Fibril-forming segments of IAPP
To confirm and extend earlier work indicating that the segment of IAPP between residues 23 and 37 is crucial to fibril formation, we used several methods. We began with the 3D profile method to computationally predict segments from IAPP that have high fibril-forming potential (Thompson et al. 2006). The hexameric segments NNFGAI and SSTNVG, spanning residues 21 to 33, are located toward the C terminus of the 37-residue IAPP and score highly with this method (data not shown). The computational predictions that these segments drive fibrillation are reinforced by our experimental finding that while a maltose binding protein (MBP)-IAPP fusion protein forms fibrils under physiological conditions, the MBP–IAPP 1–22 fusion does not (Fig. 1A,B). MALDI-TOF mass spectrometry analysis of purified fibrils confirmed that they contain the full-length MBP–IAPP construct (data not shown).
Figure Figure 1.. IAPP residues 23–37 are necessary for fibril formation of IAPP. (A) The MBP–IAPP construct forms amyloid fibrils as visualized by electron microscopy. The scale bar represents 100 nm. These fibrils were determined to contain the full-length construct by mass spectrometry. (B) The MBP–IAPP 1–22 construct failed to form fibrils. (C) The fibril-forming sequence of human IAPP is compared to that of the nonfibril-forming mouse IAPP, with residue differences noted in red. (D) The effects of residue replacements on the rate of fibrillation of IAPP. Histidine 18 of human IAPP is the only residue difference between human and mouse IAPP in the N-terminal region. This residue difference appears to have no effect on the fibril formation of IAPP, whereas the subtle F23L replacement delays fibrillation. The double mutant (H18R/F23L) shows an even greater lag time, suggesting cooperativity between these sites during fibril formation. The H18R/S28P/S29P triple mutant shows that although IAPP 22–27 may be required for fibril formation, the downstream proline substitutions can prevent conversion to amyloid fibrils. (E) Insulin increases the lag time prior to fibrillation. This affect appears to be slightly less potent with the H18R substitution.
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As a further guide to understand how IAPP forms fibrils, we compared fibril-forming human IAPP to mouse IAPP, which does not fibrillize (Westermark et al. 1990). As shown in Figure 1C, mouse IAPP differs from human IAPP in only six residues, five of which are in the segment 23–29 implicated above in fibril formation. The following fibrillation experiments were performed on an IAPP 8–37 construct, omitting the first seven residues which do not affect the de novo kinetics of fibril formation (Koo and Miranker 2005). Note that these experiments were performed with 1% hexafluoroisopropanol (HFIP). HFIP and other helical promoting agents are known to accelerate the kinetics of IAPP fibril formation (Larson and Miranker 2004; Jayasinghe and Langen 2007). The residue replacements shown in Figure 1D demonstrate that residue 18, the most N-terminal of these six positions, does not affect de novo fibrillation. Insulin is a competitive inhibitor of IAPP fibrillation (Larson and Miranker 2004), and Figure 1E shows that the H18R substitution slightly reduces this insulin-induced inhibition. The conservative F23L substitution increases the lag to fibrillation, supporting a role for phenylalanine in the nucleation of fibril formation (Gazit 2002; Tracz et al. 2004). The H18R/F23L replacements display an even greater lag time, suggesting cooperativity between residues 18 and 23 during fibrillation. The H18R/S28P/S29P substitutions failed to form fibrils in the time frame of our experiment, suggesting that although the human residues within the 22–27 (NFGAIL) segment are required for fibril formation, the proline substitutions can prevent conversion. The H18R/F23L/A25P/I26P substitutions also failed to form fibrils, showing that a construct containing human residues 28–33 (SSTNVG), but not the human residues in the 22–27 segment, also fails to maintain normal human 8–37 fibril formation kinetics. In short, both the 3D profile method and the data of Figure 1 confirm that segment 22–29 (NFGAILSS) is important for fibril formation.
Atomic structures of the amyloid-like segments NNFGAIL and SSTNVG
The atomic structure of IAPP in its fibrillar form is not known. Our attempts to crystallize full-length IAPP in the fibrillar state have been unsuccessful, so we approached this problem by crystallizing hepta- and hexapeptide segments from the C-terminal, fibril-forming portion of IAPP. We selected these segments based both on our 3D profile method and the fibril-forming assays described above. Both NNFGAIL and SSTNVG segments formed amyloid-like fibrils as well as microcrystals, and we determined their atomic structures as shown in Figure 2.
