Chiral mutagenesis of insulin. Foldability and function are inversely regulated by a stereospecific switch in the B chain

Abstract

How insulin binds to its receptor is unknown despite decades of investigation. Here, we employ chiral mutagenesis-comparison of corresponding d and l amino acid substitutions in the hormone-to define a structural switch between folding-competent and active conformations. Our strategy is motivated by the T --> R transition, an allosteric feature of zinc-hexamer assembly in which an invariant glycine in the B chain changes conformations. In the classical T state, Gly(B8) lies within a beta-turn and exhibits a positive phi angle (like a d amino acid); in the alternative R state, Gly(B8) is part of an alpha-helix and exhibits a negative phi angle (like an l amino acid). Respective B chain libraries containing mixtures of d or l substitutions at B8 exhibit a stereospecific perturbation of insulin chain combination: l amino acids impede native disulfide pairing, whereas diverse d substitutions are well-tolerated. Strikingly, d substitutions at B8 enhance both synthetic yield and thermodynamic stability but markedly impair biological activity. The NMR structure of such an inactive analogue (as an engineered T-like monomer) is essentially identical to that of native insulin. By contrast, l analogues exhibit impaired folding and stability. Although synthetic yields are very low, such analogues can be highly active. Despite the profound differences between the foldabilities of d and l analogues, crystallization trials suggest that on protein assembly substitutions of either class can be accommodated within classical T or R states. Comparison between such diastereomeric analogues thus implies that the T state represents an inactive but folding-competent conformation. We propose that within folding intermediates the sign of the B8 phi angle exerts kinetic control in a rugged landscape to distinguish between trajectories associated with productive disulfide pairing (positive T-like values) or off-pathway events (negative R-like values). We further propose that the crystallographic T -->R transition in part recapitulates how the conformation of an insulin monomer changes on receptor binding. At the very least the ostensibly unrelated processes of disulfide pairing, allosteric assembly, and receptor binding appear to utilize the same residue as a structural switch; an "ambidextrous" glycine unhindered by the chiral restrictions of the Ramachandran plane. We speculate that this switch operates to protect insulin-and the beta-cell-from protein misfolding.

Figure 1

Overview of insulin structure. (A) Sequence of human insulin indicating invariant glycine in B chain (Gly^B8; arrow). Substitutions in monomeric DKP-insulin template are shown in magenta. Disulfide bridges are indicated by lines. (B) Cylinder model of insulin showing the T state (left) and R state (right) in the crystallographic dimer. Black balls indicate positions of Gly^B8. (C and D) Ribbon models of T₆ (C) and R₆ (D) zinc hexamers. Asterisks indicate positions of Gly^B8; the variable B1–B8 segment is highlighted in silver. The central zinc ions are coordinating side chains of His^B10 and are also shown. (E and F) Molecular surfaces of T state (E) and R state (F) protomers indicating B8 site (boxes) and model of d-Ala^B8 substitution (methyl group in white and H_α in yellow). Also shown are Leu^A13 (magenta) and Leu^B6 (green). The A and B chains are otherwise shown in red and blue, respectively. 4INS (1) and 1EV3 (39) were used in panels C and D.

Figure 2

Glycine is invariant at position B8 among insulin sequences and also conserved among insulin-like growth factors (IGF–I and IGF–II). Comparison of representative B chain or B domain sequences is shown; B8 residues are boxed (asterisk).

Figure 3

Structural relationships in crystal structures of insulin surrounding Gly^B8. (A and B) In T state protomers Gly^B8 participates in a β turn (red asterisk in A) whereas in R state protomers, B8 is part of an α-helix (red asterisk in B). In the T state protomer, B8 is near Tyr^B26 of same molecule (B8 C_α–O_ζ B26 distance 5.3 Å) and dimer-related Tyr^B16 (green); in the R state protomer, B8 is near these residues and in addition Val^B12 (an (i, i +4) contact in α-helix) and A chain of adjoining molecule in hexamer (Ile^A2 and Val^A3; blue). For clarity, C_β atoms of aromatic side chains are as indicated. Coordinates were obtained from T₃R₃^f zinc hexamer (Protein Databank 1TYL; ref 75). (C and D) Ramachandran maps derived from crystal structures (Protein Databank 4INS (1), 1TYL (75), and 1TRZ (36)). Whereas in T state protomers Gly^B8 has ϕ dihedral angles (red; C), in the R state protomers, Gly^B8 belongs to an extended α-helix and so exhibits negative ϕ values (green; D).

