Structure of insulin and domain organization of the insulin receptor. A, ribbon model of insulin (as T-state protomer; Protein Data Bank code 4INS) with the A-chain in red and the B-chain in blue. Selected side chains are shown: Glu^A4 and Tyr^A14 (red); Gln^A5 and Thr^A8 (green); Leu^A13, Leu^A17, and Val^B17 (magenta); His^B10 (black); and Phe^B24 and Phe^B25 (gray). B, domain organization of the IR as an (αβ)₂-dimer. Color-coded segments indicate structural domains; at left are selected sites of limited proteolysis of photocross-linked complexes: trypsin (tr, bracket at the L1-CR domain junction) and chymotrypsin (ch, arrow within the CR domain), and tryptic and chymotryptic sites at the Fn2a-ID-N junction (asterisk). Beige arrowheads indicate sites of N-linked glycosylation in the extracellular portion of the IR. This figure was adapted from Ref. 5 with the permission of the authors. Dashed lines outline domains (light gray) not present in the crystal structure of the IR ectodomain (Fig. 7); these span the transmembrane α-helix (TM), the juxtamembrane segment (JM), tyrosine kinase (TK), and the C-terminal tail of the β-subunit (βCT). C, space-filling model of the insulin protomer (with residues B27-B30 removed) showing the functional epitope (gray) and its extended structural epitope (green). D, view of the insulin protomer rotated by 90° about the vertical axis with classification of binding sites 1 (gray) and 2 (magenta) as proposed by De Meyts (18, 73). Residues B27-B30 were deleted from the coordinate file to enable better visualization of Ile^A2 and Val^A3 in accord with the detachment model (preceding article (31)).

Sites of photoprobes in insulin and summary of cross-linking efficiencies. A, sequence of human insulin showing the A-chain (upper) and B-chain (lower); in each case, the N-terminal residue is at left, and C-terminal residue is at right. The three disulfide bridges (cysteines A6-A11, A7-B7, and A20-B19) are shown as black lines. Sites of Pmp derivatives are highlighted in light blue (within A- or B-chains) or magenta (B-chain extensions B0 and B31). Gray shading (residues B5 and B17) indicates failed syntheses due to impaired chain combination. The Pmp^B8 analog contained the d-isomer; both l- and d-Pmp derivatives were individually prepared at positions A1 and B24. Not shown are DKP substitutions in the B-chain to yield a monomeric template (Asp^B10, Lys^B28, and Pro^B29) and biotin tags at possible attachment sites (B0, B1, A1, or via the ε-NH₂ moiety of a d-Lys^A1 substituent; see “Experimental Procedures” and supplemental Table S1). B, front and back surfaces of insulin color-coded by efficiency of photocross-linking. The front surface is predominantly composed of B-chain residues, and the back surface of A-chain residues. Sites of high, medium, and low photocross-linking efficiency are shown in red, green, and black, respectively; sites not tested are shown in gray. The structure shown is based on a T₆ crystallographic protomer (2Zn molecule 1; Protein Data Bank code 4INS).

