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. 2008 May 23;283(21):14703–14716. doi: 10.1074/jbc.M800313200

FIGURE 2.

FIGURE 2.

Model of induced fit and design of interchain tethers. A, whereas the free conformation of insulin is closed (left panel), upon receptor binding, detachment of the C-terminal B-chain β-strand is proposed to expose the hidden non-polar surface of the A-chain (right panel), including IleA2 and ValA3 (green). The aromatic side chain of PheB24 (red) may function as a structural switch. GlyA1 is shown as an open circle, and B29 and B30 are shown as purple and back circles, respectively. B, tethering the A- and B-chains was effected by bifunctional cross-linking (dashed blue line) between the ε-amino group of LysB29 (purple circle) and the α-amino group of GlyA1 (open circle). C, a histogram showing the relative (rel.) activity of tethered insulin analogs is plotted as a function of the number of atoms between the α-carbons of B29 and A1 (horizontal axis), including the B29 side chain and, when present, the side chain of d-LysA1 (magenta). Bar heights indicate receptor-binding affinities relative to wild-type human insulin. Black bars indicate linkage to the α-amino group of GlyA1, whereas magenta bars indicate linkage to the ε-amino group of d-LysA1. The asterisk indicates substantial relative activity (35 ± 3%) exhibited by NεA1,NεB29-ethylene glycol bissuccinoyl d-LysA1-insulin. D, shown is a schematic design of representative cross-linked insulin derivatives. Upper panel, NαA1,NεB29-ethylene glycol bissuccinoyl insulin; lower panel, NεA1,NεB29-ethylene glycol bissuccinoyl d-LysA1-insulin. In the upper construct, the total number of atoms between the α-carbons of B29 and A1 is the sum of the lengths of the linker (dashed blue line; 12 atoms) and the LysB29 side chain (magenta; 5 atoms) plus the Nα of GlyA1:12 + 5 + 1 = 18. In the lower construct, the tether is extended by the side chain of d-LysA1:12 + 5 + 5 = 22. E, shown is the design of three single-chain analogs. Upper panel, PIP, a 53-residue analog (SCI-53) with a dipeptide-connecting domain (AK); middle panel, SCI-57; lower panel, 58-residue analog (SCI-58) with connecting domain AAAAAAK. In SCI-57, B30 (Thr) and a 6-residue linker (sequence GGGPRR) interpose 23 atoms between the α-carbons of B29 and A1; the corresponding numbers of intervening atoms in PIP and SCI-58 are 11 and 26, respectively.