FIGURE 1.

Insulin sequence and structure. A, sequence of WT insulin and modification sites. A and B chains are shown in white and gray. Conserved aromatic residues Phe^B24 and Phe^B25 are highlighted as black circles. This study focused on substitutions of Tyr^B26 (red circle); additional substitutions were made at position B29 (Nle; arrow) to facilitate semi-synthesis. B, ribbon model of insulin monomer (T state extracted from T₆ zinc hexamer) (2). The A chain is shown in yellow and the B chain in black (B1–B19) and green (B20-B30). C, environment of Tyr^B26 (stereo). The side chain of Tyr^B26 is highlighted in red; Ile^A2, Val^A3, Val^B12, Leu^B15, and Phe^B24 are as labeled. D, stick representation of residues B20–B27 (carbon atoms (green), nitrogen atoms (blue) and oxygen atoms (red)) packed between αCT and the L1-β₂ sheet. B chain residues B8–B19 are shown as a black ribbon and the A chain as a yellow ribbon; residues A1–A3 are concealed behind the surface of αCT. Key contact surfaces of αCT with B24–B26 are highlighted in magenta, and L1 with B24–B26 are highlighted in cyan; L1 and αCT surfaces not in interaction with B24–B26 are shown in lighter shades. E, orthogonal view to D, showing interaction of the side chain of Phe^B24 with the nonpolar surface of the L1-β₂ sheet. Tyr^B26 is hidden below the surface of αCT. Engagement of conserved residues A1–A3 against the nonpolar surface of αCT is shown at top. F, environment of Tyr^B26 within site 1 complex (stereo). Neighboring side chains in L1 and αCT are as labeled. Coordinates were obtained from PDB code 4OGA (39). G, model of wild-type insulin in its receptor-free conformation overlaid onto the structure of the insulin-bound μIR (71). The L1 domain and part of CR domain are shown in powder blue; αCT is shown in purple. Residues Phe^B24 and Tyr^B26 are as in A. The B chain of μIR-bound insulin is shown in dark gray (B6–B19); the brown tube indicates classical location within the overlay of residues B20–B30 of insulin in its receptor-free conformation, highlighting steric clash of B26–B30 with αCT. In the μIR co-crystal structure, insertion of the insulin B20–B27 segment between L1 and αCT peptide is associated with a small rotation of the B20–B23 β-turn and changes in main-chain dihedral angles flanking Phe^B24 (39). H, inverted V-shaped assembly of IR ectodomain homodimer. One monomer is in ribbon representation (labeled), the second in surface representation. Domains are labeled as follows: L1, first leucine-rich repeat domain; CR, cysteine-rich domain; L2, second leucine-rich repeat domain; FnIII-1, -2, and -3, first, second, and third fibronectin type III domains, respectively; ID, insert domain; and αCT, α-chain C-terminal segment. Coordinates were obtained from PDB code 4ZXB (42).