Contribution of Residue B5 to the Folding and Function of Insulin and IGF-I

Proinsulin exhibits a single structure, whereas insulin-like growth factors refold as two disulfide isomers in equilibrium. Native insulin-related growth factor (IGF)-I has canonical cystines (A6—A11, A7–B7, and A20—B19) maintained by IGF-binding proteins; IGF-swap has alternative pairing (A7–A11, A6—B7, and A20—B19) and impaired activity. Studies of mini-domain models suggest that residue B5 (His in insulin and Thr in IGFs) governs the ambiguity or uniqueness of disulfide pairing. Residue B5, a site of mutation in proinsulin causing neonatal diabetes, is thus of broad biophysical interest. Here, we characterize reciprocal B5 substitutions in the two proteins. In insulin, HisB5 → Thr markedly destabilizes the hormone (ΔΔGu 2.0 ± 0.2 kcal/mol), impairs chain combination, and blocks cellular secretion of proinsulin. The reciprocal IGF-I substitution ThrB5 → His (residue 4) specifies a unique structure with native 1H NMR signature. Chemical shifts and nuclear Overhauser effects are similar to those of native IGF-I. Whereas wild-type IGF-I undergoes thiol-catalyzed disulfide exchange to yield IGF-swap, HisB5-IGF-I retains canonical pairing. Chemical denaturation studies indicate that HisB5 does not significantly enhance thermodynamic stability (ΔΔGu 0.2 ± 0.2 kcal/mol), implying that the substitution favors canonical pairing by destabilizing competing folds. Whereas the activity of ThrB5-insulin is decreased 5-fold, HisB5-IGF-I exhibits 2-fold increased affinity for the IGF receptor and augmented post-receptor signaling. We propose that conservation of ThrB5 in IGF-I, rescued from structural ambiguity by IGF-binding proteins, reflects fine-tuning of signal transduction. In contrast, the conservation of HisB5 in insulin highlights its critical role in insulin biosynthesis.

The vertebrate insulin-related superfamily consists of insulin and insulin-related growth factors (IGF-I 5 and IGF-II) (1, 2), relaxin (3)(4)(5), and relaxin-related factors (6 -9). Insulin and IGFs function as ligands for receptor tyrosine kinases (the insulin receptor and type 1 IGF receptor; IR and IGF-1R) (10), whereas relaxin and related factors bind to G-protein-coupled receptors (11). Interest in the evolution and folding properties of insulin-related polypeptides has recently been invigorated by the discovery of mutations in the human insulin gene associated with permanent neonatal-onset diabetes mellitus (12). These dominant mutations impair the foldability of variant and (in trans) wild-type proinsulin, leading to ␤-cell dysfunction, endoplasmic reticular (ER) stress, and impaired ␤-cell viability (13). One such mutation occurs at position B5 (12,14). Insulin contains a conserved His at B5, whereas IGF-I contains a conserved Thr (Table 1). Here, we investigate reciprocal substitutions in these proteins, Thr B5 in insulin and His B5 in IGF-I, as probes of competing evolutionary constraints among otherwise homologous sequences. 6 IGFs are single-chain polypeptides containing A-and B-domains, an intervening connecting (C)-domain, and a C-terminal D-domain ( Fig. 1A) (1,2); insulin (like relaxin and related factors) contains two chains (designated A and B; Fig. 1B) as a consequence of proteolytic processing in the trans-Golgi network (3)(4)(5)15). Crystal structures of IGF-I and insulin exhibit similar ␣-helical domains (Fig. 1, A and B) (1)(2)(3)(4)(5)(6)(7)(8)(9). Protein folding is linked to specific disulfide pairing. The canonical cystines in insulin are A6 -A11, A7-B7, and A20 -B19, and the corresponding cystines in IGF-I are at polypeptide positions 47-52, 6 -48, and 18 -61. Whereas the six cysteines and selected core residues are broadly conserved throughout the vertebrate insulinrelated superfamily (Table 1; see also supplemental Table S1), other residues are restricted to particular proteins. An example is residue B5; its restricted conservation suggests distinct IGF-and insulin-specific contributions to structure or function.
B5 side chains occupy similar but not identical structural environments in IGF-I and insulin. The side chains project in each case into an inter-domain crevice adjoining cystine A6 -A11 and buttressing the solvent-exposed A7-B7 disulfide bridge (Fig. 1, C and D) (15)(16)(17). The respective N-terminal arms of IGF-I and insulin (residues 1-3 and B1-B4, respectively) exhibit different orientations (Fig. 1, A and B, arrows). An imidazolic NH of His B5 in insulin (Fig. 1D, green ball) donates one or bifurcating hydrogen bonds to A-chain carbonyl oxygens (red balls; see also supplemental Table S2), proposed to facilitate the folding of proinsulin (18,19). An Ala B5 substitution in insulin impairs chain combination, whereas binding to the insulin receptor (IR) is decreased by less than 2-fold (18). Although the side chain of Thr B5 in IGF-I is not well ordered in solution (20,21) and variably positioned in crystal structures (16,17), its ␤-OH group (Fig. 1C, green ball) can be oriented to donate an analogous hydrogen bond to an A-domain carbonyl oxygen (red) (see supplemental Table S2). His B5 -IGF-I exhibits native interactions with IGF-binding proteins (IGFBP) with 2-fold enhanced binding to the IGF-1R (22). IGF sequences anomalously encode two alternative structures in equilibrium ( Fig. 2A) (23)(24)(25)(26)(27). Native IGF-I retains canonical disulfide pairing, whereas IGF-swap exhibits the alternative pairing A7-A11, A6 -B7, and A20 -B19 (IGF-I positions 46 -52, 6 -47, and 18 -61). The non-native isomer is at least 20-fold less active (26). 1 H NMR studies have demonstrated that IGF-swap possesses a well organized three-dimensional structure with salient differences from that of native IGF-I (26,28). The alternative structure is as stable (or more stable) than native IGF-I as probed by thiol-catalyzed disulfide exchange (26) and chemical denaturation (29). Proinsulin in contrast refolds to form a unique ground state (30); disulfide isomers exist only as metastable kinetic traps (29,31,32). Strik-ing similarities are nonetheless observed between the solution structures of IGF-swap and insulin-swap ( Fig. 2B) (28). Such structural similarity suggests that one or a few sites of sequence difference might account for the contrasting refolding properties of proinsulin and IGF-I. The defective folding properties of IGF-I are circumvented in vivo by selective binding of native IGF-I to IGFBPs (33,34).
Studies of inactive mini-IGF-I models (lacking C-and Ddomains) and corresponding mini-proinsulin analogues suggest that this distinction is mediated by differences between B-domains (35)(36)(37). Construction of chimeric models has highlighted the proposed role of residue B5; substitution of Thr 4 in mini-IGF-I by His led to unique canonical pairing (19). Interpretation of these studies has remained uncertain, however, as the crystal structure of mini-IGF-I-swap (38) (Fig. 2C) was found to exhibit marked and unexpected differences from the structures of either IGF-swap (28) or insulin-swap ( Fig. 2B) (32). 7 These structural differences imply that the conformational equilibrium between mini-IGF-I and mini-IGF-I-swap may not be representative of the intact proteins. 8 It is therefore of interest to investigate the effects of His B5 in native IGF-I and the effects of Thr B5 in insulin. To this end, we have introduced reciprocal B5 substitutions into insulin and IGF-I. Our studies yield asymmetrical outcomes. In insulin Thr B5 profoundly destabilizes the native state but does not promote isomer formation. In IGF-I His B5 enables unique disulfide pairing (native IGF-I) by selectively destabilizing the competing fold (IGF-swap).
