Molecular basis for insulin fibril assembly

Insulin Segments Form Fibrils and Alter Lag Time of Insulin Fibril Formation.

The rationale behind our experiments is that segments that form fibrils on their own and alter the rate of fibril formation when co-incubated with full-length protein are likely to participate in the spine of full-length fibrils. Based on previous studies (24), there are two segments of insulin that form fibrils in isolation from the rest of the protein; these are VEALYL from the B chain and LYQLEN from the A chain.

To ascertain whether either or both of these segments form the spine of insulin fibrils, we studied their effects on the rate of fibrillation of full-length insulin. Both of these segments are located between the two interchain disulfide bonds of full-length insulin (Fig. 1A). In our first experiments, we tested the effect on insulin fibril formation of nine six-residue segments with sequences spanning the A and B chain segments between the two interchain disulfide bonds of insulin [Supporting Information (SI) Table S1]. None of the nine six-residue segments affected the fibrillation of full-length insulin. These results suggest that the spine of insulin fibrils is longer than six residues.

Searching for the smallest segment of insulin that affects the kinetics of fibrillation of full-length insulin, we synthesized two eight-residue segments: SLYQLENY (A chain) and LVEALYLV (B chain) (Fig. 1A). Both SLYQLENY and LVEALYLV form fibrils in solution (Fig. 1D). As seen in Fig. 1B, the A chain segment SLYQLENY does not affect insulin fibrillation. In contrast, the B chain segment LVEALYLV slows insulin fibrillation when incubated in molar concentrations down to 10 times less than that of insulin (Fig. 1C). The strongest inhibitory effect of LVEALYLV is observed when incubated in equimolar concentrations with insulin. The lag time of fibril formation with LVEALYLV at a 1:1 molar ratio is more than 10 times that of insulin alone (Fig. 1C). In addition, we observed few fibrils in electron micrographs of the sample of insulin with LVEALYLV (1:1) compared with the densely packed fibrils observed in the sample containing only insulin (Fig. 1E). We also observed that LVEALYLV nucleated fibril formation when incubated in molar concentrations 40 times less than insulin (Fig. 1C). Thus, depending on the concentrations used, LVEALYLV either inhibits or nucleates fibril formation.

Given that the six-residue segments do not affect the rate of insulin fibril formation and that the eight-residue segment LVEALYLV does, we wanted to know whether either of the two seven-residue peptides within LVEALYLV affected fibril formation. The two seven-residue peptides LVEALYL and VEALYLV formed fibrillar aggregates (Fig. 2A and Fig. S1A). However, only LVEALYL inhibited fibril formation of full-length insulin (Fig. 2C and Fig. S1B), and there were only sparse fibrils present in the sample of insulin incubated with LVEALYL (Fig. 2B). Also, depending on its molar ratio relative to that of insulin, LVEALYL, like LVEALYLV, either delays or nucleates fibril formation (Fig. 2C).

Structure of LVEALYL.

The segment LVEALYL forms microcrystals diffracting to 1 Å resolution, and we were able to determine its fibril-like structure. Extended strands of LVEALYL pack in register into parallel β-sheets, which run the entire length of the needle crystal (Fig. 3A). Alternating side chains L, E, L, and L extend toward a second, identical β-sheet (Fig. 3B). The same set of side chains extending from the second sheet intermesh with those from the first, forming a dry, highly complementary interface of the type termed “steric zipper” (16). Each pair of sheets forming the dry interface packs against two other identical steric zippers, separated by wet interfaces containing six water molecules (Fig. 3C). The dry interface of the LVEALYL steric zipper buries a larger surface area and displays higher surface complementarity than does the steric zipper of the structure of VEALYL, which we determined earlier (16).

Mass per Unit Length of Insulin Fibrils.

