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. 2009 Dec 11;284(50):35259-72.
doi: 10.1074/jbc.M109.046888. Epub 2009 Oct 22.

Crystal structure of a "nonfoldable" insulin: impaired folding efficiency despite native activity

Ming Liu  1 , Zhu-Li Wan, Ying-Chi Chu, Hassan Aladdin, Birgit Klaproth, Meredith Choquette, Qing-Xin Hua, Robert B Mackin, J Sunil Rao, Pierre De Meyts, Panayotis G Katsoyannis, Peter Arvan, Michael A Weiss
Affiliations

Crystal structure of a "nonfoldable" insulin: impaired folding efficiency despite native activity

Ming Liu et al. J Biol Chem. .

Abstract

Protein evolution is constrained by folding efficiency ("foldability") and the implicit threat of toxic misfolding. A model is provided by proinsulin, whose misfolding is associated with beta-cell dysfunction and diabetes mellitus. An insulin analogue containing a subtle core substitution (Leu(A16) --> Val) is biologically active, and its crystal structure recapitulates that of the wild-type protein. As a seeming paradox, however, Val(A16) blocks both insulin chain combination and the in vitro refolding of proinsulin. Disulfide pairing in mammalian cell culture is likewise inefficient, leading to misfolding, endoplasmic reticular stress, and proteosome-mediated degradation. Val(A16) destabilizes the native state and so presumably perturbs a partial fold that directs initial disulfide pairing. Substitutions elsewhere in the core similarly destabilize the native state but, unlike Val(A16), preserve folding efficiency. We propose that Leu(A16) stabilizes nonlocal interactions between nascent alpha-helices in the A- and B-domains to facilitate initial pairing of Cys(A20) and Cys(B19), thus surmounting their wide separation in sequence. Although Val(A16) is likely to destabilize this proto-core, its structural effects are mitigated once folding is achieved. Classical studies of insulin chain combination in vitro have illuminated the impact of off-pathway reactions on the efficiency of native disulfide pairing. The capability of a polypeptide sequence to fold within the endoplasmic reticulum may likewise be influenced by kinetic or thermodynamic partitioning among on- and off-pathway disulfide intermediates. The properties of [Val(A16)]insulin and [Val(A16)]proinsulin demonstrate that essential contributions of conserved residues to folding may be inapparent once the native state is achieved.

