The Structure of a Mutant Insulin Uncouples Receptor Binding from Protein Allostery

Abstract

The zinc insulin hexamer undergoes allosteric reorganization among three conformational states, designated T₆, , and R₆. Although the free monomer in solution (the active species) resembles the classical T-state, an R-like conformational change is proposed to occur upon receptor binding. Here, we distinguish between the conformational requirements of receptor binding and the crystallographic TR transition by design of an active variant refractory to such reorganization. Our strategy exploits the contrasting environments of His^B5 in wild-type structures: on the T₆ surface but within an intersubunit crevice in R-containing hexamers. The TR transition is associated with a marked reduction in His^B5 pK_a, in turn predicting that a positive charge at this site would destabilize the R-specific crevice. Remarkably, substitution of His^B5 (conserved among eutherian mammals) by Arg (occasionally observed among other vertebrates) blocks the TR transition, as probed in solution by optical spectroscopy. Similarly, crystallization of Arg^B5-insulin in the presence of phenol (ordinarily a potent inducer of the TR transition) yields T₆ hexamers rather than R₆ as obtained in control studies of wild-type insulin. The variant structure, determined at a resolution of 1.3Å, closely resembles the wild-type T₆ hexamer. Whereas Arg^B5 is exposed on the protein surface, its side chain participates in a solvent-stabilized network of contacts similar to those involving His^B5 in wild-type T-states. The substantial receptor-binding activity of Arg^B5-insulin (40% relative to wild type) demonstrates that the function of an insulin monomer can be uncoupled from its allosteric reorganization within zinc-stabilized hexamers.

Insulin is a small globular protein containing two chains, A (21 residues) and B (30 residues). In pancreatic β-cells, the hormone is stored as Zn²⁺-stabilized hexamers, which form microcrystalline arrays within specialized secretory granules (1). The hexamers dissociate upon secretion into the portal circulation, enabling the hormone to function as a zinc-free monomer. Structure-function relationships have been inferred from patterns of sequence conservation (2) and extensively probed by mutagenesis (2-10). A variety of evidence suggests that insulin undergoes a change in conformation on binding to the insulin receptor (IR)2 (11). A model for induced fit is provided by the TR transition, a long range allosteric reorganization of zinc insulin hexamers (12). The structural basis of the TR transition has been extensively investigated by x-ray crystallography (13-15). In this paper, we investigate the relationship between such allosteric reorganization and biological activity. Experimental design exploits the contrasting structures of classical hexamers to introduce an electrostatic block to the TR transition.

The TR transition encompasses three families of zinc insulin hexamers, designated T₆, , and R₆ (Fig. 1). Interconversion among these families is regulated by ionic strength (13, 16) and the binding of small cyclic alcohols (14). NMR studies have established that the structure of an insulin monomer in solution resembles the crystallographic T-state (Fig. 2, left) (17-19). The TR transition is characterized by a change in the secondary structure of the B-chain (Fig. 2, right).3 Although extensive contacts stabilize the hexamer, within an individual protomer the TR transition is associated with a loss of interchain contacts. Such R-state features have been observed only in zinc hexamers; monomeric R-like conformers have not been detected in solution by NMR (18-20). Dodson and colleagues (14) have nonetheless proposed that an insulin monomer may adopt an R-like conformation upon receptor binding. Such induced fit could extend the potential receptor-binding surface of insulin by exposing side chains otherwise inaccessible in the T-state. Indirect evidence favoring this hypothesis has been obtained by nonstandard mutagenesis (21, 22). d-Amino acid substitutions at Gly^B8 (black circles in Fig. 2) markedly impair receptor binding in association with a shift in the conformational equilibrium among T₆, , and R₆ hexamers favoring the T-state. The low biological activity of such analogs (reduced by 10² to 10³-fold) (21) has been ascribed to thermodynamic stabilization of a native-like but inactive T-state conformation (22).

FIGURE 1.

The TR transition among insulin hexamers. A, schematic representation of the three structural families, designated T₆, , and R₆. T-state protomers are otherwise shown in red, and R-state protomers are shown in blue. B, corresponding ribbon representation of crystal structures. Axial zinc ions are shown in blue-gray. Chameleon segments B1-B8 are shown in black. Coordinates were obtained from Protein Data bank entries 4INS (2), 1TRZ (71), and 1ZNJ (M. G. W. Turkenburg, J. L. Whittingham, J. P. Turkenburg, G. G. Dodson, U. Derewenda, G. D. Smith, E. J. Dodson, Z. S. Derewenda, and B. Xiao, manuscript in preparation), respectively.

FIGURE 2.

Cylinder models of T- and R-state protomers. Shown is the crystal structure of dimer comprising T-state (left) and R^f-state (right) as extracted from hexamer. The A-chains are shown in black, and B-chains are shown in green. In each panel, the positions of residues B5 and B8 (C_a atoms) are shown as filled red and black circles, respectively. Dimerization is mediated by an anti-parallel β-sheet (central ribbons) and nonpolar interactions between central B-chain α-helices. In the T state conformation, the A-chain contains an N-terminal α-helix (residues A1-A8) followed by a noncanonical turn, second helix (A12-A18), and C-terminal segment (A19-A21); the B-chain contains an N-terminal segment (residues B1-B6), type II′ β-turn (B7-B10), central α-helix (B9-B19), type I β-turn (B20-B23), and C-terminal β-strand (B24-B28), extended by less well ordered terminal residues B29 and B30. In the R-state, the N-terminal portion of the B-chain participates in a single long α-helix. The resulting B1-B19 α-helix (or B3-B19 in the frayed R^f state) projects from the globular core of the protomer to make extensive hexamer contacts, including formation of a specific phenol-binding pocket at a trimer interface (14). The side chain of His^B5 packs within an R-state-specific subunit interface in and R₆ hexamers; the side chain neither contacts a zinc ion nor contacts a bound phenolic ligand.

