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New Model for Crystalline Polyglutamine Assemblies and Their Connection with Amyloid Fibrils Pawel Sikorski*,†,‡ and Edward Atkins‡,§ Department of Physics, The Norwegian University of Science and Technology, NTNU, NO-7491 Trondheim, Norway, Physics Department, University of Bristol, Tyndall Avenue, Bristol BS8 1TL, United Kingdom, and Department of Polymer Science and Engineering, University of Amherst, Massachusetts 01003-9263 Received September 9, 2004; Revised Manuscript Received October 11, 2004
Based on the interpretation of X-ray diffraction data reported for crystals of the poly-L-glutamine-rich 19peptide D2Q15K2, Perutz et al. (Proc. Natl. Acad. Sci. USA 2002, 99, 5591-5595) proposed that hollow, water-filled nanotubes are the basic structural motif of amyloid fibers. We are able to offer an alternative interpretation for the same X-ray diffraction data. Our proposed structure consists of β-sheets, limited in size in the chain direction that stack at an intersheet distance of 0.83 nm to form cross-β crystallites. The β-sheets are composed of individual D2Q15K2 molecules hydrogen bonding together in the a direction. The relatively linear interchain amide hydrogen bonds in this growth direction occur at two sites: (i) between neighboring backbone amides and (ii) between adjacent (glutamine) side chain amides decorating both surfaces of the β sheet. The polyQ sub-lattice unit cell is orthorhombic with parameters a )0.950 nm, b ) 1.660 nm, and c ) 0.695 nm; contains two β-sheet segments; and has a calculated density of 1.54 g cm-3. A key ingredient in the proposed structure is the locking of the Q side chains by hydrogen bonding, which allow high-density packing. In addition, there is evidence suggesting that the D2Q15K2 molecules adopt a oncefolded hairpin conformation. Introduction and Background Expansion of the glutamine segments in unrelated proteins causes at least nine progressive neurodegenerative disorders, including Huntington’s disease. Currently, the exact mechanism of the neurotoxicity is not clear, though the ability of the proteins with elongated Q segments to form insoluble stable aggregates is one of the possible explanations.1 Therefore, the aggregating behavior of proteins with elongated Q segments and the structures of the aggregates formed are important in understanding the role that polyQ segments play in neurodegenerative disorders. Recently, Perutz et al.2 proposed that the structures of amyloid fibers are hollow, water-filled nanotubes with an external diameter of 3.1 nm and internal core diameter of 1.2 nm. They suggested that the nanotube walls are fabricated from protein chains curving to form a slowly advancing helix (pitch angle around 4°) with approximately 20 amino acid units per turn and successive turns hydrogen-bonded together via both the main chain and side chain amide units. The model is based on the interpretation and analysis of X-ray diffraction patterns obtained from crystalline fibers and films of a 19-amino acid polypeptide with the sequence D2Q15K2 and influenced, presumably, by electron microscope images of twisting fibrils from a heterogeneous 22-peptide fragment * To whom correspondence should be addressed. E-mail: pawel.sikorski@ phys.ntnu.no. Phone: +4773598393. † The Norwegian University of Science and Technology. ‡ University of Bristol. § University of Amherst.