Figure Figure 2.. Structures of the NNFGAIL and SSTNVG amyloid-like segments of IAPP. The NNFGAIL (A) and SSTNVG (B) segments, which abut in IAPP, are capable of forming amyloid-like fibrils (left panels) and microcrystals (right panels), respectively. The scale bars are 100 nm for the electron microscopy images (left panels) and 50 μm for the light microscopy images (right panels). (C) The structure of NNFGAIL, consisting of two close-packed β-sheets, viewed down the sheets. Notice the pronounced bend in the backbone, but not the usual interpenetration of side chains found in the steric zipper. Instead, NNFGAIL displays a dry, main chain–main chain zipper-like interface. Water molecules are shown as yellow spheres, and are outside the intersheet interface in both NNFGAIL and SSTNVG. (D) The structure of SSTNVG, viewed down the β-sheets shows the interdigitated side chains between adjacent β-sheets across a dry, steric zipper interface. (E) Two layers of the NNFGAIL structure, shown as stick molecules, showing the hydrogen bonds between layers. (F) Five layers of the SSTNVG structure viewed from the wet interface. Notice that E and F are 90° from the views of C and D.
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The crystal structure of SSTNVG resembles that of other fibril-forming segments from fibril-forming proteins (Nelson et al. 2005; Sawaya et al. 2007). Each SSTNVG segment forms hydrogen bonds with identical segments 4.8 Å above and below it, in an extended parallel β-sheet. Two such sheets face each other with their protruding side chains interdigitating (Fig. 2D), and extend the whole length of the fibril-like structure in the needle-shaped crystals. The interface between the two sheets is devoid of water; thus SSTNVG forms a Class I steric zipper (Sawaya et al. 2007), characteristic of several segments of other protein segments associated with amyloid and prion diseases. The substantial shape complementarity (Sc = 0.85) and area buried (234 Å2) are also characteristic of other steric zippers (Lawrence and Colman 1993; Sawaya et al. 2007). An unusual feature of this structure is that the side chains of Ser29 residues extend across the interface to form a hydrogen bond with one another, the “kissing serines” seen in Figure 2D.
The structure of the NNFGAIL segment is not a typical steric zipper. It contains a pronounced bend in the backbone facilitated by a Gly in the fourth position. This bend allows the side chain of Asn in the second position to turn inward and to hydrogen bond to the backbone carbonyl of the Gly residue. The structure also lacks the interdigitated side chains of the steric zipper motif. This peptide instead has a tight main chain–main chain interface formed by Phe, Gly, and Ala residues from opposing sheets separated by ∼3 Å (Fig. 2C). This interface has a shape complementarity of 0.90 and an area buried of 230 Å2. The main-chain carbonyl of the Phe is tilted within the backbone and is hydrogen bonding with a neighboring main-chain amide across this dry interface. The Phe side chain adopts a rotamer that favors this main-chain packing. In short, the NNFGAIL segment may form a turn leading into a steric zipper, formed from two sheets made up of SSTNVG segments. This model for full-length IAPP is discussed below.
IAPP fibrillar structure
IAPP forms numerous distinct fibril morphologies, with each polymorph believed to be a consequence of a unique atomic packing of the spine (Goldsbury et al. 1997; Tycko 2004; Kodali and Wetzel 2007). Consistent with this view, we have discovered several overlapping segments of IAPP that each form fibrils and needle-shaped microcrystals typical of steric zipper structures (data not shown) (Sawaya et al. 2007). A steric zipper formed by one of these segments within an IAPP molecule would preclude the formation of a steric zipper within another segment of the same molecule. For example, the R18H single-residue substitution in mouse IAPP renders this construct capable of forming fibrils (Green et al. 2003). This construct retains all five mouse residues in the putative amyloidogenic domain (Fig. 1C) including the three prolines at positions 25, 28, and 29. In this construct, we believe that one of the other segments is forming the zipper spine. Thus, the multiplicity of fibril-forming segments suggests a multiplicity of fibrillar polymorphs of IAPP, as has been observed by others.
In Figure 3 we present an atomic model for the most common IAPP fibril polymorph observed by Goldsbury et al. (1997). This fibril has a width of ∼6.4 nm and crossover distance of 25 nm. Our model is based on our SSTNVG structure, and also incorporates the bent backbone of our structure for the NNFGAIL segment. Each IAPP molecule is in an extended β-conformation with a hairpin turn, and is stacked on the molecule below to form two β-sheets. In our model, SSTNVG segments contributed by pairs of IAPP molecules are interdigitated about a 21 axis, which runs along the axis of the fibril, bisecting the line between the second Ser residues of each SSTNVG segment (Fig. 3A). The facing SSTNVG segments create a steric zipper that extends into the four C-terminal residues of IAPP, which interact with the termination of the NNFGAIL turn of the opposing molecule. This places Phe 23 and Tyr 37 of opposing molecules well within the 10.2 Å threshold established by fluorescence resonance energy transfer (FRET) (Padrick and Miranker 2001). The side chains of the Phe and Tyr residues stack as predicted by Gazit (Azriel and Gazit 2001; Gazit 2002). Thus, the fibril contains four parallel β-sheets, the central two constituting the dry steric zipper (Fig. 3C).