The present study focuses on the role of residue B5, conserved as His among eutherian mammals. Previous studies have shown that His^B5 contributes to the foldability of human proinsulin but is not required for biological activity (22). Experimental design exploits the contrasting environments of His^B5 in wild-type structures: on the T₆ surface but within an intersubunit crevice in R-containing hexamers. In the T-state, His^B5 lies within the N-terminal extended strand (Fig. 2, left, filled red circle). In the T₆ hexamer (Fig. 3A, red side chains), His^B5 is surrounded in part by water molecules (blue spheres); the imidazole ring packs against an interchain crevice near Ile^A10 and the solvent-exposed A7-B7 disulfide bridge (shown in stereo view in Fig. 3C).4 Participation of His^B5 in the R-specific α-helix (Fig. 2, right, red circle) causes its side chain to move from the T₆ surface to pack within a trimer interface; the B5 side chain does not participate in zinc coordination or the immediate binding of phenol. The structural environment of His^B5 within an R-specific trimer interface is shown in Fig. 4 (see also supplemental Figs. S1 and S2).

FIGURE 3.

Structural environment of His^B5 in T₆ hexamer and T-state protomers. A, space-filling model of T₆ hexamer showing side chain of His^B5 (red) lying along protein surface near bound water molecules (blue). B, stick model of a single T-state protomer with B-chain in gray and A-chain in black. The red box encloses the environment of His^B5 (red) in an interchain crevice near A7-B7 and A6-A11 disulfide bridges (sulfur atoms shown in gold). C, expansion of the boxed region in B, providing a stereo view of packing of His^B5. D, corresponding stereo view of Arg^B5 (red) in the crystallographic T-state protomer (see Fig. 8A). Analogous side-chain NH functions of His^B5 and Arg^B5 are near the main chain of the A-chain. Cystine A7-B7 in each case lies on the protein surface, whereas cystine A6-A11 packs within the core of the promoter. Bound water molecules near respective B5-related crevices are shown as blue spheres.

FIGURE 4.

Environment of His^B5 in R-related subunit interface in R₆ zinc insulin hexamer. A and B, structure of an R₆ hexamer (Protein Data Bank code 1ZNJ). A, space-filling representations showing (left to right) front, back, and side views. The six B5 side chains are shown in green, and zinc ions are shown in yellow. The hexamer contains six independent molecules. The corresponding A and B chains of molecules 1, 3, and 5 are shown in black and gray, respectively; the A and B chains of molecules 2, 4, and 6 are shown in light and dark blue, respectively. B, stereo pair, stick model of the intersubunit crevice. The position of His^B5 (green) is shown in relation to the B-chains of molecule 1 (M1-B; gray), molecule 2 (M2-B; blue), and molecule 4 (M4-B; purple) and to the A-chain of molecule 4 (M4-A; black). The analogous trimer interface in a hexamer is shown in supplemental Fig. S1. Specific residue labeling and corresponding space-filling models are provided in supplemental Fig. S2.

Our study has two parts. We first describe pH-dependent ¹H NMR studies of the wild-type R₆ hexamer. R-specific burial of His^B5 is shown to be associated with a marked reduction in its side chain pK_a. These results in turn predict that a positive charge at B5 would destabilize the associated R-specific trimer interface. To test this prediction, a human insulin analog was prepared in which His^B5 was substituted by Arg, a variant observed in some hystricomorph mammals, fish, and reptiles (23).5 Although the variant hormone retains substantial activity, Arg^B5-insulin is unable to undergo the TR transition in solution. Similarly, crystals of Arg^B5-insulin, grown under conditions leading to formation of wild-type R₆ hexamers, contain only T₆ hexamers. The variant structure, determined by molecular replacement at a resolution of 1.3 Å, closely resembles the wild-type T₆ hexamer. The side chains of His^B5 and Arg^B5, despite their differences in size and shape, pack within corresponding interchain crevices and participate in part in analogous networks of van der Waals interactions and hydrogen bonds. Despite such similarities, Arg^B5-insulin is less stable than the wild-type protein, as probed by chemical denaturation studies, presumably due either to loss of weakly polar interactions associated with the imidazole ring (24) or to less efficient packing of the linear Arg^B5 side chain within the crevice.

Together, the properties of Arg^B5-insulin uncouple the mechanism of receptor binding from the choreography of conformational changes in the classical TR transition. Impairment of hexamer reorganization by introduction of a charged side chain, reminiscent of classical mutations in hemoglobin that impair its cooperativity (25-28), demonstrates the utility of “electrostatic engineering” in studies of protein allostery.

EXPERIMENTAL PROCEDURES

Synthesis of Insulin Analogs—Human insulin and Asp^B28-insulin were obtained from Lilly and Novo-Nordisk (Bagsværd, Denmark), respectively. Synthesis of Arg^B5-insulin was performed as described (9, 10); see also the supplemental materials.

Receptor Binding Assays—The human IR was expressed and purified as described (29). Competitive IR binding assays were performed by a microtiter plate antibody capture assay. Microtiter strip plates (Nunc Maxisorb) were incubated overnight at 4 °C with anti-FLAG IgG (100 ml/well of a 40 μg/ml solution in phosphate-buffered saline). Washing, blocking, receptor binding, and competitive binding assays with labeled and unlabeled peptides were performed as described (29). Binding data were analyzed by a two-site sequential model with homologous or heterologous labeled and unlabeled ligands to obtain dissociation constants. The percentage of tracer bound in the absence of competing ligand was <15% to avoid ligand depletion artifacts.

Visible Absorption Spectroscopy—To probe the TR transition of Co²⁺-substituted insulin hexamers, the d-d optical absorption bands of Co²⁺ (a characteristic feature of a tetrahedral complex) (30, 31) were monitored as described (32). Solutions contained 0.2 mm insulin or insulin analog in a buffer consisting of 0.07 mm CoCl₂ and 50 mm phenol in 50 mm Tris-HCl (pH 8). Absorption spectra were obtained in the presence of 0.8 m NaSCN; the thiocyanate anion contributes a fourth ligand (in addition to three symmetry-related His^B10 side chains) to the coordination of each axial metal ion and so enhances the d-d band intensity (30, 31).