of a sup35 yeast prion protein.2 Proposed nanotubes, or variations and combinations of them, are argued, to be the basis of the protein fibers of various fragments of Alzheimer’s extracellular Aβ plaques, synuclein deposits in Parkinson’s disease, human transthyretin, and Huntington’s disease. The X-ray diffraction diagram, on which the model is based, is reproduced in Figure 1a from a paper by Perutz et al.2We believe an entirely different protein structure can account for the diffraction data shown in Figure 1a. In this manuscript, we will explain the basic features of our proposed model, utilizing published X-ray diffraction data.2 The important aspects of the structure will then be discussed. Finally, we will touch on what we believe to be some of the flaws in the paper by Perutz et al.2, but we will not, in this contribution, engage ourselves in a systematic scrutiny of the original model or undertake detailed interrogation of the original interpretation of the X-ray diffraction data. As shown by the recent study of the SOD1 mutant proteins,3 variations on the water filled nanotubes concept are a possible contender to describe the structure of some amyloids. Our analysis of the diffraction data from D2Q15K2 peptide show, however, that this type of structure is not formed by high-glutamine (highQ) peptides. Instead, a compact and highly ordered cross-β chain organization is observed. Due to distinctive differences between typical X-ray fiber diffraction pattern observed for amyloids and the diffraction pattern observed for D2Q15K2 peptide assemblies, we do not believe that the proposed model offers a general solution for the structure of amyloids. However, it
10.1021/bm0494388 CCC: $30.25 © 2005 American Chemical Society Published on Web 12/04/2004
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Figure 1. (a) Wide-angle X-ray diffraction pattern, (wavelength Cu KR) reproduced from ref 2. The incident beam is parallel to the surface of a film (horizontal) obtained on drying down an aqueous solution of the D2Q15K2 oligopeptide. (b) Schematic diagram of the texture of the polyglutamine crystallites. Dotted lines represent hydrogen bonds; random rotation of ribbons about the chain c axis. (c,d) Calculated X-ray diffraction patterns for the proposed crystal structure based on (c) apβ-sheets and (d) pβ-sheets. The main diffraction arcs are indexed. These patterns can be compared with the experimental pattern shown in (a). In the top half of (c) and whole of (d) the crystal size (csize) in the chain c axis is 7 nm, the length of a nonfolded D2Q15K2 molecule and some line broadening of the 002 is evident. When the (csize) is reduced to 3 nm, equivalent to the once-folded hairpin structure, the line broadening of the 002 naturally increases (see lower half of (c)) and is commensurate with that observed experimentally.
shows that the basis on which helical and universal model for structure of amyloids was proposed is not correct. Protein β-Sheet and Cross-β Structures. In the protein β structure,4 relatively extended polypeptide chains are in a 2-fold helical conformation (21) with an axial advance per peptide unit of 0.35 nm, i.e., with a crystallographic c-repeat of 0.70 nm.4-6 The amide units in such a polypeptide chain conformation can hydrogen bond in an orchestrated and repetitive fashion to form self-sustaining hydrogen-bonded β-sheets.4-6 The average hydrogen bond direction (a axis) is orthogonal to the chain axis and the adjacent chains can be aligned antiparallel (apβ-sheet) or parallel (pβ-sheet). The interchain, intrasheet distance is 0.47 nm, and this repetitive distance would be indexed as the 200 diffraction signal for an antiparallel chain arrangement, and 100 for a parallel chain arrangement. In β-sheets, the amino acid side groups are directed orthogonal to the sheet plane (ac plane) and systematically decorate both surfaces. The intersheet stacking distance (b axis) is controlled by the size of the amino acid side groups and can vary from 0.37 nm for polyglycine to 1.4 nm as reported for the calcium salts of polyglutamic acid.7
There is an inherent tendency for adjacent sheets to slip relative to each other by around 0.47/2 nm (so-called quarterstagger position7) in the a direction. If the a axis slip is recuperative the unit cell is orthorhombic and the b value is twice the average intersheet distance. If the chain (c axis) direction is parallel to long axis of the crystal, the arrangement is known as the β-sheet structure.4-6 Such an architecture is found in the naturally occurring β-silks from silk worms and spiders. However, if the chain axis is orthogonal to the axis of the crystal, which is instead elongated in the hydrogen-bond direction (a axis), we have the cross-β structure. Rudall8-10 established that the cross-β structure could be obtained in a number ways by denaturing proteins and also discovered it occurred naturally, for example in the insect silk of the egg stalks of the green lace-wing fly Chrysopa.11 He suggested that the cross-β structure was created by either (i) long protein chains regularly folding via hairpin-like turns or (ii) short peptides associating side-by-side. In both cases, the crystals are elongated in the a direction. Amyloids, which are in most cases formed by short peptides and are elongated
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Crystal Structure of Polyglutamine Assemblies Table 1. Miller Indices (hkl), Estimated Relative Intensities (Iobs) and Measured and Calculated d Spacings
dobs [nm] a
Iobsb
h
k
l
dcal [nm]c
0.830 0.516 0.475 0.45d 0.415 0.38d
m, sh vw, br s, sh vw, sh s, sh w, br
0.36 0.32 0.277
w, br w, br w, s
0 0 2 2 0 2 0 0 0 0
2 2 0 1 4 0 4 0 2 6
0 1 0 0 0 1 1 2 2 0
0.830 0.533 0.475 0.457 0.415 0.392 0.356 0.348 0.321 0.277
a Observed d spacings as included in Figure 4 in ref 2. b s ) strong; m ) medium; w ) weak; vw ) very weak; sh ) sharp; br ) braod. c dhkl has been calculated based on a orthorhombic unit cell with a ) 0.95 nm, b ) 1.66 and c ) 0.695 nm. d This diffraction signal has not been labeled on the original figure, however it is clearly visible and has been measured on the reproduced X-ray diffraction pattern.