Why is a folding-defective residue excluded in insulin but conserved in IGF-I? On the one hand, we demonstrate that Thr B5 blocks the secretion of proinsulin in a human cell line and so presumably would cause ␤-cell dysfunction in vivo (39). On the other hand, IGF-IGFBP co-evolution has cir-7 Mini-IGF-I-swap exhibits an additional ␣-helix in the A-domain and elongated B-domain ␣-helix, which includes His B5 (magenta in Fig. 2C). It is possible that these anomalous structural features reflect conditions of crystallization. 8 The solution structure of mini-IGF-I retains three canonical ␣-helices (105) but with relative orientations different from those in the solution structures of insulin, mini-proinsulin, or native IGF-I (20, 106 -108).

MATERIALS AND METHODS
Synthesis of Thr B5 -Insulin-The protocol for solid-phase synthesis is as described previously (40). The wild-type A-chain was obtained by oxidative sulfitolysis of human insulin as described previously (18,41). Thr B5 B-chain was prepared by automated Fmoc (N-(9-fluorenyl)methoxycarbonyl)-based synthesis and derivatized by S-sulfonation. Insulin chain combination was achieved as described previously (41). Purification of Thr B5 -insulin was accomplished by cation-exchange chromatography (CM52, 24 ϫ 1.2 cm, Whatman) using acetate buffer (pH 3.3) and a NaCl gradient. Final purification was by reverse-phase high-performance liquid chromatography (rp-HPLC) using a C18 column with a 0.1% trifluoroacetic acid/acetonitrile solvent system. Combination of 40 mg of A-chain and 20 mg of the variant B-chain resulted in a final yield of 1.1 mg. The corresponding yield of wild-type chain combination under these conditions is ϳ4.0 mg. The purified analogue was observed to be a single component on analytical rp-HPLC (C18 column, 25 ϫ 0.46 cm) using two independent elution gradients, acetonitrile in 0.1% trifluoroacetic acid and methanol in 0.1% trifluoroacetic acid. Because insulin isomers exhibit very low affinity for IR, native disulfide pairing was implied by the higher residual receptor-binding affinity of the analogue ( Fig. 3C; see "Results"). The yield of chain combination was reduced by 4-fold relative to wildtype chain combination; no nonnative isomers were encountered in the course of chain combination. The matrix-assisted laser desorp- . The A-domain is shown in red, B-domain in blue, and C-and D-domains in gray. The three disulfide bridges are shown in gold (canonical numbering A7-B7, A6 -A11, and A20 -B19; IGF residues 6 -48, 47-52, and 61-18); balls represent sulfur atoms. Side chain of Thr 4 (canonical position B5) is also shown (green). B, ribbon model of human insulin (T-state protomer; PDB accession code 4INS). The coloring scheme is as in A. His B5 is shown in green. C, position of Thr 4 (canonical position B5) adjoining cystine A6 -A11 (IGF residues 47-52) in inter-domain crevice of IGF-I. The ␤-OH group and ␥-CH3 of Thr 4 (Thr B5 ) are shown as green and black spheres; neighboring A-chain carbonyl oxygens are shown as red spheres. D, position of His B5 in inter-chain crevice of insulin. Imidazolic NH is shown as green sphere, and neighboring A-chain carbonyl oxygens in red spheres. Whereas His B5 consistently engages in hydrogen bonding to the A-chain in crystal structures of insulin (T-state protomers), Thr 4 in IGF-I engages in analogous hydrogen in only a detergent-stabilized crystal structure and not in multiple other structures (see supplemental Table S2).
tion-ionization (MALDI) mass spectrum of the product was in accord with its predicted value (5770.4 Da).
Cellular Expression and Folding of Proinsulin-Plasmids encoding human proinsulin or B5 variants in mammalian cell culture were constructed to enable analysis of protein folding and secretion as described previously (18,46). A control for an uncleaved preproinsulin was provided by a cleavagesite mutation associated with neonatal diabetes mellitus (Asp S24 in the signal sequence (12)). In brief, HEK293T cells (human) were cultured in high glucose Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 0.1% penicillin/streptomycin at 37°C with 5% CO 2 . For metabolic labeling, cells were plated into 6-well plates 1 day before transfection. Plasmid DNA (2 g) was transfected into each well using Lipofectamine (Invitrogen). At 40 h post-transfection, cells were preincubated in methionine/cysteine-deficient medium with or without 10 g/ml tunicamycin (TM) as indicated for 30 min, metabolically labeled in the same medium containing 35 Slabeled Met and Cys for 30 or 60 min, washed once with complete medium, and chased in complete medium with or without 10 g/ml TM at different time points as indicated (35). After chase, media were collected, and cells were lysed in 100 mM NaCl, 1% Triton X-100, 0.2% sodium deoxycholate, 0.1% SDS, 10 mM EDTA, and 25 mM Tris-HCl (pH 7.4) with protease inhibitors. Lysates were immunoprecipitated with guinea pig anti-insulin antiserum (Linco Diagnostic) and analyzed by Tris/ Tricine-urea-SDS-PAGE under nonreducing conditions or reducing conditions as indicated (18,47). For treatment with peptide N-glycosidase F (PNGase F), the anti-insulin immuno- Folding and Function of IGF-I FEBRUARY 12, 2010 • VOLUME 285 • NUMBER 7 precipitates were incubated with PNGase F at 37°C for 1 h prior to gel analysis.
Refolding of IGF-I and His B5 -IGF-I-Redox-coupled folding was effected in the following conditions: ϳ0.5 mg/ml IGF-I(SH) 6 or His B5 -IGF-I, 20 mM Tris-HCl (pH 7.7), 8 mM cysteine, 1 mM cystine⅐HCl, and 0.5 M guanidine hydrochloride. Folding, as monitored by liquid chromatography-mass spectrometry, was almost complete in Ͻ1 min (to yield native IGF-I and IGFswap) for wild-type sequence, whereas folding of the variant sequence (to yield a single product) was approximately halfcomplete at Ͻ1 min; folding was thus extended for 5 h. Whereas refolding of the native polypeptide yielded two predominant products, refolding of the variant yielded a single dominant product (see "Results"). Following oxidation, the mass of the protein decreased by 6.6 Ϯ 0.7 Da in each case, indicating the formation of three disulfide bonds. Principal components of the folding reaction were purified by preparative C18 rp-HPLC, enabling native IGF-I, IGF-swap, and His B5 -IGF-I to be obtained. Respective overall yields of native IGF-I and His B5 -IGF-I were 6.7 and 8.3% from peptide segment IGF-I[Cys 48 -70]. Disulfide pairing in the variant protein was established by NMR fingerprinting (see "Results").