A measurement that tells much about the molecular structure of a fibril is its mass per unit length (MPL), which can be determined by STEM. The STEM observations were made on insulin fibrils grown under the same conditions used in the fibril kinetic assays. The most common MPL values of the narrowest fibrils clustered around a mean of 2.85 ± 0.35 kDa/Å (Fig. 4A). This MPL value of 2.85 ± 0.35 kDa/Å corresponds well with the value of 2.47 kDa/Å expected for two insulin molecules (Mr = 2 × 5808 Da) per 4.7 Å rise of an amyloid fibril (2 × 5.81 kDa/4.7Å = 2.47 kDa/Å). Other common MPL values clustered around two and three times this value, suggesting that these fibrils consist of two and three of the narrowest measured filaments. Also observed was a clustering around an MPL value of about four times that of the narrowest measured filament. As seen in the Fig. 4B, the morphologies of the fibrils with different MPLs are quite similar with nearly equal cross-over distances. For example, the cross-over distances of fibrils with MPL values of 2.85 kDa/Å and 5.43 kDa/Å is ≈1,200 Å. This value was used in building our model of insulin fibrils shown in Fig. 5. This cross-over distance of ≈1,200 Å implies that each layer of the steric zipper of the fibril spine is not only separated by 4.7 Å from the layer below but is also rotated by ≈0.71° ([4.7 Å/2400 Å] × 360°).

Insulin Segments Forming the Spine of the Fibril.

The work presented here shows that the seven-residue B-chain segment LVEALYL can either delay or accelerate insulin fibril formation in a molar ratio–dependent manner. In equimolar ratios with insulin, LVEALYL inhibits fibrillation, and in much lower concentrations than insulin LVEALYL accelerates it. This dual mode of action of the B-chain segment LVEALYL on insulin fibril formation suggests that this segment is important for the formation of the spine of the insulin fibrils. In contrast, segments from the A chain lack this dual mode of action. The experiments represented in Fig. 1B show that the eight-residue A-chain segment SLYQLENY does not affect the rate of insulin fibril formation. Apparently this segment, although bound to the B-chain through two disulfide bonds, is peripheral to the spine. In short, our experiments on fibrillation indicate that the spine of insulin fibrils includes the segment of the B chain between the interchain disulfide bonds, and model building of the fibril was based on this basic observation. In addition, the A chain is covalently bound to the B chain via two interchain disulfide bonds and contributes peripherally to the fibril. The crystal structure of LVEALYL shows how the A chain can be accommodated at the periphery of the spine, as explained below.

Dual Action of LVEALYL on the Rate of Fibrillation.

Our observation that the segment LVEALYL is an effective inhibitor of insulin fibrillation, and yet when present in low molar ratios to insulin can accelerate fibril formation, has precedents. In two separate studies, α₁-antichymotrypsin has been observed to both accelerate and inhibit β-amyloid (Aβ) fibrillation. For example, α₁-antichymotrypsin, when incubated with Aβ peptides at ratios lower than 1:100, accelerates Aβ fibrillation (36). However, when the α₁-antichymotrypsin to Aβ1–40 ratio is raised to 1:10, fibril formation is inhibited (37). Apoliprotein E4 (Apo E4) is another example in which fibrillation of Aβ peptide is both inhibited and accelerated. In contrast with LVEALYL and α₁-antichymotrypsin, the apo E4 dual effect on Aβ fibrillation is reversed: Apo E4 inhibits fibrillation when incubated at concentrations lower than those that accelerate fibril formation (36, 38, 39). The dual effect of action of apo E4 and α₁-antichymotrypsin on fibrillation of Aβ was reported in separate studies. This study shows that a short peptide segment can also nucleate and inhibit fibril formation.

The molecular mechanism of the dual effect is unknown. One possibility is that, at low concentrations, LVEALYL nucleates the spine of full-length insulin fibrils more rapidly than do insulin molecules. This is reasonable because the side chains of the segment LVEALYL are exposed, and hence a small group of molecules can interact with each other by intermeshing their side chains, thereby forming a nucleus. In full-length insulin molecules, there must be conformational changes for the LVEALYL side chains of the segment to be exposed and to interact with each other. Once nuclei are formed, full insulin molecules can add to the growing fibril. At high concentrations, LVEALYL can compete for sites on the nucleus with full insulin molecules and can slow fibril growth.