along the hydrogen-bonding direction, exhibit diffraction fingerprint that emanates from the cross-β structure.12-14 It is important to note that this X-ray fingerprint (but not the structure itself) might change if the sample is prepared in a form of a thin film, rather than in the form of the usual fiber.15-20 Results Structural Model for Polypeptide D2Q15K2 Crystals. Sublattice Unit Cell. The wide-angle X-ray diffraction pattern shown in Figure 1a was obtained from a film prepared by drying down an aqueous solution of D2Q15K2; the incident X-ray beam is parallel to the film surface, and the film normal is vertical. On the equator, there are a series of sharp diffraction signals; the most prominent, at a d spacing of 0.475 nm, can be associated directly with the characteristic β-sheet interchain repeat in the hydrogen bond direction (a axis). In the first instance, we will envision an apβ-sheet and therefore choose a ) 0.950 nm. The innermost equatorial diffraction arc is at a d spacing of 0.830 nm and two additional orders occur at 0.415 and 0.277 nm. We associate the 0.830 nm diffraction signal with the intersheet stacking distance, but because of anticipated a axis recuperative slippage between contiguous sheets, as mentioned above and also discussed again later, we shall double this value to give the unit cell b parameter. All observed diffraction signals index on an orthorhombic unit cell with parameters a )0.950 nm, b ) 1.660 nm, and c ) 0.695 nm, and the measured and calculated d spacings, together with the indexing, are given in Table 1. One would expect to find the 002 diffraction signal on the meridian at a spacing of 0.348 nm. Experimentally, a broad meridional arc is reported centered at a d spacing of 0.360 nm. This 3% difference is a puzzle. Our c ) 0.695 nm value (estimated errors ≈(1%) is central in the range (0.690-0.700 nm) reported for β structures.7 At this stage, we are reluctant to stretch our β conformation by 3% to make c ) 0.720 nm until we consider in more detail the diffraction from the whole D2Q15K2 molecule in case there are other diffraction effects that can perturb the c value slightly. It is not a sufficient discrepancy to affect the calculations to the resolution intend in this analysis. A second
(weaker) meridional arc is reported, centered at a d spacing of 0.320 nm. This diffraction signal could emanate from the overlapping tails of the 102 (0.326 nm) or possibly the 022 (0.322 nm) or a combination of both. The calculated density for this sub-lattice based on polyQ is 1.54 g cm-3 and compares with the calculated values of 1.37 and 1.45 g cm-3 for polyA and polyAG, respectively.7 The reason for this higher calculated density in polyQ will be discussed later. Other Diffraction Features and Texture. The equator represents the hk0-reciprocal plane and there is cylindrical averaging about the directionally coincident c,c* axis (meridian), as illustrated in Figure 1b. The relative sharpness of the 0.475 nm diffraction signal suggests that the crystals are long in the hydrogen bond or a direction. The broadness of diffraction signals incorporating a l index suggests a relatively short coherence length in the chain or c direction; a feature which can be anticipate for short chain molecules. An interesting aspect in the X-ray diffraction pattern of D2Q15K2 is the relative sharpness of the series of 0k0 (e.g., the 020 at 0.830 nm) diffraction signals the emanate from the intersheet stacking; usually the 0k0 signals in protein β sheet structures are relatively broad due to limited crystal growth in this direction and the less demanding van der Waals interactions. In this case, the localized intersheet interaction is more precise and exhibits sustained long range order in the b direction. This intersheet interaction has to relate to the side group engagements and we shall discuss this facet in more detail later; indeed it will be an important feature in proposed structure for D2Q15K2. The experimental data from D2Q15K2 suggest that the entities that have crystallized or self-assembled are thin (∼7 nm, the maximum length of the 19-peptide D2Q15K2 molecule) lamellae (size along a and b axes much greater than the size along c axis). Many of these lamellae are arranged within the specimen film with