¹H NMR pH Titrations—Spectra were obtained at 700 MHz with a high sensitivity cryogenic probe (Bruker Biospin, Inc., Billerica, MA). Free histidine (Sigma-Aldrich, St. Louis, MO), human insulin (calculated pI 5.4), or Asp^B28-insulin (calculated pI 4.9) were first dissolved in 500 μl of 99.0% D₂O containing 10 mm deuterated Tris-HCl (pH^* 8.5, direct meter reading; Isotec, Miamisburg, OH) at respective sample concentrations of 10 mm, 1 mm, and 1 mm. After ∼12 h of hydrogen-deuterium amide proton exchange at room temperature, samples were lyophilized and redissolved in 500 μl of 99.9% D₂O containing 50 mm deuterated phenol (Sigma-Aldrich); 0.33 mm ZnCl₂ was then added to the protein solutions to form phenol-stabilized R₆ hexamers. The apparent pD of the samples (designated pH^*; uncorrected for isotope effects) was in each case measured with a microelectrode (Ingold 6030; Toledo-Mettler, Columbus, OH) at 40 °C and adjusted to pH^* 8.3 with DCl or NaOD. ¹H NMR spectra were acquired at 40 °C; chemical shift values are shown relative to 2,2-dimethyl-2-silapentane-5-sulfonic acid (DSS). Successive pH^* values were obtained by adding an aliquot of dilute DCl; serial spectra were obtained at each pH^* tested between pH^* 8.5 and 2.0. Titration curves were fitted to a modified Henderson-Hasselbalch equation using Kaleida-Graph, Synergy Software (Reading, PA) by nonlinear least squares analysis as follows,

(Eq.1)

in which δ_obs is the chemical shift observed at each pH^* value, and δ_AH+ and δ_A are the chemical shifts of the protonated and deprotonated histidines, respectively. The relationship,

(Eq.2)

was applied to convert the apparent pK_a estimates obtained in D₂O to corresponding values in H₂O as described (33).

Circular Dichroism—Far-UV CD spectra were obtained as described (34). Spectra, acquired with an Aviv spectropolarimeter (Aviv Biomedical Inc., Lakewood, NJ), were normalized by mean residue ellipticity. Samples were dissolved in 10 mm potassium phosphate (pH 7.4) and 50 mm KCl at a protein concentration of ∼25 μm. ZnCl₂ was added to provide 2.2 zinc ions/insulin hexamer. For equilibrium denaturation studies, samples were diluted in the same buffer to 5 μm; guanidine HCl was employed as denaturant (34). Data were obtained at 4 °C and fitted by nonlinear least squares to a two-state model (35).

X-ray Crystallography—Crystals were grown by hanging drop vapor diffusion under intended R₆ conditions (see supplemental materials). Data were collected from single crystals mounted in a rayon loop and flash-frozen to 100 K. Reflections from 30.6 to 1.3 Å were measured with a CCD detector system using synchrotron radiation at the Advanced Light Source (Lawrence Berkeley National Laboratory, Berkeley, CA). Data were processed with the program DTREK. Crystals belong to space group R3 with unit cell parameters a = b = 81.39 Å, c = 34.00 Å, α = β = 90, and γ = 120°. These dimensions are typical of T₆ crystals under cryoconditions. The structure was determined by molecular replacement using the CNS suite of programs (36). A model was obtained using the native T₂ dimer (Protein Data Bank identifier 4INS) following removal of all water molecules and zinc ions. A translation-function search was performed using coordinates from the best solution for the rotation function following analysis of data between 15.0 and 4.0 Å resolution. Rigid body refinement using CNS, employing overall anisotropic temperature factors and bulk solvent correction, yielded respective values of 0.31 and 0.30 for R and R_free for data between 19.2 and 3.0 Å resolution. Between refinement cycles, 2F_o - F_c and F_o - F_c maps were calculated using data to 3.0 Å resolution; zinc ions were then built into the structure using the program O (37). The geometry was monitored with PROCHECK (38); zinc ions and water molecules were built into the difference map as the refinement proceeded. Calculation of omit maps (of particular importance in relation to the N-terminal segment of the B-chain) and further refinements were carried out using CNS (36), which implements maximum likelihood torsion angle dynamics and conjugate-gradient refinement. X-ray diffraction and refinement statistics are provided in supplemental Table S1.

RESULTS

¹H NMR Studies of the R₆ Hexamer—We first describe pH-dependent ¹H NMR studies of the wild-type R₆ zinc insulin hexamer as a probe of the electrostatic environment of His^B5. Due to isoelectric precipitation of the wild-type hexamer near pH 6, these studies were extended through the use of a variant R₆ hexamer with lower isoelectric point (Asp^B28-insulin) (39, 40). Crystallographic studies have established that Asp^B28-insulin (a fast acting analog in clinical use for treatment of diabetes mellitus) forms R₆ hexamers that (with the exception of a local distortion at the dimer interface) are essentially identical to those of wild-type insulin. Human insulin contains two histidine residues (positions B5 and B10). Whereas His^B10 mediates the binding of axial zinc ions in each family of insulin hexamer, the environment of His^B5 varies among T- and R-states.