Model for the Insulin Fibril, Based on the LVEALYL Crystal Structure.

Taken together, the x-ray fiber diffraction pattern of aligned insulin fibrils and MPL measurements suggest important features of fibrils of full-length insulin. The x-ray fiber diffraction pattern reveals that the fibrils consist of β-sheets running parallel to the fibril axis with their β-strands running perpendicular (13). Thus, the 4.7 Å reflection in the x-ray diffraction pattern (Fig. 6A) corresponds to the distance between the strands in the β-sheets. The two broad reflections centered at 11.7 Å and 9.0 Å on the equator (horizontal axis) correspond to the distances between the sheets in the insulin fibril. These two reflections might imply that there are two pairs of sheets with different sheet-to-sheet distances, which form the fibrils. The MPL measurements suggest that there are two insulin molecules per 4.7 Å along the fibril (Fig. 4A).

Based on the findings above, the atomic structure of the B chain segment LVEALYL was used as the molecular basis for the steric zipper spine of the insulin fibril. Insulin fibrils are primarily composed of β-sheets (40), which implies that LVEALYL undergoes a conversion from its native α-helical structure (shown in dark blue in Fig. 5, left panel) into an extended β-strand, as in the crystal structure of LVEALYL (shown in dark blue in Fig. 5, middle panel). In our insulin fibril model, two strands of LVEALYL from two insulin molecules interdigitate to form one layer of the steric zipper spine of the fibril. Thus, in the fibril of full-length insulin, LVEALYL retains its conformation from the crystal structure (Fig. 5, right panel).

The covalent constraints of the disulfide bonds linking the B chain to the A chain (41) force the A chain also to convert to an extended β-strand upon extension of the LVEALYL α-helix of the B chain (Fig. 5, right panel). The crystal packing of LVEALYL molecules also provides a structural template for the extended A chain segment LYQLENY. Thus, this A chain segment adopts the backbone conformation of the β-strands (colored in gray Fig. 5, middle panel) which lies astride the β-strands forming the dry steric zipper interface. These two outer β-sheets of the spine of insulin fibril are formed by two stacks of LYQLENY segments of the A chain (Fig. 5, right panel). The two B chain LVEALYL β-sheets, which form the dry steric zipper interface, lie between the two sheets formed by LYQLENY. Because LYQLENY contains a Tyr residue in the second position, this side chain superimposes on a Tyr from LVEALYL in the crystal structure, preserving the “kissing tyrosine” interaction observed across the wet interface of the crystal of LVEALYL (Fig. 5, middle panel) in the fibril model of full-length insulin. These four β-strands complete the model of the fibril spine.

The remaining segments of the A and B chains outside of the fibril spine were modified from their native structure to fit within the constraint of two molecules per 4.7 Å layer. Furthermore, to be compatible with the 1,200 Å cross-over distance observed in insulin fibrils, each layer of our model is given a left-hand twist of ≈0.71° with respect to the layer below. The result is the fibril model shown in Fig. 5 (right panel).

In summary, the spine of our model contains four β-sheets, the inner pair forming a steric zipper that superimposes on our crystal structure of LVEALYL from the B chain. The outer two β-sheets are formed from the segment LYQLENY of the A chain, also found to contribute to the fibril spine. At the periphery of the model, the N- and C-termini retain the native-like structure of the insulin molecule. A similar model, with a steric zipper spine and native-like structure on the periphery, was proposed for a designed amyloid of ribonuclease A (42).

A comparison of our model with the density of the 3D cryo-EM reconstruction of insulin fibrils, determined by Jimenez et al. (18), is given in Fig. S2. The overall shape of the electron density is captured well in most parts by our model (Fig. S2, right). The parts of the model that do not fit the density map are outside the spine of the fibril and possibly disordered, which could explain why they are not observed in the low-resolution density map. Moreover, the calculated MPL of insulin fibrils, including only the residues from the core of the fibrils, is within experimental error of the value obtained by Jimenez et al. (18) (for more details, see SI Text). Thus, we can conclude that our model agrees with the cryo EM density (18).