Figure Figure 3.. Model of an IAPP fibril based on our crystal structures of NNFGAIL and SSTNVG. (A) View down the fibril axis (shown by the black dot). Two molecules of IAPP mate in a dry steric zipper around the fibril axis. The segment NNFGAIL, shown in blue in each IAPP molecule, is part of the hairpin turn and the start of the long steric zipper interface. The extension of the central strand, and its mating strand, contain the SSTNVG zipper interface, shown in green in each IAPP molecule. The final four residues of IAPP are modeled to complete the zipper interface. The initial 20 residues of IAPP are modeled to complete the outer strands of the two sheets, and to form the cyclic disulfide bridge near the N terminus. (B) Space-filling representation shows the tight steric zipper interface between residues 23 and 37 on the two IAPP molecules. (C) View down the axis of the fibril with a diameter of 64 Å. The modeled residues are shown in white and the residues corresponding to our crystal structures are shown in blue for NNFGAIL and green for SSTNVG. (D) View perpendicular to the fibril axis of the same fibril with a length of 125 Å (one-quarter of a full turn), showing the 4.8 Å spacing that gives rise to the strong meridional reflection of the fibril diffraction pattern. This model has a mass per unit length and crossover distance that agrees closely with the experimental value for the most commonly observed polymorphic fibril.
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We have chosen the SSTNVG segment as the centerpiece of the model because its intersheet distance in the crystal structure corresponds more closely with the observed equatorial reflections in the fiber diffraction pattern. This packing, observed in the SSTNVG crystal structure, can be extended throughout the 23–37 segment, which is consistent with the FRET data (Padrick and Miranker 2001). We did not choose NNFGAIL for the center of the fibril because the spacing in the crystal structure between NNFGAIL sheets is not in agreement with the fiber diffraction data, and the close main chain packing in NNFGAIL would not allow for extension of side chain packing beyond this segment.
We modeled the N terminus of IAPP (residues 8–17) as a short β-strand on the periphery of the fibril in the absence of other information about its structure. The model was optimized to reduce steric clashes and improve hydrogen-bonding geometry. The model has a diameter of 64 Å and incorporates a left-handed twist of 3.4° per layer, consistent with the observed crossover distance of 25 nm for a fibril of IAPP (Green et al. 2004). The simulated X-ray fiber diffraction pattern of our model is in reasonable agreement with the full-length experimental diffraction pattern of IAPP and in excellent agreement with the fiber diffraction pattern of fibrils composed of IAPP 23–37 (Fig. 4A,B; Supplemental Fig. S1; Sumner Makin and Serpell 2004). Moreover, the calculated mass per unit length of our fibril of 16.3 kDa/nm is in reasonable agreement with the STEM measured values of Goldsbury et al. (1997) of 20 kDa/nm, and of Luca et al. (2007) of 13–19 kDa/nm.
Figure Figure 4.. Comparison of the simulated fibril diffraction patterns of fibril models with the experimental diffraction images of IAPP 23–37. The 23–37 segment represents the steric zipper spine of the fibril and is the segment of highest confidence in the model. Space-filling representations are shown for our models (A,C). The simulated fiber diffraction patterns, obtained by Fourier-transforming these models, are shown on the left halves and the experimental diffraction pattern for the IAPP 23–37 segment are shown on the right halves of B and D. The hallmark 4.78 Å meridional reflection characteristic of the hydrogen bonding axis is observed. The arrows in B indicate equatorial reflections at ∼15 Å (red) and ∼8.5 Å (white) that match between the simulated model and experimental diffraction patterns.
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Recently, two other atomic models have been proposed for IAPP fibrils. Both, like ours, stack IAPP molecules in β-conformation on top of one another, perpendicular to the fibril axis, to form extended β-sheets parallel to the fibril axis. Kajava et al.'s (2005) “serpentine” model is for a protofibril with the narrow cross section of 45–55 Å. It features one IAPP molecule per 4.7 Å layer, folded into three segments, thus producing three β-sheets that run the length of the fibril. Kajava et al. (2005) argue that this model is consistent with the mass-per-unit-length measurements for higher order fibrils. The serpentine model differs from ours also in that its three sheets do not closely interdigitate into one another, as found in the steric zippers of known structure.
A very recent model proposed by Tycko and coworkers was based on distance constraints from solid-state NMR (Luca et al. 2007). The Tycko model is similar to ours in proposing two IAPP molecules per 4.8 Å layer, also organized into four sheets. The principal difference is that the sheets of the Tycko model do not interdigitate as closely as the sheets in our model, based on the SSTNVG structure. The second main difference is in the registration of the two inner sheets. The Tycko model obeys a distance constraint of 6 Å between the side chains of either Asn14 or Tyr15 and Ile26 and Leu27, whereas in our model these two residues are spaced further apart (12 Å). After the recent publication of the Tycko model, we computed an alternative model (Fig. 4C), which is similar to the model of Figure 3 in adhering to our experimental SSTNVG and NNFGAIL structures, but which introduces the Tycko distance constraint. The central steric zipper of the alternative model remains the same, but the two strands within the same molecule no longer align as well as in our original model (as shown in the space-filling representations of Fig. 4A and C). In short, solid-state NMR and X-ray approaches have yielded similar models, which, however, differ in the details of the packing of side chains.