Synthesis of His B5 Two-chain Hybrid-The wild-type insulin A-chain S-sulfonate was combined with a 29-residue B-chain derived from the B-domain of IGF-I in which Thr 4 was substituted by His (canonical position B5). The IGF-I-derivated peptide was synthesized and purified as in the synthesis of human insulin analogues (41). Combination of 30 mg of A-chain and 15 mg of the variant B-chain resulted in a final yield of 0.9 mg. The corresponding yield of wild-type chain combination under these conditions is ϳ3.0 mg. The MALDI-mass spectrum of the product was in accord with its predicted value (5562.8 Da).
Receptor Binding Assays-Dissociation constants for binding of insulin, IGF-I, and respective B5 analogues to IR were determined in competitive radioligand binding assays with 125 I-Tyr A14 human insulin. The assay employed the B isoform of IR (IR-B). Experimental details are provided as supplemental material. In brief, assays were performed with the isolated IR-B with a C-terminal FLAG tag using a microtiter plate antibody capture technique as described previously (48). Microtiter strip plates (Nunc Maxisorb) were incubated overnight at 4°C with FLAG M2 IgG (100 l/well of 40 g/ml in phosphate-buffered saline). In all assays, the percentage of tracer bound in the absence of competing ligand was less than 15% to avoid ligand-depletion artifacts. Dissociation constants of analogues were obtained by nonlinear regression analysis of binding data by the method of Wang (49); this employs an exact mathematical expression to describe the competitive binding of two different ligands to a receptor. A corresponding microtiter plate antibody assay using IGF-1R was employed to assess binding of insulin, IGF-I, and respective B5 analogues to this homologous receptor using 125 I-Tyr31 IGF-I as tracer (50). Results are summarized in Table 2. Control studies of cellular extracts in the absence of prior transfection of the epitope-tagged receptor constructs (either IR or IGF-1R) demonstrated that background binding to endogenous cellular proteins was negligible.
Cellular Studies of IGF-I Signaling-Mouse anti-Akt antibody was from Cell Signaling Technologies (Beverly, MA); rabbit anti-phospho-Akt (Ser(P) 493 ) was from Invitrogen; mouse Cy5-labeled IgG and rabbit Cy3-labeled IgG secondary antibodies were from GE Healthcare. An IGF-1R-deficient mouse embryo fibroblast cell line expressing the human IGF-1R was provided by Prof. R. Baserga (see Ref. 51). Akt phosphorylation as a parameter for activation of the IGF-IR signaling pathway was assayed essentially as described previously (52). In brief, cells were treated with 0.1-10 nM ligand for 5 min and lysed in  Table 2. C, receptorbinding studies employing IR (isoform B). Competitive displacement assay in which pre-bound 125 I-labeled insulin is displaced by successive concentrations of unlabeled Thr B5 -insulin () or wild-type insulin (OE) is shown. Ratio of bound-to-total tracer counts are plotted as a function of the logarithm (base 10) of the ligand concentration (nanomolar). D, analogous receptor-binding studies employing IGF-1R. Competitive displacement assay in which pre-bound 125 I-labeled IGF-I is displaced by successive concentrations of unlabeled Thr B5insulin () or wild-type insulin (OE).
SDS sample buffer without dithiothreitol or bromphenol blue and boiled immediately to inhibit protease and phosphatase action. Protein concentration was determined with a detergent-compatible protein assay kit (Bio-Rad). Dithiothreitol (100 mM) and bromphenol blue (0.1%) were then added. Whole-cell lysates (20 mg) were subjected to reducing SDS-PAGE on 10% Criterion gels (Bio-Rad), and resolved proteins were transferred to Immobilon FL polyvinylidene difluoride membranes (Millipore Corp., Billerica, MA). Blots were probed simultaneously with the Akt and phospho-Akt antibodies (1:2500 and 1:1000 dilutions, respectively), washed three times with Tris-buffered saline/Tween 20 (TBS-T), and then probed with a combination of the Cy-3-and Cy-5-labeled secondary antisera (1:5000 dilution each). Membranes were washed again three times with TBS-T buffer and dried, and fluorescent images were captured and quantified using a Fluorchem Q imaging system (Alpha Innotech, San Leandro, CA). Paired Student's t tests were used for all statistical analyses. Significance was accepted at p Ͻ 0.05.
Disulfide Reassortment-␤-Mercaptoethanol-catalyzed disulfide exchange was assessed as described previously (26). In brief, purified IGF-I or His B5 -IGF-I was dissolved in 50 mM sodium phosphate (pH 7.5 at 20°C) containing 1 mM EDTA at a protein concentration of 100 g/ml (13 M). The solutions were incubated at 20°C after initiating the disulfide exchange reaction. To catalyze exchange, a freshly made stock solution of 0.02% (v/v) ␤-mercaptoethanol was added to a final concentration of 0.001% (v/v) (0.17 mM). Aliquots were removed after overnight incubation, quenched with 10-fold excess 0.1% trifluoroacetic acid, and analyzed by rp-HPLC. Samples were applied through an autosampler onto a Vydac RP-C4 column (214TP54, 250 ϫ 0.46 cm; Grace Vydac, Hesperia, CA) at a flow rate of 1 ml/min. The proteins were eluted with an acetonitrile gradient using a solvent system consisting of 0.1% aqueous trifluoroacetic acid (solvent A) and acetonitrile containing 0.1% trifluoroacetic acid (solvent B). Proteins were also eluted with an alternative gradient system consisting of 0.1% trifluoroacetic acid and methanol. Protein elution was monitored at 215 nm using a dual-2487 absorbance detector. Data acquisition and processing utilized the Waters Breeze HPLC software. A parallel sample set was also analyzed at 37°C.
Circular Dichroism-Far-ultraviolet CD spectra were acquired using an Aviv spectropolarimeter equipped with thermistor control. Samples were made 30 -50 M in 140 mM NaCl and 10 mM sodium phosphate at pH 7.4 (phosphate-buffered saline) and observed at 4 or 25°C in quartz cuvettes with a path length of 1 mm. Samples were diluted to 5 M for protein denaturation studies. Guanidine-induced denaturation data at 4 or 25°C were fitted by nonlinear least squares to a two-state model as described previously (53).
NMR Studies of IGF-I and His B5 -IGF-I-One-and two-dimensional 1 H NMR spectra of IGF-1, IGF-swap, and His B5 -IGF-I were obtained at 700 MHz in 10% deuterated acetic acid at 40°C as described previously (21,28,54). These conditions have been employed previously in NMR studies of IGF-I and its analogues (55). Two-dimensional nuclear Overhauser enhancement spectroscopy (mixing time of 200 ms) and total correlation spectroscopy (mixing time of 55 ms) spectra were obtained in 10% deuterated acetic acid and 90% D 2 O. Resonance assignment of the 1 H NMR spectrum of native IGF-I was independently obtained based on 15 N-based heteronuclear NMR studies (56); results were consistent with prior studies (21,54). Presumptive assignment of the spectrum of His B5 -IGF-I was obtained by analogy.

RESULTS
Our results are presented in two parts. We first describe the synthesis of Thr B5 -insulin and the effects of the substitution on structure, stability, activity, and cellular biosynthesis. Investigation of the reciprocal IGF-I analogue is then described in relation to native IGF-I and IGF-swap. His B5 -IGF-I exclusively exhibits native disulfide pairing.