Assessment of Our Insulin Fibril Model.

The model has been built to be consistent with several structural and biochemical observations. First, its structure with two insulin molecules per 4.7 Å layer of the fibril is consistent with our MPL measurements (Fig. 4). It was also found that three dimers of insulin comprise the fibril precursors (43). Thus the fibril growth proceeds via stacking of these precursors along the fibril axis, suggesting that insulin fibrils are formed of dimers repeating along the length of the fibrils in agreement with our MPL measurements. Second, the simulated fiber diffraction pattern (Fig. 6B) computed from our model corresponds well to the observed cross-β pattern of oriented insulin fibrils (Fig. 6A). Of note, two equatorial reflections, which arise from sheet-to-sheet interactions, are present in both the simulated and observed diffraction patterns at 11.7 Å and 9.0 Å. (Fig. 6). Third, our previous finding that crystals of LVEALYL can accelerate fibril formation of insulin (16) implies that the crystal structure of the peptide resembles the structural organization of the fibril spine. This is the basis for our use of the structure of LVEALYL as a template for the spine of the full-length insulin fibrils. Fourth, the model is consistent with previous findings (4, 17, 20, 21) and our observation that the B chain is important for fibril formation: the B chain forms fibrils independently and can also delay and accelerate fibril formation. Fifth, our model incorporates the LYQLENY segment of the A chain on the periphery of the spine. This is consistent with finding that segments from the A chain (residues A13–A19) and B chain (residues B9–B19) are protected against proton exchange (25). Sixth, inclusion of the Glu residues of LVEALYL and LYQLEN in the spine of our model may explain the more rapid fibrillation of insulin at pH 2.5, where the Glu is expected to be largely uncharged, than at neutral pH, where it carries a negative charge. Thus our model for the insulin fibril is supported by much structural and biochemical data.

Polymerization Assays.

Insulin was purchased from Sigma-Aldrich. Peptides were purchased from CS Bio and Celtek Bioscience Peptides. Insulin fibrillation assays were performed with 0.25 mM insulin in 50-mM glycine buffer pH 2.5. All reactions were performed with four or more replicates. The progress of the reaction was monitored by ThioflavinT (ThT) fluorescence (for more information, see the SI Text).

Electron Microscopy.

For details on EM, see the SI Text.

Crystallization of LVEALVL.

Crystals of LVEALYL grew in a hanging drop after mixing 2 μl of 1.82-mM peptide with 1 μl crystallization solution containing 20% MPD/0.1 M sodium citrate pH 5.5 at room temperature.

X-Ray Crystallographic Data Collection and Processing.

X-ray diffraction data sets were collected at the Swiss Light Source beamline X10SA, equipped with a MAR CCD detector. Data were collected in 5° wedges at a wavelength of 0.97645 Å using a 5 μm beam diameter. For more details see Table S2 and SI Text.

Preparation of Oriented Samples and X-Ray Diffraction.

For details on preparation of oriented samples and x-ray diffraction, see the SI Text

Model Construction.

The model of the insulin fibril was built directly from the crystal structure of the segment LVEALYL (insulin B chain residues 11–17). Model building was performed with the graphics program O (44). This crude model was energy minimized using the program CNS (45) with van der Waals, electrostatic, and hydrogen bonding terms (46) (for more details, see the SI Text).

Simulation of Fibril Diffraction.

Simulated fibril patterns of fibril models were produced by cylindrical averaging of the single crystal diffraction intensities (for more information, refer to the SI Text).

STEM Sample Preparation and Data Processing.

The samples were prepared at the STEM facility at the Brookhaven National Laboratory (for more information, see the SI Text and Figs. S3 and S4).