Solution Structure of Proinsulin

Bacterial Expression

DKP-human proinsulin, containing substitutions His^B10 → Asp, Pro^B28 → Lys, and Lys^B29 → Pro, was expressed in Escherichia coli and purified as described (7). The protein was labeled by fermentation in M9 minimal medium containing [¹³C]glucose and [¹⁵N]ammonium chloride as sole carbon and nitrogen sources. ¹³C,¹⁵N-labeled C-peptide was obtained from DKP-proinsulin by tryptic digestion (12). Receptor binding activity and stability were characterized as described (13).

NMR Spectroscopy

Samples were prepared in H₂O (7% D₂O) in 50 mm sodium phosphate (pH 7.1) and 50 mm NaCl; the protein concentration was 0.3 mm. Spectra, acquired at 25 °C and 700 MHz, were processed with nmrPipe (14) and analyzed with pipp (15). Assignments were obtained from three-dimensional HNCACB, CBCA(CO)NH, C(CO)NH, and H(CCO)NH spectra⁴ and extended by analyses of three-dimensional ¹³C- and ¹⁵N-separated NOE spectroscopy-heteronuclear single-quantum coherence spectra (6). Interproton distance constraints were derived from ¹H-¹H nuclear Overhauser effects (NOEs) (16). To probe subnanosecond fluctuations, hetNOEs were measured as described (8, 17) with a relaxation delay of 6 s. Secondary-structure propensity (SSP) scores were calculated based on ¹³C_α and ¹³C_β chemical shifts (18).

Structure Calculations

Structures were calculated using XPLOR-NIH (19, 20) (see supplemental Methods). Root mean square deviations (r.m.s.d. values) were calculated with respect to both mean coordinates and insulin structures in the Protein Data Bank.

Proinsulin Processing

To probe sequence requirements of prohormone processing, 14 variants of DKP-proinsulin were expressed and purified (7). Human SPC3 (PC1/3) was purified as described (supplemental Methods and supplemental Figs. S1 and S2). Following incubation of enzyme and substrate, junctional cleavages were analyzed by reverse-phase HPLC and mass spectrometry (supplemental Methods and supplemental Fig. S3).

Insulin Moiety

The ¹H-¹⁵N heteronuclear single-quantum coherence spectrum of DKP-proinsulin resembles that of DKP-insulin with the addition of C-domain cross-peaks (supplemental Fig. S4). Further insight was provided by analysis of secondary ¹H_α, ¹³C_α, and ¹³C_β chemical shifts (differences between observed and random-coil values), which are sensitive to secondary structure (22). Trends in such shifts indicate that DKP-proinsulin and DKP-insulin exhibit similar structural elements within the A- and B-domains (Fig. 2A, blue and red; supplemental Table S3). These include N- and C-terminal A-domain α-helices (residues A1–A8 and A12–A20), central B-domain α-helix (B9–B18), and B-domain β-strand (B24–B27). In accord with canonical features of an α-helix, the C-terminal A-domain α-helix and central B-domain α-helix each exhibit large positive secondary ¹³C_α shifts and negative secondary ¹³C_β shifts (Fig. 2A). Helical secondary structure was corroborated by characteristic NOEs (H_N(i, i+1) and H_N(i, i+3) contacts; Ref. 23). The N-terminal A-domain α-helix by contrast exhibits attenuated ¹³C secondary shifts (Fig. 2A) (24) despite retention of helix-related NOEs. Residue-specific SSP scores of DKP-insulin and DKP-proinsulin are illustrated by color-coded ribbon models (Fig. 2B).⁵

C-domain

The BC junction and residues C1–C26 exhibit near random-coil chemical shifts (Fig. 2A; supplemental Table S4) and motional narrowing (supplemental Fig. S5). The absence of medium and long range contacts (despite retention of sequential and intraresidue NOEs) suggests disorder. Near the CA junction, however, residues C27–C31 (sequence ALEGS) exhibit helix-related NOEs. ¹³C_α/β secondary shifts and SSP scores are similar to those of the A1–A8 α-helix (Fig. 2, A and B; supplemental Tables S3 and S4). No segmental substructure was observed in control NMR studies of the isolated C-peptide.