Studies of an Insulin Analogue
Characterization of Thr B5 -Insulin-Insulin chain combination recapitulates the native folding of proinsulin (57); the combination of IGF-I-derived A-and B-domain peptides likewise yields disulfide isomers corresponding to native IGF-I and IGFswap (58). Combination of the Thr B5 insulin B-chain with the wild-type A-chain yields a single predominant product with an efficiency 4-fold lower than that of wild-type chain combination. Analysis of the crude chain combination mixture by rp-HPLC revealed several side products (cyclic A-and B-chains and B-chain dimer) but none with the mass of the insulin analogue. This purified product exhibited the predicted mass (5770.4 Da) and was found to be a single component following rp-HPLC using two independent elution co-solvents in either 0.1% trifluoroacetic acid/acetonitrile or 0.1% trifluoroacetic acid/methanol. These HPLC systems readily resolve insulin disulfide isomers (29).
The Thr B5 substitution perturbs the structure of insulin as probed by CD spectroscopy (Fig. 3A) and causes a marked Folding and Function of IGF-I FEBRUARY 12, 2010 • VOLUME 285 • NUMBER 7 reduction in thermodynamic stability as probed by guanidine denaturation at 4°C (Fig. 3B). Application of a two-state model provided estimates of respective free energies of unfolding (⌬G u ) of 4.4 Ϯ 0.1 kcal/mol (wild-type insulin) and 2.4 Ϯ 0.1 kcal/mol (Thr B5 -insulin). The extent of destabilization (⌬⌬G u 2.0 Ϯ 0.2 kcal/mol) is more severe than ordinarily observed in studies of mutant insulins but similar to that previously observed in studies of an Ala B5 analogue (18). Relative to Ala B5 , introduction of a longer side chain and potential hydrogen bond donor and acceptor (the ␤-OH group of Thr) into the B5-related inter-chain crevice does not augment stability. These analogues each exhibit attenuated helix-associated CD features, presumably representing transmitted perturbations in structure or dynamics. The affinity of Thr B5 -insulin for IR (isoform B) is reduced by ϳ5-fold relative to human insulin (Fig. 3C). Wild-type and variant hormone receptor dissociation constants in vitro are 0.06 Ϯ 0.01 and 0.33 Ϯ 0.04 nM, respectively ( Table 2). The cross-binding of Thr B5 -insulin to IGF-1R is reduced by ϳ6-fold relative to that of wild-type insulin (respective dissociation constants were 53.8 Ϯ 5 and 8.7 Ϯ 1.3 nM; Fig. 3D). Within statistical uncertainties, the extent of reduction in affinities to IR and IGF-1R is congruent. The activity of Thr B5 -insulin is at least 2-fold lower than that of an Ala B5 analogue (18) but 4-fold higher than that of non-native disulfide isomers of wild-type sequence (18).
Effects on Proinsulin Biosynthesis-Insulin undergoes a complex pathway of biosynthesis in pancreatic ␤-cells (Fig. 4A). Folding and trafficking of variant proinsulins were evaluated in live HEK293T cells transfected with human preproinsulin cDNA bearing or lacking mutations. Following transfection, cells were pulse-labeled with 35 S-amino acids for 1 h and then chased for 1 h to examine expression, disulfide isomer formation, and secretion of newly synthesized proinsulin (Fig. 4, B and C). Denaturing PAGE (Tris/Tricine-urea-SDS-PAGE) in the absence of reducing agent permitted examination of distinct proinsulin disulfide isomers as formed in the ER. The wild-type construct (Fig. 4B, lanes 3 and 4) gave rise to robust expression, primarily of a fast-migrating band; previous studies have established that this is the native species (46,59). This is efficiently secreted from transfected cells ("C" in Fig. 4) to medium ("M"). Two minor species are also present as slower migrating isomers with mispaired disulfide bonds; these exhibit lower secretion efficiency (Fig. 4B, brackets). Substitution of His B5 by Met or Glu markedly impairs expression (Fig. 4B, lanes  5 and 7) and increases the fraction that forms mispaired disulfide isomers in the ER (Fig. 4B, lanes 5 and 7); moreover, secretion is blocked (lanes 6 and 8). Arg B5 -proinsulin exhibits efficiencies of folding and secretion similar to those of wild-type proinsulin (Fig. 4, lanes 11 and 12).
Strikingly, folding of Thr B5 -proinsulin leads to a product of higher molecular mass (Fig. 4, lane 9, asterisk) that is not secreted (lane 10). The marked shift in electrophoretic mobility might be the result of three possible molecular perturbations as follows: (i) aberrant aggregation of the mutant proinsulin leading to inter-molecular disulfide bridges; (ii) failure of cleavage of the signal peptide leading to expression of a mutant preproinsulin; or (iii) anomalous post-translational modification giv-ing rise to increased molecular mass and decreased mobility. The latter mechanism was suggested by the fortuitous creation of a potential N-linked glycosylation site (Asn B3 -Gln A4 -Thr B5 ) (60) following substitution of His B5 . To distinguish between these possibilities, SDS-PAGE analysis was performed under nonreducing or reducing conditions (Fig. 5A, left-and righthand panels. respectively). On reduction, proinsulin and proinsulin analogues behave as random coil polypeptides, and so their electrophoretic mobilities on denaturing SDS-PAGE reflect molecular mass. Dithiothreitol reduction of wild-type insulin led to the collapse of discrete disulfide isomers (Fig. 5A,  lane 2) to a single band of reduced mobility (lane 7). Dithiothreitol reduction of the Thr B5 -associated set of bands (Fig. 5A, lane A, cellular pathway of insulin biosynthesis. Nascent proinsulin folds as a monomer in the ER (left) wherein the zinc ion concentration is low; in post-Golgi granules proinsulin is processed by cleavage of the connecting peptide to yield mature insulin, and zinc-stabilized hexamers begin to assemble. Zincinsulin crystals are observed in secretory granules. On secretion into the portal circulation (right), hexamers dissociated to yield bioactive insulin monomers. B, pulse-chase studies in HEK293T cells as analyzed under nonreduced conditions (46). Cells were pulse-labeled with 35 S-labeled amino acids for 1 h and chased 1 h. Chase media (M, even-numbered lanes) and cell lysates (C, odd-numbered lanes) were immunoprecipitated with anti-insulin antiserum and analyzed by nonreducing Tris/Tricine-urea-SDS-PAGE. Whereas wildtype human proinsulin (lanes 3 and 4) and Arg B5 -proinsulin (lanes 11 and 12) exhibited efficient folding in the ER and secretion to the medium, Met B5 and Glu B5 variants exhibited impaired folding associated with non-native disulfide isomers (lanes 5 and 7) and a block to secretion (lanes 6 and 8). Thr B5proinsulin forms a larger molecular weight intracellular aggregate (lane 9; asterisk) also with absent secretion (lane 10). An empty vector control (pcDNA3) is provided in lanes 1 and 2. C, control studies of unstable protein variants in transfected HEK293T cells suggest that cellular foldability is not under thermodynamic control. Folding and secretion of a molten two-disulfide analogue (containing paired substitutions Cys A6 3 Ser and Cys A11 3 Ser; lanes 17 and 18) and partially folded analogue (Ile A2 3 Gly; lanes 19 and 20) are similar to wild type (lanes 15 and 16); an empty vector control is provided in lanes 13 and 14. 3) likewise led to a single band (lane 8) whose mobility is proportionately reduced relative to the reduced wild-type polypeptide (lane 7). The relative mobility of Thr B5 -proinsulin-associated polypeptide (Fig. 5A, lane 8) is slower than either that of wild-type proinsulin (lane 7) or that of Asp S24 -preproinsulin (lane 12); the latter is a clinical variant, recently discovered in a patient with neonatal-onset diabetes mellitus (12), that we and others predicted to be defective in signal-peptide processing. Failure to match the mobility of either proinsulin or preproinsulin indicates that anomalous mobility of biosynthetic Thr B5proinsulin is not due to either aberrant inter-molecular disulfide bridges or failure of signal-peptide cleavage.