Photocross-linking studies of B-chain derivatives. A, Western blots showing subunits of the holoreceptor (α₂β₂, 460 kDa; α, 135 kDa; β, 95 kDa) cross-linked with biotin-labeled insulin derivatives containing Pap at positions B24-D (lanes 1-4), B24-L (lanes 5-8), B25 (lanes 9-12), B26 (lanes 13-16), B27 (lanes 17-20), and B29 (lanes 21-24) or at extended site B31 (lanes 25-28). Samples were treated without (odd-numbered lanes) or with (even-numbered lanes) UV irradiation. After cross-linking and reduction with DTT, samples were resolved by SDS-PAGE and detected either with alkaline phosphatase-conjugated NeutrAvidin (NAv; upper panel) or with anti-receptor antibody (IR_α-N; middle panel); the latter demonstrates equal amounts of IR. Control blots probed with NeutrAvidin (lower panel; without DTT reduction) demonstrate equal amounts of insulin analog. In the control lanes, in each case, photocross-linking was not observed in the absence of the IR and UV irradiation (lanes 1, 5, 9, 13, 17, 21, and 25), in the absence of the IR and presence of UV irradiation (lanes 2, 6, 10, 14, 18, 22, and 26), or in the presence of the IR and absence of UV irradiation (lanes 3, 7, 11, 15, 19, 23, and 27). B, limited chymotryptic digestion of Pap^B24-, Pap^B25-, Pap^B26-, and Pap^B27-photocross-linked hormone-receptor complexes (6). At the indicated time points, aliquots were mixed with an equal volume of Laemmli sample buffer containing 100 mm DTT, heated at 95 °C for 5 min, and resolved by SDS-PAGE. Photoproducts and their fragments were blotted onto nitrocellulose membrane and probed with NeutrAvidin (left panel) or IR_α-N (right panel). The apparent molecular masses were as follows: α(N)-B, 47 kDa; α(C)-B, 34 kDa; and α(N)/α(N)-B, 47/50 kDa (Table 1). C, deglycosylation of B25 and B27 chymotryptic fragments as probed with NeutrAvidin (left panel) or IR_α-N (right panel). The IR was cross-linked to Pap^B25 or Pap^B27 derivatives (lanes 13 and 16; - indicates the absence of protease), digested with chymotrypsin (lanes 14, 15, 17, and 18; +), and then subjected to enzymatic deglycosylation (lanes 15 and 18; labeled d) (25). The apparent molecular masses were as follows: α(C)^*-B, 23 kDa; and ID-N^*/B, 17 kDa (Table 1).

Photocross-linking studies of A-chain derivatives. A, IR cross-linking of insulin derivatives containing Pap at positions A1-D (lanes 7-10), A2 (lanes 11-14), A3 (lanes 15-18), A4 (lanes 19-22), and A8 (lanes 23-26) in relation to B-chain derivatives B25 (lanes 1 and 2) and B24 (lanes 3-6). Samples were resolved by SDS-PAGE prior to (odd-numbered lanes) or following (even-numbered lanes) UV irradiation. A-chain Pap derivatives (except A1) had a biotin tag attached to the ε-NH₂ moiety of a d-Lys^A1 substituent; A1 d- and l-Pap derivatives were labeled with biotin at N^α. The format is as described for Fig. 3A. Cross-linked adducts were detected after DTT reduction by NeutrAvidin (NAv; upper panel). Control blots probed by IR_α-N (middle panel) demonstrate equal amounts of IR. Control blots probed with NeutrAvidin (lower panel; without DTT) demonstrate equal amounts of insulin derivative. In the control lanes, in each case, the photocross-linking band was not detected in the absence of the IR and UV irradiation (lanes 3, 7, 11, 15, 19, and 23), in the absence of the IR and presence of UV irradiation (lanes 4, 8, 12, 16, 20, and 24), or in the presence of the IR and absence of UV irradiation (lanes 1, 5, 9, 13, 17, 21, and 25). B, Western blots showing A2-specific IR photocross-linking at successive concentrations of Pap^A2 derivative (lanes 3-7, corresponding to ligand concentrations of 200, 240, 320, 400, and 600 nm). Pap^B25-specific photocross-linking is also shown (lane 1; lane 2 is empty). C, competition experiment. A2-specific photocross-linking could be competed by unmodified insulin (Ins) or IGF-I, added prior to UV irradiation. Successive concentrations of insulin (lanes 1-4) or IGF-I (lanes 5-8) were 0, 3, 30, and 300 times that of Pap^A2 (600 nm).

Chymotryptic mapping of A-chain-specific photocross-linked complexes. Photoadducts were obtained at the indicated positions and digested with chymotrypsin as described (6). At successive time points, aliquots were reduced by treatment with DTT, heat-denatured, and resolved by SDS-PAGE. Fragments were blotted onto nitrocellulose membrane and detected by NeutrAvidin (NAv; A and C) or antiserum IR_α-N (B and D). Dotted boxes in A and C indicate the 19-kDa N-terminal L1-derived tryptic fragment estimated to contain ∼100 residues (and three N-linked carbohydrates). Molecular masses and domain assignments are otherwise as indicated.