Subnanosecond fluctuations were probed by analysis of {¹H}-¹⁵N hetNOEs (Fig. 2C; supplemental Table S5). Residues B3, B8, B21, B27–B30, and A9 are less well ordered than the remainder of the insulin moiety.⁶ The V-shaped pattern of C-domain hetNOEs (Fig. 2C, black) is a signature of a flexible loop tethered at each end. Residues immediately adjoining the BC and CA junctions (including the dibasic cleavage sites) exhibit hetNOE values lower than those of adjoining residues in the insulin moiety in accord with their motional narrowing and absence of non-local interproton contacts.

Structure

A DG/SA ensemble (Fig. 3A) was calculated based on 939 distance restraints and 97 dihedral angular restraints (supplemental Table S6). The number of restraints per residue in the insulin moiety (A1–A21 and B1–B28) and C-domain nascent α-helix (C27–C31) was 14. There were no distance violations >0.3 Å and no dihedral angle violations >5°. r.m.s.d. values within the insulin moiety are 0.24 Å (main chain) atoms and 0.89 Å (all heavy atoms); the C-domain α-helix exhibits segmental precisions 0.47 Å (main chain) and 0.91 Å (all heavy atoms) (supplemental Table S6). The A- and B-domains closely resemble the cognate chains of crystallographic T-state protomers (supplemental Fig. S6 and supplemental Table S7) (4). The structure of the C-domain is not well defined. The position of its nascent α-helix appears uncorrelated with that of the insulin moiety (Fig. 3B). Flexible tethering of the C-domain helix to the insulin moiety is shown in schematic form in Fig. 3C (dashed lines); a representative ribbon model is shown in Fig. 3D.

SPC3 Cleavage

To establish a model of prohormone processing, we investigated sequence determinants of cleavage by SPC3. Variant C-domains were introduced within DKP-proinsulin and tested as substrates (supplemental Table S8). Each variant retained native dibasic sites (Arg³¹-Arg³² and Lys⁶⁴-Arg⁶⁵; respective positions C1–C2 and C34–C35). These sites exhibit complementary structural contexts within a flexible extended strand (BC) or flanked by α-helices (CA). SPC3 cleaves the BC junction with higher efficiency than the CA junction (9). An HPLC/mass spectrometry assay enabled independent assessment of BC and CA cleavage.

Major determinants of substrate recognition by subtilisin/Kex-related proteases are mediated by residues N-terminal to the scissile bond (9). Surprisingly, however, deletion of residues C-terminal to the BC junction (residues C3–C6; sequence EAED) completely blocked BC cleavage; CA cleavage was impaired by less than 2-fold. The deletion shifted uncharged residues to positions C3–C6 (LQVG). Impaired BC cleavage is unlikely to be due to foreshortening of the C-domain as an extensive deletion (ΔC8–C32; lacking residues 38–62) impaired cleavage of either junction by <2-fold in accord with past studies (25). Deletions ΔC3 and ΔC3–C4 (leaving adjoining sequences AEDL and EDLQ) impaired BC cleavage by ∼4-fold. By contrast, activity was regained with successive deletion ΔC3–C5 (adjoining sequence DLQV). These results suggested that the P2′ residue (underlined above) contributes to SPC3 site selection. Indeed, a single acidic substitution at this position (Ala^C4 → Glu; position 34) completely blocks BC cleavage. Ala substitutions at positions P1′, P3′, or P4′ (positions 33, 35, or 36) are by contrast well tolerated (supplemental Fig. S7A). The specific structural role of Ala^C4 in the SPC3-proinsulin complex may enhance substrate binding as its substitution by Ser (in the context of HAED derived from glicentin; supplemental Table S8) likewise impairs BC cleavage (supplemental Fig. S7B). These findings suggest that the structural mechanism of prohormone processing has constrained C-domain sequences independently of its role in nascent protein folding.

Dynamics of Proinsulin

{¹H}-¹⁵N hetNOEs provide a sensitive probe for subnanosecond fluctuations. Such studies revealed a V-shaped pattern in the C-domain, indicating progressive flexibility from its tethered ends. Although the isolated C-peptide is a random coil (supplemental Table S4), the intact C-domain contains a short α-helix within its C-terminal subsegment. Its position is likely to be poorly correlated with that of the insulin moiety. {¹H}-¹⁵N hetNOE-detected sites of flexibility within the insulin moiety are consistent with patterns of crystallographic B-factors (4).