The expressed intracellular proteins were further characterized for possible N-linked glycosylation in the ER either by digestion of the extracted proteins with PNGase F (which removes N-linked carbohydrates) or by synthesis in cells treated with TM (which inhibits the addition of N-linked glycans (61)). Following digestion with PNGase F, the gel mobility of Thr B5 -proinsulin (Fig. 5A, lanes 4 and 9) reverted to that seen for wild-type proinsulin (lanes 2 and 7). This finding indicates that the anomalous mobility of Thr B5 -proinsulin is due to a novel N-linked glycosylation. Further support for this conclusion was obtained in cells treated with TM in which an additional set of Thr B5 -associated bands (Fig. 5A, lanes 6 and 11) was observed similar to those of wild-type proinsulin (lanes 2  and 7).
To address whether the impaired secretion of Thr B5 -proinsulin was strictly a result of aberrant glycosylation, we per-formed pulse-chase experiments in the absence and presence of TM. Intracellular (C) and secreted fractions of proinsulin (M) were analyzed by SDS-PAGE under reducing conditions (Fig. 5B). The key result was obtained on comparison of secreted proteins after a chase period of 2 h. Whereas wild-type proinsulin is efficiently secreted regardless of the presence of TM (Fig. 5B, lanes 4 and 10), the secretion of Thr B5 -proinsulin is blocked even when produced as an unglycosylated species (Fig. 5B, lane 13).
Because Thr B5 markedly impairs the stability of insulin in vitro (see above), we further compared the intracellular expression of Thr B5proinsulin to that of two partially folded mutant proinsulins as follows: a two-disulfide analogue containing pairwise substitution of Cys A6 and Cys A11 by Ser (and so lacking cystine A6 -A11; Fig. 4C, lanes 17 and 18), and an analogue containing core substitution Ile A2 3 Gly (lanes 19 and 20). Biophysical studies of these analogues have previously been described in the context of an engineered monomer (DKP-insulin) (62). The two-disulfide analogue is a molten globule of marginal stability lacking the A1-A8 ␣-helix; Gly A2 -insulin also exhibits segmental unfolding of this ␣-helix (63)(64)(65)(66). Denaturation studies indicate that the stabilities of these analogues are reduced to an extent similar to or greater than that of Thr B5 -insulin, 9 and yet the corresponding mutant proinsulins are efficiently folded and secreted by HEK293T cells. These results suggest that, even in the absence of aberrant glycosylation, Thr B5 -proinsulin exhibits an altered conformation that fails a quality control checkpoint in the biosynthetic pathway and so undergoes proteosomal degradation (67). Possible molecular mechanisms include increased exposure of nonpolar surfaces (ordinarily hidden in the B5-related crevice) and/or non-native aggregation mediated by such surfaces. These quality control criteria may not be reflected in the in vitro thermodynamic impairment of the monomer in guanidine solutions.

Studies of an IGF-I Analogue
Characterization of His B5 -IGF-I-The two competing ground states of wild-type IGF-I are shown in schematic form in Fig. 6A, panel a. Redox-coupled refolding was conveniently monitored by rp-HPLC. In accord with prior studies (24, 26), 9 Substitution of Ile A2 by Gly causes a decrement in stability with a ⌬⌬G u of Ϫ1.8 Ϯ 0.09 kcal/mol (65). Pairwise substitution of Cys A6 and Cys A11 by Ser leads to a molten partial fold not amenable to two-state thermodynamic modeling. Estimates of its decrement in stability are Ͼ2 kcal/mol (89). IGF-swap elutes just before native IGF-I, whereas intermediates containing one or two disulfide bridges elute after native IGF-I (Fig. 6A, panels b and c). The early eluting isomer was not observed on refolding of His B5 -IGF-I (Fig. 6B, chromatogram  panels d-f). In addition, folding of the variant was slower than that of native IGF-I as probed by appearance of the native elution peak and disappearance of later eluting intermediates. The predominant product was purified and verified as a mono-component by analytical rp-HPLC (see supplemental Fig. S2). The molecular mass of this fraction indicated formation of three disulfide bridges. Physical evidence that the eluted His B5 -IGF-I fraction contains a single pairing scheme (i.e. is not a co-eluted mixture of isomers) and that this pairing is canonical (i.e. is like native IGF-I) was provided by NMR fingerprinting (below). Although we cannot exclude that His B5 -IGF-I-swap was formed at a low level but with an anomalously delayed elution time, the instability or inaccessibility of the putative His B5 isomer was demonstrated by studies of disulfide exchange. Starting with either purified wild-type IGF-I or IGF-swap, an equilibrium distribution of the two forms can readily be catalyzed within 60 min at roomtemperaturebyasub-stoichiometric concentration of ␤-mercaptoethanol (26). This protocol by contrast leads to no change in rp-HPLC elution profile (using 0.1% trifluoroacetic acid/acetonitrile cosolvent) of folded His B5 -IGF-I, which elutes as a single peak (data not shown). After a 24-h incubation, the equilibrium elution of IGF-I was at 22.7 min, whereas IGF-swap eluted at 20.9 min. Under the same conditions, His B5 -IGF-I eluted as a single peak at 21.2 min. To address the possibility that kinetic barriers preclude disulfide exchange at room temperature, the protocol was repeated for 12 h at 37°C with the same outcome. In addition, the single peak status of His B5 -IGF-I was maintained in the presence of ␤-mercaptoethanol as monitored by another co-solvent elution gradient (methanol/trifluoroacetic acid), making unlikely the fortuitous coelution of disulfide isomers. In this co-solvent system the His B5 -IGF-I also eluted as a single peak at 28.5 min.