Mapping of photo contacts and relationship to induced fit. A, schematic model of insulin fit. The insulin monomer is proposed to undergo a change in conformation from its closed unbound state (left) to a more open state (right) in which detachment of the C-terminal B-chain β-strand (residues B24-B28) exposes conserved aliphatic side chains Ile^A2 and Val^A3. B, alternating pattern of photo contacts by the C-terminal B-chain β-strand in which its even-numbered face (Phe^B24 and Tyr^B26) contacts the L1 domain of the IR (α-subunit residues 1-158), whereas the odd-numbered face (Phe^B25 and Thr^B27) contacts the C-terminal insert domain (ID)-derived tail of the α-subunit. C, space-filling model of insulin depicting its front and back surfaces (left and right). Putative L1 contact residues Tyr^B16, Phe^B24, and Tyr^B26 are shown in blue, and insert domain contact residues (Gly^A1, Ile^A2, Val^A3, Glu^A4, Thr^A8, Tyr^A14, Phe^B25, and Thr^B27) are shown in yellow (A2 and A3) or gold. The A-chain is otherwise shown in light gray, and the B-chain in dark gray. D, corresponding molecular surfaces following removal of residues B26-B30 to simulate exposure of Ile^A2 and Val^A3 upon detachment of the C-terminal B-chain β-strand in the hormone-receptor complex. The color code is the same as described for C.

Structure of the IR ectodomain and possible proximity of L1 and ID-N. A, ribbon model of the component protomer (Protein Data Bank code 2DTG). Individual domains L1, CR, L2, Fn1, Fn2, and Fn3 are shown in gray, black, light blue, red, purple, and dark blue, respectively. The insert domain (ID) exhibits missing or discontinuous electron density (residues 655-755; IR isoform A); only respective N- and C-terminal subsegments of ID-N and ID-C are well defined (orange). The color code is in accord with the schematic model in Fig. 1B. B, inverted V-shaped dimer. The position of the plasma membrane is indicated at the bottom in a schematic lipid bilayer. C, stereo model showing the L1 domain (gray ribbon) in relation to unassigned and discontinuous electron density (green), potentially from αCT; density may represent ∼20 residues of the insert domain. αCT- and ID-N-related density may reorganize upon insulin binding. Residues in L1 critical to hormone binding (as inferred from Ala scanning mutagenesis) (62) are shown as red sticks. C was kindly provided by C. Ward.

Classical insulin allostery and induced fit. A, conformational equilibria among three families of zinc insulin hexamers: T₆ (left), T₃R^f₃ (middle), and R₆ (right). In each structure, the A-chains are shown in light gray; residues B9-B30 in dark gray; the side chain of Phe^B24 in tawny; and axial zinc ions (overlaid) in magenta. The adjustable conformations of residues B1-B8 in the T- and R-states are highlighted in green and blue, respectively. Coordinates were obtained from Protein Data Bank codes 4INS, 1TRZ, and 1TNJ. B, T-state-specific B7-B10 β-turn and adjoining B7-A7 disulfide bridge (gold). Substitution of the invariant Gly^B8 (C^α in red) by d-amino acids stabilizes the T-state but markedly impairs receptor binding (33, 70). The corresponding l-amino acid substitutions destabilize the T-state but can be highly active. The arrow indicates pro-d-H^α of Gly^B8. A- and B-chain residues (B6-B10 and A6-A8) are otherwise shown in light and dark gray, respectively; selected residue numbers are provided. C, schematic model of insulin T-state showing proposed sites of conformational change upon receptor binding: an R-state-related B8-related switch (red, right) within the N-terminal segment of the B-chain (green) and a B24-related switch (tawny, right) as revealed by stereospecific unfolding of the C-terminal segment of the B-chain (see preceding article (31)). The A-chain is represented as a gray rectangle, and the central B-chain α-helix (cylinder) and C-terminal β-strand (arrow) are shown in black.

Sites of diabetes-associated mutations in human insulin. A, sequence of human insulin with the A-chain (upper) in red and the B-chain (lower) in blue. Disulfide bridges are shown in orange. Black circles represent mutations associated with neonatal DM due to presumed folding defects in proinsulin (78-81); gray circles represent mutations that permit disulfide pairing in the ER with subsequent circulation of mutant proteins. Whereas substitutions at positions A3, B24, and B25 lead to variant hyperinsulinemias, the B10 substitution interferes with protein trafficking, leading to mutant hyperproinsulinemia (77). B, positions of mutation sites in the T-state of insulin (crystallographic protomer 1 of 2Zn insulin; Protein Data Bank code 4INS) (1). Black spheres indicate C^α atoms of Gly at A1, B8, and B23.