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Figures

FIGURE 1.
FIGURE 1.
Proinsulin and its biosynthetic pathway. A, pathway of insulin biosynthesis beginning with preproinsulin (top): signal peptide (gray), B-domain (blue), dibasic BC junction (green), C-domain (black), dibasic CA junction (green), and A-domain (red). In the ER the unfolded prohormone undergoes specific disulfide pairing to yield native proinsulin (middle panels). Cleavage of BC and CA junctions (by prohormone convertases PC1 and PC2 and by carboxypeptidase E) leads to mature insulin and the C-peptide (bottom). B, structural model of insulin-like moiety and disordered connecting peptide (dashed line). The A- and B-domains are shown in red and blue, respectively; the disordered connecting domain is shown in dashed black line. Cystines are labeled in yellow boxes. C, cellular pathway of insulin biosynthesis: nascent proinsulin folds as a monomer in ER (left) wherein zinc-ion concentration is low; in post-Golgi granules proinsulin is processed by cleavage of connecting peptide to yield mature insulin, and zinc-stabilized hexamers begin to assemble. Zinc-insulin crystals are observed in secretory granules. On secretion into the portal circulation (right), hexamers dissociated to yield bioactive insulin monomers. rER, rough endoplasmic reticulum; SRP, signal recognition particle.
FIGURE 2.
FIGURE 2.
Structural environment of LeuA16 in insulin monomer. A, stereo space-filling model showing limited exposure of internal A16 side chain (red) between B-chain (gray, overlying surface) and A-chain (black). The solvent-exposed A7–B7 disulfide bridge is shown in gold (top); internal cystines A6–A11 and A20–B19 are not visible. B, corresponding ribbon model in same orientation showing LeuA16 in relation to TyrA19 (blue) and the internal side chains of IleA2 (black), LeuB11 (gray), and LeuB15 (gray). The A and B main chains are shown as gray and black ribbons, respectively. The three disulfide bridges (labeled at left) are shown as gold spheres. Coordinates were obtained from 2-Zn insulin molecule 1 (Protein Data bank code 4INS).
FIGURE 3.
FIGURE 3.
Biosynthesis and degradation of proinsulin variants in cell culture. A and B, analysis of folding and secretability by Tris-Tricine/urea-SDS-PAGE under nonreducing (A, lanes 1–10) and reducing (B, lanes 1′–10′) conditions. CLA14 cells were transfected to express wild-type proinsulin (pro, lanes 3 and 4, 3′ and 4′) or variants ThrA8 → His (lanes 5 and 6, 5′ and 6′), LeuA16 → Val (lanes 7 and 8, 7′ and 8′), or both HisA8,ValA16 (lanes 9 and 10, 9′ and 10′). Lanes 1 and 2, 1′ and 2′ provide an empty-vector control (con). The A8 substitution enhances overall expression and secretion; secretion of non-native isomers is also increased. At 48 h, cells were pulse-labeled with 35S-labeled amino acids for 1 h and chased for 1 h. Chase media (M) were collected, and cells (C) were lysed; each fraction was immunoprecipitated with anti-insulin antiserum. Prior to transfections, 15 mm isopropyl β-d-1-thiogalactopyranoside was added to induce the expression of ER chaperone ATF6 as described previously (54). C, corresponding pulse-chase studies in HEK293T cells as analyzed under nonreduced conditions (33). For ValA16 mutant (lanes 15 and 16, cellular (C) and medium (M)), a higher fraction of nascent polypeptide migrated as misfolded disulfide isomers relative to wild-type proinsulin (lanes 13 and 14); an empty-vector control is provided in lanes 11 and 12. D, effect of proteosome inhibitor lactacystin (lactacys) on intracellular proinsulin expression in transfected HEK293T cells expressing newly synthesized 35S-pulse-labeled wild-type proinsulin (pro, lane 18), diabetes-associated variant CysA7 → Tyr (Akita mutant (75); designated AK, lanes 19, 21 and 23), or ValA16 variant (lanes 20, 22 and 24); an empty-vector control is provided in lane 17. Cells were chased with or without 20 μm lactacystin for 4 h. Akita and ValA16 variants exhibit enhanced degradation relative to wild type, in each case partially blocked by the proteasome inhibitor. E, control studies of unstable protein variants in transfected HEK293T cells suggest that cellular folding efficiency is uncorrelated with native-state thermodynamic stability. Folding and secretion of a molten two-disulfide analogue (containing paired substitutions CysA6 → Ser and CysA11→Ser; lanes 29 and 30) and partially folded analogue (IleA2 → Gly; lanes 31 and 32) are similar to wild type (lanes 27 and 28); an empty-vector control is provided in lanes 25 and 26. Cells were pulse-labeled with 35S-labeled amino acids for 1 h and chased 1 h. Chase media (M) and cell lysates (C) were immunoprecipitated with anti-insulin antiserum and analyzed by nonreducing Tris-Tricine/urea-SDS-PAGE. F, effect of proteosome inhibitor MG132 on intracellular proinsulin expression in HEK293T cells transfected with wild-type proinsulin (pro, lane 33) or ValA16 variant (lanes 34–36). Cells were pulse labeled for 1 h and lysed without chase, or chased 4 h in the presence (lanes 35) or absence (lane 36) of 20 mm MG132.