1 H NMR studies of His B5 -IGF-I were conducted under conditions previously well characterized in studies of wild-type IGF-I and IGF-swap (10% deuterated acetic acid and pH 2.0 at 40°C). Under these conditions, the proteins are monomeric, exhibit well resolved 1 H NMR resonances, and retain insulin-like folds (28). The spectrum of His B5 -IGF-I closely resembles that of native IGF-I (Fig. 7, A and B) and is unrelated to the spectrum of IGF-swap (supplemental Fig. S3 and S4). Aliphatic spin systems (Fig. 7, C and D) and aromatic spin systems (Fig.  7, E and F) exhibit similar patterns of chemical shifts. Published assignments of the wild-type spectrum immediately suggested corresponding assignments in the spectrum of the variant. Small shifts are observed in the chemical shifts of aliphatic side chains in the hydrophobic core (IGF-I residues L14 and I43 in Fig. 7D; canonical positions B15 and A2, respectively); these may be due to either adjustments in core structure or the introduced aromatic ring current of His B5 . Evidence for structural similarity between the core structures of wild-type IGF-I and His B5 -IGF-I was provided by comparison of nuclear Overhauser enhancement spectra (Fig. 8). These include contacts between ␣-helices 1 and 3 (Tyr-60/Leu-14, canonical positions A19 and B15), between ␣-helices 1 and 2 (Tyr-60/Ile-43, positions A19 and A2), and the ␣/␤ B-domain super-secondary structure (Phe-23/Leu-14, positions B24 and B15). These framework residues are conserved among both vertebrate insulin and IGF sequences (supplemental Table S1). No nuclear Overhauser enhancements are observed between the ring of His-4 (His B5 ) and methyl resonances (Fig. 8B, dashed line) in accord with the structure of IGF-I. 10 The crowded nature of this spectra region precludes definitive assignment.
The far-ultraviolet CD spectrum of His B5 -IGF-I (Fig. 9A, filled circles) is also similar to that of native IGF-I (solid line) and distinct from that of IGF-swap (dotted line). The latter exhibits attenuated ␣-helical features consistent with the absence of ␣-helix 2 (IGF residues 42-49; canonical positions A1-A8) in the solution structure of IGF-swap (28). CD-detected guanidine denaturation studies indicate that native IGF-I and His B5 -IGF-I exhibit similar stabilities at 25°C (Fig. 9B, solid  line and circles). Application of a two-state model implies respective free energies 2.9 Ϯ 0.1 kcal/mol (wild type) and 3.1 Ϯ 0.1 kcal/mol (variant). Stabilization by the B5 substitution is at the limit of significance (⌬⌬G u 0.2 Ϯ 0.2 kcal/mol). The corresponding denaturation transition of IGF-swap is more sharply sigmoidal (Fig. 9B, dashed line), leading to a higher estimate of ⌬G u (3.7 Ϯ 0.1 kcal/mol). Direct comparison to native IGF-I to estimate ⌬⌬G u (which would differ in sign from that obtained by studies of disulfide exchange) may be confounded by the difference in respective denatured states in the two guanidine titrations.
Biological Activity of IGF-I Analogue-Functional studies of native IGF-I and His B5 -IGF-I demonstrated that the substitution augments IGF-IR binding (Fig. 9C) in accord with past studies (22). Respective ligand-receptor dissociation constants are 0.040 Ϯ 0.005 nM (wild-type) and 0.022 Ϯ 0.002 nM (variant). Studies of IR demonstrated that the B5 substitution also enhances cross-binding of the IGF-I analogue by 2-fold (Fig.  9D). Respective ligand-IR dissociation constants are 13.9 Ϯ 2.2 nM (wild type) and 5.7 Ϯ 1 nM (variant). Thus, as in reciprocal 10 In the nuclear Overhauser enhancement spectroscopy spectrum of insulin, the C 4 H proton of His B5 exhibits a prominent contact to the methyl groups of Ile A10 ; the corresponding residue is Ser 54 in IGF-I.  (58) demonstrated that isolated A-and B-chain peptides derived from IGF-I undergo chain combination to yield native and isomeric two-chain products analogous to those observed on refolding of IGF-I. These observations first demonstrated that information is missing in the IGF A-or B-domains. Combination of the IGF-I-derived B-chain (29 residues) and insulin A-chain likewise yield two isomeric products as in single-chain chimeric models (68). To test whether residue B5 is sufficient in this model to restore the fidelity of chain combination, we synthesized the His B5 -IGF-I B-chain and attempted chain combination with the insulin A-chain. Yield was reduced 3-fold relative to wild-type insulin chain combination. Unlike reciprocal chain combination of Thr B5 -insulin, which exhibited low yield but no isomers (above), the hybrid reaction gave rise to a major product with three minor isomeric contaminants (each resolved on rp-HPLC with longer elution times than that of the major product and relative peak intensity Ͻ10%). These studies suggest that the differences in folding information between the isolated B-chains of IGF-I and insulin are not confined to residue B5 alone.

DISCUSSION
The insulin-related superfamily provides a model for the evolution and divergence of a structural motif (15, 69 -71). The A-and B-domains encode an ␣-helical fold that is shared by vertebrate insulins, growth factors, and relaxin-related factors. These polypeptide sequences contain five classes of residues as follows: (a) conserved in the superfamily; (b) conserved among insulin, IGF-I, and IGF-II but not only relaxin-related factors; (c) conserved among IGF-I and IGF-II but not more broadly; (d) restricted to one protein type; and (e) not conserved. Invariant in the superfamily is a pattern of six cysteines (supplemental Table S1). Gly A1 -Ile A2 -Val A3 provides a conserved element within vertebrate insulins and IGFs required to bind a cognate family of receptor tyrosine kinases (IR and IGF-1R); this element is not characteristic of relaxin-related factors, which bind to an unrelated class of receptors (G-protein-coupled receptors) (72). The evolutionary history of the insulin superfamily extends to invertebrate insulin-like proteins exhibiting further sequence variation, including the pattern of disulfide bridges (71,73).
In this study, we have investigated reciprocal substitutions in human insulin and IGF-I at position B5. This residue is conserved as His in insulin and as Thr in the IGFs; relaxin-related factors contain a basic residue (Lys or Arg). We discuss our results in relation to general principles of protein folding and biological constraints governing the evolution of the insulin-IGF family. The importance of residue B5 is underscored by the recent discovery of a mutation at this site in human proinsulin causing permanent neonatal-onset diabetes mellitus (12).
Principles of Protein Folding-Protein folding represents a major unsolved problem. Anfinsen and co-workers (74) demonstrated that the native state of a globular protein is encoded by its sequence. Although this view has undergone recent modification (embracing, for example, the cellular roles of chaperones and of "folding enzymes" such as peptidylprolyl isomerase and protein-disulfide isomerase (75)), it is nevertheless believed that the protein structures are thermodynamically (rather than kinetically) determined as the ground state of a multidimensional energy landscape (76). Fundamental to protein biophysics, these ideas have broad biological implication as folding defects underlie diverse human diseases (77,78). IGF-I with its two alternative ground states provides an example of a polypeptide sequence that is missing information.
Oxidative protein folding pertains to a major class of secreted and membrane proteins whose folding is coupled to disulfide pairing in the endoplasmic reticulum (79,80). In vitro, the relative reactivities of thiol groups have long provided site-specific kinetic probes, enabling reaction intermediates to be trapped (81). As demonstrated by Creighton and co-workers (82,83), the time course of formation and disappearance of free cysteines and specific pairing arrangements provides a chemical map of the oxidative folding pathway. An experimental paradigm has been provided by bovine pancreatic trypsin inhibitor (BPTI) (84 -86). The folding of BPTI is remarkable for intermediates that are well populated at neutral pH, exhibit native structures, and face large kinetic barriers to reach the ground state (84 -86). The preferred final step is formation of an external disulfide bridge between solvent-exposed loops.
