 residues) prevent induced fit upon receptor binding and so are essentially without biological activity. Substantial but incomplete activity can be regained with increasing linker length. Here, we describe the design, structure, and function of a single-chain insulin analog (SCI-57) containing a 6-residue linker (GGGPRR). Native receptor-binding affinity (130 +/- 8% relative to the wild type) is achieved as hindrance by the linker is offset by favorable substitutions in the insulin moiety. The thermodynamic stability of SCI-57 is markedly increased (DeltaDeltaG(u) = 0.7 +/- 0.1 kcal/mol relative to the corresponding two-chain analog and 1.9 +/- 0.1 kcal/mol relative to wild-type insulin). Analysis of inter-residue nuclear Overhauser effects demonstrates that a native-like fold is maintained in solution. Surprisingly, the glycine-rich connecting segment folds against the insulin moiety: its central Pro contacts Val(A3) at the edge of the hydrophobic core, whereas the final Arg extends the A1-A8 alpha-helix. Comparison between SCI-57 and its parent two-chain analog reveals striking enhancement of multiple native-like nuclear Overhauser effects within the tethered protein. These contacts are consistent with wild-type crystal structures but are ordinarily attenuated in NMR spectra of two-chain analogs, presumably due to conformational fluctuations. Linker-specific damping of fluctuations provides evidence for the intrinsic flexibility of an insulin monomer. In addition to their biophysical interest, ultrastable SCIs may enhance the safety and efficacy of insulin replacement therapy in the developing world.

Selected figure(s)

Figure 3.
FIGURE 3. IR binding assay. Representative displacement data illustrate the activity of the present single-chain analog with a 6-residue linker (SCI-57; ), human insulin ( ), and PIP (an SCI-53; ). Whereas binding of SCI-53 (PIP) is reduced by >500-fold, a slight enhancement of affinity by SCI-57 is seen as a leftward shift of its displacement curve. The y axis (B/B[o]) indicates percent receptor-bound ^125I-labeled human insulin; the x axis (logarithmic scale to base 10) indicates the concentration of unlabeled competing insulin analog.

Figure 9.
FIGURE 9. Solution structure of SCI. A, ensemble of DG/restrained molecular dynamics models of SCI (Protein Data Bank code 2jzq). The A-chain is shown in red, the B-chain in blue, and the linker in gray. Structures were aligned according to the main chain atoms of A2-A7, A13-A19, and B9-B26. B, ribbon model of des-Thr^B30-SCI-50, an inactive 50-residue single-chain analog in which a peptide bond links Lys^B29 to Gly^A1 (Protein Data Bank code 1pid) (15). C and D, front and back views, respectively, of SCI-57 with selected side chains shown relative to the ribbon model of DKP-insulin (black) (Protein Data Bank code 2jmn) (7). The side chain coloring scheme is as follows: Val^A3, green; Tyr^A19, green; Phe^B24, dark purple; Tyr^B26, blue; and linker residue Pro^C4, magenta.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2008, 283, 14703-14716) copyright 2008.

Figures were selected by an automated process.