FIGURE 4.
FIGURE 4.
CD studies of protein stability. A, guanidine-induced unfolding of insulin analogues as monitored by mean residue ellipticity at 222 nm: wild-type insulin (a, ■), [HisA8,ValA16]insulin (c, ◩), and control analogue [HisA8]insulin (b, formula image). B, corresponding studies of DKP-insulin analogues: parent monomer (d, ●), GlyA2 analogue (e, ▾), and SerA6,SerA11 two-disulfide analogue (f, ▴). Fit to a two-state model by nonlinear least squares regression was in each case characterized by R-factors greater than 0.99, justifying extrapolation of free energies (ΔGu) to zero denaturant concentration (supplemental Table S2) (58). GuHCl, guanidine-HCl.
FIGURE 5.
FIGURE 5.
1H NMR studies of insulin analogues. Aromatic resonances provide probes of dimerization. A, spectra of [AspB10]insulin in the protein concentration range 0.05–0.30 mm at neutral pH and 25 °C (spectra a–c). B, corresponding dimerization of [ValA16,AspB10]insulin (spectra d and e). C, reference spectrum of ValA16 analogue of engineered monomer DKP-insulin (spectrum f).
FIGURE 6.
FIGURE 6.
Crystal structure of [HisA8,ValA16]insulin. A, overview of wild-type T3R3f zinc hexamer. The A-chain is shown in light gray with A16 side chain in red. The B1–B8 segments are shown in blue (T-state) or green (R-state); the remainder of the B-chain is dark gray. Also shown is the coordination of the central zinc ions (magenta) by HisB10 side chains (black). B, corresponding view of variant hexamer. C and D, regions of electron density maps (2Fo − Fc) showing A16 side chain and its environment in T-state protomer (C) and R-state protomer (D).
FIGURE 7.
FIGURE 7.
Comparison of the crystal structure of [HisA8,ValA16]insulin with prior crystal structures. A, superposition of T-state protomer of variant with reference structures (PDB entries 1APH, 1DPH, 1BEN, 1MPJ, 1TRZ, 1TYL, 1TYM, 1RWE, 1G7A, 1ZNI, 2INS, and 4INS). The A- and B-chains of the A16 variant are red and blue (thick sticks), respectively, whereas the A- and B-chains of the reference structures are dark and light gray (thin lines). The B1–B8 segment of the variant is powder blue. B, corresponding alignment of R-state protomers. The A-chain of the variant is shown in red, and the B-chain in green (B1–B8) or blue (B9–B30). The A- and B-chains of the reference structures are shown in dark and light gray (PDB entries 1BEN, 1G7A, 1RWE, 1EV3, 1EV6, 1MPJ, 1TRZ, 1TYL, 1MPJ, 1ZEG, 1ZNJ, and 1ZNI). Structures were aligned with respect to the main-chain atoms of residues A1–A21 and B3–B28.
FIGURE 8.
FIGURE 8.
Structural environment of ValA16 in relation to LeuA16 in control structure of [HisA8]insulin. A, stereo comparison of corresponding side chains in T-state protomers. Position of ValA16 (gray) in T-state protomer in relation to selected residues in A-chain (IleA2 and TyrA19 (red); CysA6 and CysA11 (gold)) and B-chain (PheB1, LeuB11, AlaB14, LeuB15, and ValB18 (green)). [HisA8]Insulin (black; PDB entry 1RWE, molecule 1). B, stereo comparison of corresponding side chains in R-state protomers of [ValA16,HisA8]insulin (color scheme as in A with addition of R-state-specific ligand (phenol; blue and asterisk) and [HisA8]insulin (black; PDB entry 1RWE, molecule 2).
FIGURE 9.
FIGURE 9.
Energy landscape view of proinsulin folding and disulfide pairing. A, formation of successive disulfide bridges may be viewed as enabling a sequence of folding trajectories on a succession of steeper funnel-shaped free-energy landscapes. B, preferred pathway of disulfide pairing begins with cystine A20–B19 (left), whose pairing is directed by a nascent hydrophobic core formed by the central B-domain α-helix (residues B9–B19), part of the C-terminal B-chain β-strand (B24–B26), and part of the C-terminal A-domain α-helix (A16–A20). Alternative pathways mediate formation of successive disulfide bridges (middle panel) en route to the native state (right panel). The mechanism of disulfide pairing is perturbed by clinical mutations associated with misfolding of proinsulin.
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