Although ¹H NMR spectra of T₆ and hexamers are not tractable, the spectrum of the phenol-stabilized R₆ hexamer exhibits high resolution features and has been well characterized (supplemental Fig. S3) (41). To probe the pK_a values of the histidine residues, ¹H NMR spectra were obtained in D₂Oat 40 °C as a function of pD; reference spectra of free histidine were also obtained. (A temperature of 40 °C was chosen to enhance resonance line widths relative to room temperature with retention of native pattern of chemical shifts (41)). Whereas on the surface of a T₆ hexamer, His^B5 would be expected to exhibit a pK_a between 6 and 7 (in accord with past NMR studies of engineered T-like monomers and dimers) (18, 42),6 its burial within an R₆ interface predicts a shift of the pK_a to lower values. By contrast, because His^B10 is bound by zinc, its imidazole ring is unavailable for protonation, so its resonances would not be affected by changes in pH under conditions of stable self-assembly.

¹H NMR spectra of wild-type insulin and Asp^B28-insulin were obtained below pH^* 3.8 and above either pH^* 5.8 (wild type) or pH^* 5.3 (Asp^B28 variant). Spectra were not obtained within respective pH^* ranges 3.8-5.8 and 3.8-5.3 (direct meter reading in D₂O buffer at 40 °C) due to reversible pH-Dependent isoelectric precipitation (39). Although the R₆ hexamer is highly soluble under neutral and basic conditions, the wild-type and variant protein solutions each exhibited a pretransition from clear to hazy near pH^* 6.8. Below pH^* 4, the solutions reclarified as protonation of His^B10 leads to release of the axial zinc ions and disassembly of the hexamers to form soluble zinc-free insulin dimers and monomers. pH-dependent aggregation and then disassembly of the zinc insulin hexamer at low pH^* precludes conventional assessment of side-chain pK_a values by continuous pH^* titration. Crystallographic and NMR studies under acidic conditions have nonetheless demonstrated retention of a native-like fold, including in the packing of the His^B5 imidazole ring along the surface of the A-chain (43-46).

Despite these experimental limitations, upper bounds to pK_a values were estimated from the apparent pH^* dependence of side-chain imidazole C₂ H or C₄H ¹H NMR resonances under accessible conditions (Fig. 5). Because measurements were obtained in D₂O at elevated temperature in a buffer with a marked pH-dependent pK_a (Tris·DCl), care was taken with interpretation of direct pH meter readings (see “Experimental Procedures”). In accord with standard values, spectra of free His (Fig. 5A) yielded essentially identical estimates of its pK_a from analysis of either ring resonance (6.10 ± 0.07) on correction for solvent isotope effects (Fig. 5B).

FIGURE 5.

¹H NMR studies of pK_a of His^B5 in R₆ hexamers. Aromatic region of spectra enables analysis of the pH dependence of His C-2 and/or C-4 protons of the free amino acid, wild-type R₆ hexamer, and Asp^B28-insulin R₆ hexamers. A, representative ¹H NMR spectra of free His (top), Asp^B28-insulin (middle), and wild-type insulin (bottom) obtained at pH^* 7.3; the C-2 proton of histidine is labeled with a blue asterisk, and the C-4 protons in each case are labeled by red asterisks. B, top, control pH titration curve of C₂H (blue) and C₄H (green) resonances in free His; bottom, C₄H resonances of His^B5 in wild-type insulin (black) and Asp^B28-insulin (red). Two simulated points are represented as two black open circles. Titrations were performed at 40 °C in 10 mm deuterated Tris·DCl and 50 mm deuterated phenol.

¹H NMR spectra of wild-type and Asp^B28 R₆ hexamers exhibit similar but not identical patterns of chemical shift dispersion, as expected from comparison of their crystal structures (47). In such spectra, the C₄H resonance of His^B5 is upfield of its position in zinc-free insulin (and of its random coil value), in each case well resolved in one-dimensional spectra (Fig. 5A, red asterisks). Strikingly, in each case, this resonance exhibits essentially no change in chemical shift at accessible pH^* values of >6. Because a pK_a of 6.0 would predict significant changes in C₄H chemical shift between pH^* values 6.5 and 7.0, the actual pK_a of His^B5 must be lower. Despite the unavailability of chemical shift values between intermediate pH^* values due to isoelectric precipitation (see above), upper bounds on the His^B5 pK_a may be estimated by simulation as follows. (i) The limiting chemical shift of His^B5 C₄H on protonation of the imidazole ring at pH 2.0 is assumed to be similar to that observed in the zinc-free dimer (wild-type insulin) or monomer (Asp^B28-insulin). (ii) At intermediate pH^* values, 3.0 and 4.0 the same chemical shift was assumed (Fig. 5B, open circles), forcing the putative pK_a to be >4.0 to enable conservative estimate of an upper bound. The resulting curve fitting suggests that the pK_a of His^B5 in the wild-type R₆ hexamer is less than 5.1; this upper bound is tightened to less than pH 4.8 upon analysis of the Asp^B28 variant. Although these bounds depend on the above two assumptions, our central observation, that the C₄H resonance of His^B5 in R₆ hexamers does not depend on pH in the range 6-8, immediately predicts that a positively charged side chain at B5 would be unfavorable in this conformational state.

Characterization of Arg^B5-insulin—If competence to undergo the formal TR transition in zinc insulin hexamers were subject to evolutionary selection, then the above ¹H NMR studies suggest that positively charged side chains might be disallowed. Inspection of the protein sequence data base nevertheless suggests that His^B5 may functionally be replaced by Arg; indeed, Arg is observed as the only alternative to His. A variant of human insulin containing this positively charged side chain was therefore prepared. The binding affinity of Arg^B5-insulin for the IR is 40% relative to that of wild-type human insulin (Fig. 6A).

FIGURE 6.

Receptor binding and stability assays. A, human insulin and Arg^B5-insulin exhibit slightly reduced affinity compared with human insulin for the IR. Competition binding assay in which fractional bound ¹²⁵I-labeled insulin (trace) is plotted versus the log-concentration of unlabeled wild-type insulin (wt; squares) or analog (triangles). The relative affinity of Arg^B5-insulin is ∼40%. B, CD-detected protein denaturation studies demonstrate that Arg^B5-insulin is less stable than wild-type insulin; fractional change in ellipticity is plotted as a function of concentration of guanidine HCl at 4 °C. Analysis by a two-state model (35) implies ΔG_u values of 3.6 kcal/mol (Arg^B5-insulin) and 4.4 kcal/mol (wild type).