Like BPTI, IGF-I and proinsulin are small globular proteins containing three disulfide bridges. Profound differences from BPTI are observed. IGF-I and proinsulin are significantly less stable than BPTI (87,88); kinetic barriers among populated two-disulfide species are also lower (63,88). Furthermore, the conformational search is intertwined with formation of disulfide bridges: analogues of IGF-I and insulin exhibit stepwise stabilization of native structural elements with successive disulfide pairing (24,29,89). Oxidative folding of IGF-I and proinsulin thus occurs on successive energy landscapes whose topography is constrained by disulfide pairing (13,88). Evidence for nonrandom initial folding trajectories has been provided in studies of IGF-I, mini-IGF-I, and mini-proinsulin by the trapping of a unique one-disulfide intermediate containing cystine A20 -B19 (55,90). Although several two-disulfide species have been characterized, a slow subsequent step is formation of the solvent-exposed A7-B7 bridge. Diabetes-associated mutations in proinsulin cluster in residues neighboring either cystine A20 -B19 or A7-B7 (12,14).
IGF-I refolding bifurcates to yield two products (24,26). Designated native IGF-I and IGF-swap (26), the two products share the A20 -B19 disulfide bridge. The isomers exhibit near-equal thermodynamic stabilities. Proinsulin and singlechain insulin analogues by contrast refold to form a unique ground state; alternative pairing schemes are metastable and, if present, rearrange to the native state. That the number of products is precisely two demonstrates that a nonrandom folding pathway is encoded but must be "ambiguous" following formation of cystine A20 -B19. In studies of mini-protein models Feng and coworkers (37) obtained evidence that the respective B-domains of proinsulin and IGF-I, in particular His B5 or Thr B5 , are responsible for determining the relative stability of the swapped isomer. The present studies of His B5 -IGF-I supports this conclusion. The B5 substitution in IGF-I selectively destabilizes the non-native isomer. The accumulation of a two-disulfide intermediate in the refolding of His B5 -IGF-I presumably represents a non-native species containing cystines A20 -B19 and A7-A11 (IGF-I residues 18 -61 and 48 -52), ordinarily the precursor of IGF-swap but here an off-pathway intermediate. We speculate that the delayed overall time course of folding reflects the formation and rearrangement of this species.
The yield of insulin chain combination is impaired by the reciprocal substitution His B5 3 Thr. Such impairment stands in contrast to the general robustness of chain combination to substitutions, especially in the C-terminal B-chain segment and N-terminal A-chain segment (65). Sites neighboring Cys B7 define an Achilles' heel; impaired chain combination has also been observed following substitution of Leu B6 , Ile B11 , and Val B12 (41,91), which like B5 are sites of neonatal diabetesassociated mutations (12,14). It is possible that substitutions at  FEBRUARY 12, 2010 • VOLUME 285 • NUMBER 7 these sites, like Thr B5 , markedly perturb the stability of insulin. Synthetic and cell biological studies nonetheless suggest that thermodynamic criteria are insufficient to account for differences in foldability. Pairwise substitution of Cys A7 and Cys B7 by Ser was observed to rescue chain combination otherwise blocked by substitution Gly B8 3 Ala (92). Because in the absence of cystine, A7-B7 insulin exhibits a partial fold of lower stability than that of Ala B8 -insulin (18), such rescue suggests that Ala B8 imposes a kinetic block to pairing of Cys A7 and Cys B7 (92). Pairwise substitution of Cys A6 and Cys A11 by Ala (or Ser) likewise leads to a partial fold of marginal stability. Not only is chain combination preserved, but also in transfected mammalian cells a corresponding variant proinsulin exhibits efficient folding and secretion (63).

Folding and Function of IGF-I
How might His B5 contribute to chain combination and the biosynthesis of proinsulin? A clue is provided by the presence of a basic side chain (Arg or Lys) at this site in relaxin-related factors and the occasional occurrence of Arg among nonmammalian insulins (93). Whereas chain combination fails in the case of Met B5 , near wild-type efficiencies are obtained for Arg B5 (93). We imagine two possible molecular roles for His B5 or Arg B5 in the mechanism of chain combination. First, because crystal structures of insulin and Arg B5 -insulin exhibit analogous hydrogen bonds between a B5 side-chain NH donor (N␦H in His B5 and N⑀H in Arg B5 ) to A-chain carbonyl oxygens, native-like interactions might stabilize an on-pathway folding intermediate whose local structure facilitates pairing of Cys A7 and Cys B7 . Second, the positive charge of Arg B5 and potential positive charge of His B5 may stabilize a thiolate anion at A7 or B7 in the course of disulfide bonding. The latter mechanism would be consistent with the pK a of His B5 , which is shifted from 6.0 to 7.0 in native insulin (93,94). The neonatal diabetes-associated mutation at B5 is Asp (12), whose negative charge would be expected to destabilize a thiolate reaction intermediate.
Our studies of the cellular folding of proinsulin analogues on transfection in human fibroblast-derived HEK293T cells demonstrate that His B5 or Arg B5 support folding and secretion to an equivalent extent. Met B5 and Glu B5 (like Asp B5 , Gln B5 , and Phe B5 in a previous study (18)) impair folding in the ER, leading to increased formation of disulfide isomers and a block to secretion. Expression of Thr B5 -proinsulin leads to fortuitous modification by an N-linked glycan whose secretion is blocked. Experiments employing TM to impair N-linked glycosylation nonetheless demonstrated that secretion of Thr B5 -proinsulin, folded in the absence of such modification, is itself markedly impaired. Despite this block in the secretory pathway, disulfide pairing of Thr B5 -proinsulin (in the presence of absence of glycosylation) is more efficient than that of Ala B5 -, Met B5 -, Glu B5 -, or Asp B5 -proinsulin (present study and see Ref. 18) as indicated by native SDS-PAGE in the absence of reduction (i.e. the ratio of less compact non-native disulfide isomers to the most rapidly migrating band).
We speculate that the ␤-OH group of Thr B5 (unlike the above folding-defective side chains) can in principle donate a hydrogen bond to a putative thiolate intermediate and, once pairing is complete, to the A-domain within the B5-associated crevice but that these interactions are weaker (or more transient) than those of secretion-competent side chains His B5 and Arg B5 . This model is supported by the decreased thermodynamic stability of Thr B5 -insulin. We imagine that conformational fluctuations in Thr B5 -proinsulin expose nonpolar surfaces, leading to recognition of a folding defect by quality control checkpoints in the ER and Golgi apparatus. The blocked secretion of Thr B5 -proinsulin highlights in the breach the puzzling conservation of Thr at this position in IGF-I and IGF-II.