Figure 4

Foldability as probed by combinatorial peptide synthesis with MS read out. (A) Functional-selection scheme based on insulin chain combination. B chain analogue or library of analogues is mixed with wild-type A chain to initiate specific disulfide pairing. Products are detected by mass. (B–E) MALDI-TOF MS spectra of the chain-combination reactions following 24 h at 4 °C. (B and C) Control reactions of l-Ala^B8 (B) and d-Ala^B8 (C) B chains. (D and E) Combinatorial reactions of l and d libraries at B8, respectively. Whereas l analogues undergo inefficient chain combination, productive pairing is observed to yield multiple diverse d substitutions at B8. MS spectra also detect oxidized A and B chains and A chain dimers (d).

Figure 5

Activity and stability of B8 analogues. (A) Receptor-binding titrations of DKP-insulin analogues relative to native human insulin (dashed line): Gly^B8 (diamonds), l-Ala^B8 (triangles), and d-Ala^B8 (open circles). Relative affinities of these and other analogues are given in Table 1. (B) Far-UV CD spectra of DKP insulin analogues: Gly^B8 (solid line), l-Ala^B8 (triangles), and d-Ala^B8 (open circles). (C and D) Guanidine (C) and thermal (D) denaturation studies of DKP-insulin analogues (symbols as in panel B). l-Ala^B8 aggregates exhibit an anomalous thermal signature. Extent of folding was monitored by ellipticity at 222 nm. Receptor-binding and CD studies were conducted at 4 °C.

Figure 6

¹H NMR studies of DKP-insulin analogues at 600 MHz at 25 °C (pH 7.4). (A) Gly^B8, (B) d-Ala^B8, and (C) l-Ala^B8. Asterisk in panel B indicates position of d-Ala^B8 methyl resonance (see Figure 7). The spectrum of the Gly^B8 and d-Ala^B8 analogues are similar whereas the l-Ala analogue undergoes aberrant aggregation.

Figure 7

¹H NMR TOCSY spectra of (A) DKP-insulin and (B) d-Ala^B8-DKP-insulin highlighting novel d-Ala methyl resonance in analogue (asterisk in panel B). Spectra were acquired at 600 MHz at 32 °C and pD 7.6 (direct meter reading) with a TOCSY mixing time of 55 ms.

Figure 8

d-Ala^B8-DKP-insulin exhibits nativelike solution structure. (A) Collection of crystal structures showing superimposition of T state protomers (Protein Databank 4INS (1), 1TYL (75), 1PID (76), 1TRZ (36), and 1TYM (75)). The position of Gly^B8 is highlighted in yellow. The B chain is otherwise shown in blue, and the A chain is red. Structure of truncated analogue des-pentapeptide[B26–B30]-insulin (DPI) is indicated by d’s at positions B1 and B25. (B) Solution structure of DKP-insulin (PDB 1LNP; ref 28). (C) Ensemble of DG/RMD structures of d-Ala^B8-DKP-insulin showing position of d-Ala side chain (yellow). The coloring scheme in panels B and C corresponds to that in panel A.

Figure 9

Environment of d-Ala^B8 side chain at protein surface. (A and B) Ribbon and space-filling models of representative structure of d-Ala^B8-DKP-insulin showing position of d-Ala^B8 methyl group (tawny; sphere in panel A at 0.7 van der Waals radius) relative to side chains of Val^A3 (red), Val^B12 (dark blue), and Tyr^B26 (black). Leu^B11 (magenta) lies behind B8 in core. The A chain is otherwise shown in gray and the B chain otherwise in blue. (C) Stereopair showing spatial relationships among residues A3, B8, B11, B12, and B26. The ensemble was aligned on these side chains to illustrate B8 pocket. Color scheme is the same as in panels A and B.