Thermodynamic Stability—The stability of Arg^B5-insulin was inferred from CD-detected denaturation studies (Fig. 6B). Analysis of fractional unfolding as a function of guanidine concentration permitted extraction of the free energy of unfolding (ΔG_u) by a two-state model (35). Whereas the stability of wild-type insulin under these conditions is 4.4 kcal/mol, that of Arg^B5-insulin is 3.6 kcal/mol. The decrease in free energy (ΔΔG_u 0.8 ± 0.1 kcal/mol) is less than that observed in studies of an Ala^B5 analog (ΔΔG_u 1.7 ± 0.1 kcal/mol) (22). The decreased stability of Arg^B5-insulin is more modest than that observed on substitution of Val^A3 by Thr (ΔΔG_u 1.3 ± 0.1 kcal/mol) in which a polar β-OH makes unfavorable interactions within an analogous nonpolar crevice elsewhere in the protein (48).

Protein Allostery—The capacity of Arg^B5-insulin to undergo the TR transition in solution was assessed in cobalt-substituted hexamers by optical absorption spectroscopy (Fig. 7B). This method exploits a change in the coordination geometry of the axial metal ions from octahedral in each hexamer-related T-state trimer to tetrahedral in each hexamer-related R-state trimer (Fig. 7A). Whereas no such bands are observed in an octahedral coordination site, the tetrahedral-specific d-d transitions of the bound Co²⁺ ions therefore provide an intrinsic probe of the TR transition. As expected, wild-type insulin exhibits an intense tetrahedral signature between 500 and 650 nm in the absorption spectrum of the phenol-stabilized R₆ hexamer (Fig. 7B, upper trace). By contrast, this spectroscopic feature is markedly attenuated or absent in Arg^B5-insulin (lower trace).

FIGURE 7.

Substitution of His^B5 by Arg impedes the TR transition in solution. A, molecular structure (stereo pairs) of zinc-binding sites in representative T₆ hexamer and R₆ hexamer. Whereas the T₃-related site is predominantly octahedral, the R₃-related site is tetrahedral. This difference has been exploited to monitor the TR transition in Co²⁺-substituted hexamers in wild-type insulin (wt) and among insulin analogs (32, 72). hexamers contain one octahedral (T3) and one tetrahedral () metal-ion binding site. B, optical absorption studies of Co²⁺-substituted insulin hexamers in the presence of 50 mm phenol exhibit characteristic d-d transitions (a signature of R-specific tetrahedral metal ion coordination) between 525 and 650 nm in wild-type R₆ hexamer (upper trace); by contrast, this feature is markedly attenuated by the B5 substitution (lower trace). C and D, CD studies of zinc insulin hexamers in the absence (solid lines) and presence (open squares or circles) of 50 mm cyclohexanol, an inducer of the TR transition. The spectra suggest that this ligand enhances the α-helix content of wild-type insulin hexamers (C) but has no significant effect on the CD spectrum of Arg^B5-insulin hexamers (D).

Extension of these studies from cobalt insulin hexamers to zinc hexamers utilized CD as a probe of an R-state-specific increase in α-helix content; this feature is due to a conformational change in the N-terminal segment of the B-chain (Fig. 2). Whereas wild-type insulin exhibits an increase in the magnitude of helix-associated mean residue ellipticity at 208 and 222 nm upon the addition of cyclohexanol (an inducer of the TR transition whose low absorbance at these wavelengths, unlike phenol, is compatible with far-UV CD studies) (Fig. 7C), the CD spectrum of Arg^B5-insulin is unaltered upon the addition of this cyclic alcohol (Fig. 7D). CD spectra of wild-type insulin and the analog in the absence of cyclohexanol are nonetheless similar, suggesting that the substitution does not lead to a gross structural perturbation. In accord with the results of optical spectroscopy, the ¹H NMR spectrum of Arg^B5-insulin under the high phenol conditions that yield high resolution spectra of wild-type (or Asp^B28) R₆ hexamers (see above) instead exhibits broad and poorly resolved resonances (see supplemental Fig. S3), similar to those observed in the spectrum of zinc insulin hexamers in the absence of phenol or other R-specific ligands (30).

Crystal Structure—Arg^B5-insulin was crystallized under conditions that lead to crystallization of wild-type insulin as phenol-stabilized R₆ hexamers. In accord with the above spectroscopic studies, Arg^B5-insulin crystallized instead as a T₆ zinc hexamer. A classical lattice was observed in which one T₂ dimer (with protomers designated molecules 1 and 2) defines the asymmetric unit; the hexamer is generated by crystallographic symmetry (see supplemental Fig. S4). The dimensions of the unit cell (see “Experimental Procedures”) are inconsistent with standard and R₆ crystal forms.7 The structure (refined at a resolution of 1.3 Å) includes all residues in each chain and 129 water molecules per protein dimer. 2F_o - F_c electron density maps, illustrating the region near Arg^B5 in molecules 1 and 2, are shown in Fig. 8B (left and right panels, respectively); continuous density is observed throughout each side chain. The final model exhibits an R-factor of 0.206 with an R_free of 0.232. No density is observed suggestive of bound phenol molecules. The two protomers of Arg^B5-insulin are similar to each other and to those of wild-type insulin in T₆ hexamers (see supplemental Fig. S5 and Tables S2 and S3). No significant changes are observed in secondary structure, chain orientation, mode of assembly, or structure of Zn²⁺-binding sites (see supplemental Fig. S6). Comparison between the variant protomers and wild-type T₆-state structures yields root mean square differences (excluding B5) of 0.85 Å (main chain) and 1.50 Å (side chains). These values are similar to those obtained in pairwise comparison between T-state structures of wild-type insulin in different crystal forms (see supplemental Tables S2 and S3). The two axial Zn²⁺-binding sites are each well defined without evidence of multiple ligand conformations. Ligation is mediated in each case by three symmetry-related His^B10 side chains with zinc-nitrogen distances of 2.04 Å (see supplemental Fig. S6).