Co-evolution of a Regulatory Network-Why is a secretiondefective residue at B5 invariant among IGF-I and IGF-II? Such a conversation poses a seeming paradox, especially in light of the genetic susceptibility of proinsulin to misfolding-associated mutations. Specification of native IGF-I disulfide pairing in vivo is attributed to specific IGFBPs, present in equimolar proportions. Such partner proteins bind native IGF-I but not IGFswap, thus favoring formation of the native isomer by Le Chatelier's Principle (95). We propose that the availability of IGFBPs as ubiquitous heterodimeric folding partners (33) has enabled IGF-I to diverge from insulin at B5. The extent of divergence is not so marked as to preclude disulfide pairing as in vitro reduced IGF-I successfully refolds to form only 2 of the 15 possible disulfide isomers (native IGF-I and IGF-swap). Discrimination against the 13 other possible combinatoric pairing schemes demonstrates that almost all folding information (but not quite all) is retained in the polypeptide sequence of IGF-I. This subtle defect is repaired on substitution of Thr B5 by His. In this context it is noteworthy that in cellular assays of proinsulin folding, we found that the defect in disulfide pairing associated with Thr B5 is less profound (despite the subsequent block to secretion) than that of clinical mutation of Asp B5 or the other substitutions tested in cell culture. We speculate that IGFBPs enable IGF-I to overcome the block to secretion observed in Thr B5 -proinsulin; formation of a heterodimer enables quality control checkpoints to be satisfied.
Conservation of Thr B5 in IGF-I and IGF-II has presumably been enjoined by a selective advantage. His B5 -IGF-I exhibits a 2-fold increase in binding to both IGF-1R and IR. Thus, threonine subtly attenuates IGF signaling at the expense of uniqueness of folding of the isolated polypeptide. We therefore imagine that IGFBPs (in addition to sequestering circulating IGF-I and their direct signaling activities) have implicitly expanded the space of foldable sequences available to IGFs. Such co-evolution may in general enable the fine-tuning of receptor affinities of hormones and growth factors under physiological selection to coordinate the regulation of development and metabolism. We envisage that variation at B5 in IGFs enabled fine-tuning of receptor affinities without perturbations in receptor selectivity or binding to IGFBPs. Analogous variation in proinsulin was disallowed by impaired secretion and, in the case of a broader set of substitutions at B5, by proteotoxicity due misfolding of the nascent polypeptide in the ␤-cell. Whereas mutation of His B5 by Asp in the human insulin gene leads to neonatal diabetes mellitus because of ␤-cell death (12), we speculate that an analogous Thr B5 substitution might lead to decreased insulin secretion (from the remaining wild-type insulin allele) but would otherwise be well tolerated.
Why could fine-tuning of IGF-I activity, even by a factor of 2, be of biological importance? Factors of 2 can seem negligible in biochemical studies, and yet in vivo such variation may have profound consequences, especially in regulatory control systems. An instructive example in pharmacology was provided by a once-promising insulin analogue under development 20 years ago (96). Just as this study has investigated reciprocal insulin and IGF-I substitutions at position B5, a candidate rapid-acting insulin formulation (97) was proposed in which His B10 was substituted by Asp B10 (98), whose side-chain carboxylate recapitulates a feature of IGF-I (Glu B10 ; position 9 in the polypeptide sequence of IGF-I). The Asp B10 substitution was observed to enhance the stability of insulin (99) and accelerate its absorption after subcutaneous injection (97), both desirable pharmacological properties. This substitution also enhanced binding to the IR and IGF-1R by 2-fold due to increased residence times (98,100). Although seemingly minor or even advantageous, this 2-fold alteration in the receptor-binding properties of Asp B10insulin was associated with an increased incidence of mammary tumors in Sprague-Dawley rats and enhanced mitogenicity in human breast cancer cell lines (96). Human trials were therefore discontinued. By analogy, we suggest that the intrinsic activity of free IGF-I may likewise be under strict biological selection. Fine-tuning of IGF-I by co-evolution of its sequence and IGFBPs is likely to affect both growth rates and base-line risk of tumorigenesis in an organism.
The fine-tuning of IGF-I may extend to the extent of its instability to disulfide exchange. In a pioneering study, Nillson and co-workers (27) demonstrated that under the redox conditions of the Golgi apparatus, secretory granules, and bloodstream, isolated IGF-I undergoes in vitro disulfide rearrangement to form an equilibrium distribution of native and swapped species. Because, to our knowledge, the biological implications of these findings have not been explored, it would be of future interest to extend this study to molecular analysis of the state of IGF-I during cellular biosynthesis. Co-expression or RNA interference knockdown of IGFBPs might be found to modulate the fidelity of disulfide pairing. Nillson and co-workers (27) further speculated that isomerization of free IGF-I in the bloodstream and tissues might provide a mechanism to down-regulate its growth-promoting activity (27). The proposed mechanism thus exploits the instability of free IGF-I and the low activity of IGFswap as a biological defense against excessive mitogenic signaling. To our knowledge, this intriguing hypothesis has not been tested.
Extension to Invertebrate Insulin-like Sequences-The insulin superfamily includes invertebrate insulin-like polypeptides. Although these sequences are more divergent than those of the vertebrate insulin-IGF family, selected NMR structures exhibit insulin-like folds as exemplified by bombyxin (extracted from the silkworm Bombyx mori) (70) and INS-6 (found in the nematode Caenorhabditis elegans) (71). These proteins contain Thr B5 and Arg B5 , respectively. Of the 36 insulin-like genes identified in C. elegans by Ruvkun and co-workers (73), 26 encode a basic residue at B5 and 2 encode Thr. The genome of Drosophila melanogaster encodes 7 insulin-like polypeptides of which 4 contain a basic residue at B5; the remaining sequences contain Thr, Ser, or Met. The Thr B5 -containing protein (designated DILP-2) exhibits respective sequence identities of 35 and 26% with respect to human insulin and IGF-I. Conserved residues include Gly A1 -Ile A2 -Val A3 , part of a key recognition ␣-helix (101) shared by insulin and IGF-I but not critical for folding (65). Interestingly, DILP-2 has been demonstrated to bind to a partner protein in D. melanogaster (102,103). This binding protein, designated IMP-2, is a member of the immunoglobulin superfamily homologous to human IGFBP7. We speculate that, like vertebrate IGFs, folding of DILP-2 may be coupled to coexpression of this or another binding protein. Should the folding properties of human insulin and IGFs generalize to invertebrate insulin-like polypeptides, we would speculate that, on the one hand, autonomous folding requires Arg, Lys, or His at B5 and that, on the other hand, Thr B5 (or other divergent residue at B5) might indicate the presence of a corresponding binding protein.
Concluding Remarks-A major challenge is posed by the problem of how members of the insulin superfamily fold and pass quality control checkpoints en route to secretion. This problem has both biophysical and cell biological dimensions. Its subtlety is highlighted by the implicit role of His B5 in a variant IGF-I to destabilize a competing but unseen fold. Despite decades of investigation, rules governing cellular foldability remain elusive. Indeed, Arvan and co-workers (46) have found that substitutions in proinsulin that are well tolerated in vitro can be associated with disulfide mispairing in cell culture. Furthermore, mini-proinsulin analogues, although highly efficient in in vitro refolding assays (104), can quantitatively misfold in mammalian cell culture to yield a metastable disulfide isomer (presumably analogous to IGF-swap). These observations strongly suggest that cellular folding of proinsulin is under kinetic control, whereas thermodynamic control of IGF folding is imposed, at least at the final steps, by IGFBPs.
This study has demonstrated the value of reciprocal substitutions in insulin and IGF-I as probes of structure and function. These homologous systems provide a model in which kinetic and thermodynamic determinants of protein folding may be dissected. In addition to their biophysical interest, future studies may define a relationship between misfolding and ER stress in the pathogenesis of ␤-cell exhaustion in type II diabetes mellitus.