¹³C NMR resonance assignment enabled calculation of residue-specific SSP scores (18). This score predicts at each residue the percentage of folded conformers (α-helix or β-strand). Limiting SSP scores of 1 or −1 imply formation of a fully formed α-helix or β-strand, respectively. In DKP-proinsulin, the mean segment-specific SSP scores are 18% (N-terminal A-domain segment), 77% (C-terminal A-domain α-helix), 100% (central B-domain α-helix), and 27% (nascent C-domain α-helix). The similar pattern in DKP-insulin (Fig. 2B) (12) implies that the C-domain does not “tighten” the secondary structure of the insulin moiety. Segmental SSP scores of the A1–A8 A-domain α-helix and C-domain α-helix imply conformational fluctuations (leading to averaging of chemical shifts; Ref. 24) despite maintenance of helix-related NOEs. Because A1–A8 hetNOE values are unremarkable, the time scale of such fluctuations must be longer than the rotational correlation time of the protein in accord with previous observations of conformational broadening on the millisecond time scale (26). Further characterization of such motions may be obtained by analysis of rotating-frame spin relaxation spectroscopy (27).

Biological Implications

Conversion of proinsulin to insulin occurs in the course of trafficking through the trans-Golgi network (1). C-peptide excision requires cleavage at Arg³¹-Arg³² (positions C1 and C2; the BC junction) and Lys⁶⁴-Arg⁶⁴ (positions C34 and C35; the CA junction). These cleavages are effected by prohormone convertases SPC3 (PC1/PC3) and SPC2 (PC2), members of a family of calcium-dependent endoproteases that process a wide variety of precursor proteins (9). The active sites of such enzymes accommodate extended and flexible peptide substrates as observed at the BC junction. Because excision of the C-domain occurs after folding of proinsulin, however, formation of enzyme-substrate complexes may require a conformational change at the CA junction to expose an extended peptide conformation (28). The attenuated SSP profile of the A1–A8 segment, evidence of a molten conformation, implies that kinetic and thermodynamic barriers to such distortion may be low. The dibasic residues at the CA junction are themselves flexible. The nascent C-domain α-helix would not in itself be expected to interfere with formation of an enzyme-substrate complex.

The molten character of the A1–A8 α-helix may reflect the anomalous presence of three β-branched residues (sequence GIVEQCCT; β-branched residues, underlined). Ile^A2, ordinarily buried in the core, must be exposed for both prohormone processing and binding of the mature hormone to the insulin receptor (29, 30). Flexibility of the N-terminal A-domain segment may also facilitate its disulfide-coupled folding in the β-cell by reducing kinetic barriers to pairing of cysteines A7–B7 and A6–A11. Oxidative folding of proinsulin proceeds through a defined series of intermediates (31). Pairwise substitution of cysteines A6–A11 by Ala or Ser leads to segmental unfolding of the A1–A11 segment (32), and more marked flexibility occurs on pairwise substitution of cysteine A7–B7 (33). Flexibility of the A1–A8 segment in protein-folding intermediates may facilitate transient deprotonation of thiolate moieties, their alignment for disulfide pairing, and interactions with protein disulfide isomerase (34). In the future, probes of protein dynamics (such as {¹H}-¹⁵N hetNOEs and ¹³C CSI and SSP scores) may provide insight into the biophysical properties of populated disulfide intermediates (32) and perturbations caused by diabetes-associated mutations (10, 11).

A general mechanism of disease is mediated by proteotoxicity, ranging from aberrant intracellular aggregation of folding intermediates to formation of extracellular fibrils. Such pathological processes are exemplified by the toxic misfolding of insulin and proinsulin (31). Due to the presence of the C domain, proinsulin is markedly less susceptible to fibrillation than is insulin (35). We suggest that despite its flexibility, the C-domain sequence has evolved to minimize its propensity to fold or misfold. The extreme resistance of the isolated C-peptide to fibrillation (35), which is otherwise a universal property of polypeptides, highlights the special nature of such a non-folding sequence.

Concluding Remarks

The present study has demonstrated the utility of heteronuclear NMR spectroscopy in combination with protein engineering to investigate a protein refractory to crystallization. DKP-proinsulin provides a tractable model for both structural analysis and studies of prohormone processing. Although the organized insulin moiety foreshadows the structure of the mature hormone, its flexible tethering by the C-domain facilitates nascent folding. The further characterization of variant proinsulins may enable molecular rules governing dibasic target site selection to be deciphered. The solution structure of DKP-proinsulin thus provides a foundation for analysis of prohormone processing and comparative studies of non-foldable variants associated with neonatal diabetes mellitus.