FIGURE 8.

Crystal structure of Arg^B5-insulin reveals a B5-A7 hydrogen bond. A, ribbon model of T-state protomer (molecule 1) in variant T₆ zinc hexamer. The A-chain ribbon is shown in black, and the B-chain ribbon is shown in blue (B1-B8) or gray (B9-B30). The variant B5 side chain is highlighted in red; selected side chains are otherwise purple (in A-chain) or green (in B-chain). The sulfur atoms of cystine A7-B7 are gold. B, electron density maps (2F_o - F_c plotted at 1σ) of Arg^B5 side chains in molecules 1 and 2 (left and right) exhibit well defined density for the variant side chain with positioning of the N_ε atom in each case near the carbonyl function of Cys^A7. C, stereo ribbon representation of molecule 1 illustrating the interchain hydrogen bond (dashed line) between the H-N_ε group (green) of Arg^B5 (red) and the carbonyl oxygen of Cys^A7. The A-chain is shown in black, and the B-chain is shown in gray; the B1-B8 is shown in blue; the solvent-exposed A7-B7 disulfide bridge (gold) is also shown.

The environment of Arg^B5 is similar but not identical to that of His^B5 in T-state protomers. In the wild-type and variant T₆ hexamers, the B5 side chain lies at the protein surface at an interface between A- and B-chains (Fig. 9A and 10A, left-hand panels). The main-chain dihedral angles (Φ, ψ) of Arg^B5 are (-98°, 125°) in molecule 1 and (-94°, 129°) in molecule 2. These values fall within the range of dihedral angles adopted by His^B5 in multiple wild-type T₆ hexamers (see supplemental Table S4).8 The terminal guanidinium group of Arg^B5 protrudes into solvent. The B5-related crevice is less efficiently filled by the linear methylene portion of Arg^B5 (Fig. 10A, right) than by the planar imidazole ring of His^B5 (Fig. 9A, right). Whereas classical cavities within cores of globular proteins are lined predominantly by nonpolar side chains, the shallow B5-related crevice, in large part open to solvent, is lined by a combination of polar and nonpolar atoms. A complex and asymmetric electrostatic environment is created by (i) the bordering A7-B7 and A6-A11 disulfide bridges, (ii) the orientation of the A1-A8 α-helical dipole, and (iii) individual main-chain amide and carbonyl functional groups; neighboring side chains and bound water molecules in the respective structures are shown in Figs. 9B and 10B. The lower stability of Arg^B5-insulin (relative to wild-type insulin) may reflect more favorable electrostatic interactions by an aromatic heterocycle within this shallow asymmetric pocket.

FIGURE 9.

Environment of His^B5 in wild-type T-state protomer. A, left, space-filling model showing the B5 side chain (green) at the protein surface (green arrow). The A-chain is shown in light gray, and the B-chain is otherwise shown in dark gray. The position of the His^B5 side chain (green sticks) is shown relative to the PyMol-generated electrostatic surface in corresponding orientation; the color bar (below the structure) indicates calibration from negative (-15 kT/e in red) to positive (+15 kT/e in blue). B, stereo representation of side-chain packing in the B5-related crevice. The His^B5 side chain is shown in green with the N_ε atom highlighted in purple. The carbonyl oxygen of Cys^A7 is shown as a red ball; other carbonyl oxygen atoms are shown as red sticks. A- and B-chain residues are otherwise shown in gray and black, respectively. Adjoining bound water molecules are shown in blue spheres, and the solvent-exposed A7-B7 disulfide bridge is shown in gold. The corresponding structural features of Arg^B5-insulin are shown in Fig. 10.

FIGURE 10.

Environment of Arg^B5 in variant T-state protomer. A, left, space-filling model showing variant B5 side chain (green) at the protein surface (green arrow). The A-chain is shown in light gray, and the B-chain is otherwise shown in dark gray. The position of the Arg^B5 side chain (green sticks) is shown relative to the PyMol-generated electrostatic surface in corresponding orientation; color bar (below the structure) indicates calibration from negative (-15 kT/e in red) to positive (+15 kT/e in blue). B, stereo representation of side-chain packing in the variant B5-related crevice. The Arg^B5 side chain is shown in green, with the N_ε atom highlighted in pink. The carbonyl oxygen of Cys^A7 is shown as a red ball; other carbonyl oxygen atoms are shown as red sticks. A- and B-chain residues are otherwise shown in gray and black, respectively. Adjoining bound water molecules are shown in blue spheres, and the solvent-exposed A7-B7 disulfide bridge in gold. Corresponding structural features of wild-type insulin are shown in Fig. 9.

The packing of Arg^B5 is constrained by its local packing in the B5-related crevice and possibly also by hexamer-hexamer contacts in the crystal lattice.9 Whereas the guanidinium NH₂ groups of Arg^B5 interact only with bound water molecules, an interchain hydrogen bond is formed within each protomer from B5 N_εH (Fig. 10B, pink) to the carbonyl oxygen of Cys^A7 (Fig. 10B, red ball; see also Fig. 3D and supplemental Fig. S7). (Although the former hydrogen atom is not visualized, the positions of neighboring heavy atoms strongly favor such hydrogen bonding; the nitrogen-oxygen distance in molecule 1 is 2.70 Å, and the bond angle is 137.3°.) This inferred hydrogen bond recapitulates an interchain hydrogen bond in wild-type insulin from the imidizole N_ε2H of His^B5 (Fig. 9B, purple) to the carbonyl oxygen of Cys^A7 (Fig. 9B, red ball; N_ε2-O distance 2.41 Å and bond angle 143.2° in 2-Zn molecule 1) (2). In molecule 2, the N_εHofArg^B5 forms bifurcating hydrogen bonds to the carbonyl oxygens of both Cys^A7 (distance 2.80 Å and angle 129.2°) and Ser^A9 (distance 3.03 Å and angle 105.1°). Hydrogen bonds and van der Waals contacts made by Arg^B5 in molecules 1 and 2 are given in supplemental Table S5; other lattice contacts are described in supplemental Table S6.

Formation of analogous interchain hydrogen bonds by Arg^B5 and His^B5 is of interest in relation to the failure of Met^B5 to support insulin chain combination (above), thus highlighting the likely contribution of specific side-chain functional groups to foldability. We envisage that formation of B5-related interchain hydrogen bond(s) in an oxidative folding intermediate would facilitate alignment of neighboring reactive A7 and B7 thiolate moieties as a kinetic aid to disulfide pairing (see “Discussion”).

DISCUSSION

Allosteric assembly of globular proteins has long provided a model for the transmission of conformational change (13, 49-51). Might the TR transition of insulin foreshadow the mechanism of receptor binding? This intriguing possibility, first suggested by the late D. C. Hodgkin and colleagues (2, 14), has been raised anew by studies of nonstandard insulin analogs (21, 52).

Interpretation of crystal structures of insulin is limited by its state of assembly as the hormone binds to the IR as a monomer. Although studies of truncated insulin analogs have revealed only T-like features (18, 19, 53, 54), a variety of theoretical and experimental studies indicate that the monomer is flexible (41, 46, 55-57), including at the N-terminal segment of the B-chain (residues B1-B8). Stabilization of its T-state conformation has been achieved by substitution of Gly^B8 by d-amino acids. Because in the T-state Gly^B8 resides in a β-turn, with positive Φ angle in a region of the Ramachandran plane unfavorable to l-amino acids, d-analogs exhibit both greater thermodynamic stability and partial impairment of the TR transition (21, 52). Remarkably, however, such stabilized analogs exhibit very low activities. Although these observations suggest that the T-state represents an inactive conformation and indeed that B8 functions as a site of induced fit, such studies do not specify the overall features of the bound state, in particular whether the N-terminal segment of the B-chain reorganizes as an α-helix as in the hexamer-specific TR transition (13, 14).

As a further test of the relationship between the TR transition and biological activity, the present study has exploited a species variant (Arg) at position B5. We chose to focus on this substitution in human insulin because of the structural environment of His^B5 in wild-type hexamers. In the T-state, the imidazole ring packs within a solvated crevice at the edge of the hexamer surface (2), whereas in the R-state the side chain packs at an interface between dimers. Although this interface is in part solvated, NMR studies of the pK_a of His^B5 suggested that the R-specific (or R^f-specific) trimer interface would be destabilized by the uncompensated positive charge of an Arg^B5 substituent. In accord with this prediction, our spectroscopic results demonstrated that substitution of His^B5 by Arg impedes the TR transition in solution. Further, crystals of Arg^B5-insulin grown under conditions leading to stabilization of wild-type R₆ hexamers (i.e. in the presence of phenolic ligands) were observed to contain only T₆ hexamers. A similar block to the TR transition has been recently reported in a review article of an analog containing substitutions of Asn^B3 by Lys and of Lys^B29 by Glu^B29 (insulin glulisine) (58).

The crystal structure of Arg^B5-insulin as a variant T₆ hexamer is essentially identical to that of the wild-type T₆ hexamer, including analogous interactions by the variant B5 side chain. Because Arg^B5-human insulin binds well to the IR (with affinity ∼40% relative to wild type), our results collectively demonstrate that competence to undergo the TR transition in a hexamer is not required for the biological activity of an insulin monomer. We discuss these results in relation to protein stability. Because of the recent clinical finding of a mutation at B5 causing neonatal diabetes mellitus due to protein misfolding (59), we also discuss our findings in relation to the nascent folding of proinsulin in the endoplasmic reticulum of the pancreatic β-cell.

Determinants of Insulin Stability—His^B5 contributes to the thermodynamic stability of insulin: its substitution by Ala in an engineered monomer yields a nativelike structure whose unfolding free energy (ΔG_u), as inferred from chemical denaturation studies, is reduced from 4.9 to 3.2 kcal/mol at 4 °C (22). This decrement (ΔΔG_u 1.7 ± 0.1 kcal/mol relative to DKP-insulin) is presumably due to a local packing defect in the B5-related interchain crevice and possible changes in solvation. In Arg^B5-insulin, a nativelike pattern of contacts in this crevice is maintained, including interchain hydrogen bonds and a neighboring network of bound water molecules. These interactions constrain the orientation of the N-terminal segment of the B-chain against the A chain and in part define the environment of the solvent-exposed A7-B7 disulfide bridge.

The stability of Arg^B5-insulin as a zinc-free monomer is also lower than that of wild-type insulin; the decrement (ΔΔG_u 0.8 ± 0.1 kcal/mol) is less marked than that of an Ala^B5 analog. The intermediate stability of Arg^B5-insulin suggests that, relative to Ala^B5, the variant side chain in part recapitulates the native packing of His^B5 but that its net contribution is less favorable than that of an imidazole ring. Favorable electrostatic interactions may also be present as the Arg^B5 guanidinium group (with its positive charge) projects into solvent near the C-terminal (and hence negative) end of the A1-A8 α-helical dipole axis. In addition, the N_εH group of Arg^B5 donates a hydrogen bond to the carbonyl oxygen of Cys^A7 and perhaps (in molecule 2) a bifurcating second hydrogen bond to the carbonyl oxygen of Ile^A10. Although these interactions recapitulate native contacts by His^B5, the proximal aliphatic portion of the Arg^B5 side chain differs from His in shape, size, and electrostatic properties. We speculate that the packing of His^B5 within the interchain crevice uniquely optimizes an asymmetric pattern of weakly polar interactions not satisfied by Arg or other side chains.

Determinants of Foldability—Just as Arg^B5 partially reverts the thermodynamic instability of an Ala^B5 analog, this species variant also partially restores the efficiency of disulfide pairing in the course of chain combination. Whereas chemical synthesis of an Ala^B5 insulin analog encountered very low yields, the yield of Arg^B5-insulin was reduced by less than 3-fold. This improvement appears due to the guanidinium group, since an attempted synthesis of Met^B5-insulin failed to yield detectable product. A similarly profound block to chain combination was observed upon substitution of Gly^B8 by any l-amino acid (21). Such impediments seem remarkable in light of the general utility of chain combination in the synthesis of diverse insulin analogs since its development in 1966 (60).

Specific blocks to chain combination presumably reflect conformational events in a critical oxidative folding intermediate. We envisage that the conformation of Gly^B8 and interchain packing of His^B5 are each integral to stabilization of the nascent B7-B10 β-turn and in turn to formation of the A7-B7 disulfide bridge. The biological relevance of these observations is supported by the recent finding of mutations in the insulin causing neonatal diabetes mellitus (61). Remarkably, the emerging clinical data base of such mutations includes substitutions at B5 and B8 (59). Cell biological studies have shown that Ala^B5 and diverse other side chains, including the diabetes-associated mutation Asp^B5 (59), impair the folding of proinsulin in the endoplasmic reticulum of a transfected human cell line (22).

Because insulin chain combination and the in vivo folding of proinsulin reflect kinetic processes, small decrements in the thermodynamic stability of the products (the insulin analogs) cannot in themselves account for relative yields. Relative to Ala^B5, therefore, how can Arg^B5 enhance both stability and efficiency of disulfide pairing? We imagine that allowed side chains at B5 both participate in nascent interchain interactions preceding formation of cystine A7-B7 and stabilize the subsequent disulfide-bridged species, including the mature hormone, once folding has been achieved. That Arg^B5 is allowed among vertebrate insulin sequences suggests that its nativelike contacts in the B5-related crevice, including the directional interchain hydrogen bonds observed in the present crystal structure, would productively guide disulfide pairing in vivo. Efforts to test this prediction in cell culture are planned.

Why is His^B5 (rather than Arg^B5) conserved among eutherian mammals and the predominant residue among other classes of vertebrates? It seems unlikely that this preference reflects a physiological advantage of 2-fold higher receptor-binding affinity, since the activities of vertebrate insulins vary by more than 10-fold, and small decrements in affinity are readily compensated by correlated changes in clearance rates (62, 63). We suggest instead that an evolutionary constraint is imposed by requirements of protein folding and trafficking in the β-cell. Interchain interactions by His^B5 in a nascent proinsulin polypeptide may enhance its folding efficiency in the endoplasmic reticulum (as it does in chain combination in vitro), in turn protecting the β-cell from toxic protein misfolding (64, 65). Importantly, the genetic link between misfolding of proinsulin and human β-cell dysfunction (described above in relation to neonatal diabetes) (61) suggests that the foldability of proinsulin has imposed a powerful constraint on the evolution of allowed insulin sequences.

Conclusion—The TR transition of insulin provides a model of protein allostery. The classical nomenclature T and R reflects an analogy between the transmission of conformational change in insulin and the TR transition of hemoglobin, associated with the cooperative binding and release of three gases (O₂, CO, and NO) (49, 50, 66). Whereas we have characterized a novel substitution in insulin that blocks its TR transition, biochemical studies of variant human globin chains associated with defective oxygen transport have identified clinical mutations that perturb cooperativity. Interestingly, two such clinical mutations also involve substitution of His by Arg; these occur near the binding site of the allosteric effector 2,3-bis-phosphoglycerate (hemoglobins Abruzzo and Deer Lodge) (25-28). Analogous mutations have been described in the allosteric oligomeric enzyme aspartate transcarbamylase; these impair the ability of its substrates (carbamoyl phosphate and l-Asp) to induce the TR transition (67).

An essential difference between insulin and classical TR assemblies is that insulin functions as a monomer. Although the zinc insulin hexamer is of biological relevance as a storage vehicle, the relevance of its structure to the mechanism of receptor binding has long been the subject of speculation. Substitutions at subunit interfaces of the insulin hexamer that modulate its allosteric properties may thus be unrelated to the mechanism of monomer binding to the IR (i.e. monomeric Arg^B5-insulin can form the R-state and therefore bind to the IR, but it cannot stabilize the R-state in the hexamer due to the high intrinsic pK_a value for residue Arg^B5). Arg^B5-insulin retains substantial receptor-binding affinity (40% relative to wild-type human insulin), strongly suggesting that competence of an insulin analog to undergo the formal TR transition is not necessary for biological activity. We may regard such substitutions as extrinsic to the conformational repertoire of the monomer. The small decrement in activity of Arg^B5-insulin is presumably due to a change in the electrostatic or steric contours of the receptor-binding surface (see supplemental Fig. S8).

Does it remain possible that on receptor binding the active insulin monomer exhibits R-like features? This possibility is indeed suggested by studies of an intrinsic probe of the conformational repertoire: stereospecific modulation of the B8 Φ angle by corresponding d- or l-amino acid substitutions (21, 52). Substitution of Gly^B8 by d-amino acids favors the T-state by a mechanism unrelated to subunit interfaces and yet markedly impairs receptor binding. In light of these and the present results, we propose that induced fit of the insulin monomer and the hexameric TR transition exploit corresponding sites of flexibility in the molecule but as distinct processes. Identification of such sites has direct relevance to structural determinants of foldability (21) and hence the newly recognized genetics of neonatal diabetes mellitus (61). How induced fit enables this ancestral hormone to bind to and trigger the insulin receptor represents a major unsolved problem in structural biology.