random azimuthal orientation about the lamellar normals that are themselves collectively approximately parallel to the film surface normal. Now that the texture and the sub-lattice unit cell has been established, we can proceed to investigate possible structures and test them more rigorously against the X-ray diffraction data including relative intensities. Modeling. The software packages Cerius2 and InsightII (Accelrys) were used in structural modeling and X-ray diffraction simulations. Care was taken to ensure that the models were stereochemically sound and that the calculated diffraction patterns were in good agreement with the experimental data. In the computer-generated X-ray diffraction patterns, the realistic temperature factor (B ) 15 Å2) and degree of arcing were chosen to match the experimental X-ray diffraction pattern as closely as possible. Figure 2a shows a model of the D2Q15K2 peptide in the β-conformation with the side groups in a conformation that would allow stacking to form a crystal. In our structural modeling, the D and K amino acids were considered uncharged and with no associated cations. To the resolution level we shall take the analysis, this should not be a problem. With respect to the dominating Q central core, the left-hand terminal Ds demand marginally less space and the righthand terminal Ks need slightly more space, respectively.
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Figure 2. Computer-generated views, parallel to a axis (hydrogenbonded direction), of space-filling models for, (a) the 19-peptide D2Q15K2 molecule in the β-conformation and (b) the 22-peptide (H2NDDNNQQNYQQYSQNGNQQQGKK-COOH) sup35 fragment of a yeast prion protein in the β conformation. Color code: red, oxygen; blue, nitrogen; gray, carbon; white, hydrogen.
Thus in the first instance at least, we will focus on finding an appropriate structure for a polyQ sub-lattice. Figure 3a,b shows a view, parallel to the a axis, of a Q-sheet segment stacking along the b axis. Using molecular models, it was found that the closest the sheets can possibly stack without causing overlaps between side chain atoms is 0.760 nm. In reality, the experimental value is 9% greater at 0.830 nm as illustrated for two sheets in the space-filling mode in Figure 3a. This value (or within 1.2%) has been reported for other highQ polypetides, e.g. Krull et al.,21 and so it would appear that 0.830 nm is an optimum polyQ stacking value. For additional clarity, a ball-and-stick model of four sheets is shown in Figure 3b. In our proposed model, the Q side chain amides hydrogen bond together in concert running along the adirection as shown in Figure 3c,d each acting as both a donor and acceptor and systematically covering both surfaces of the β-sheet. Together with the inherent hydrogen bonds between the polyQ backbone, this model makes for a robust structure indeed. DeVelopment of the Proposed Model and Its Relationship to the Experimental X-ray Diffraction Data. Using the β-sheet model shown in Figure 3c, the sheets were stacked at 0.830 nm with recuperative 0.25a slip along the a axis, i.e., the standard quarter-stagger position described above. The best match with the relative intensities of the observed diffraction signals was obtained by increasing the recuperative a axis slip to 0.6a, as illustrated in Figure 3e. This particular a axis intersheet slip is able to occur in this polyQ structure because the side chains adopt an approximate planar geometry and lie in the ab plane with their amide units both donating and accepting hydrogen bonds (see Figure 3e). In order for the side chain amide units to hydrogen bond in this fashion, successive N-CR-Cβ-Cγ side chain torsion angles need to have values of +69° and -113°, respectively. Koetzle et al.22 found that the N-CR-Cβ-Cγ torsion angle in L-glutamine single crystals is 66°; thus, to enable the proposed continuous hydrogen bonding scheme to occur, successive side chains need to flip by 180°. Molecular modeling calculations showed that the potential energy difference between these two conformations is 12 kJ mol-1. Consequently, the total energy penalty is 6 kJ mol-1 since only 50% of the side chains need to be rotated. This value
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is significantly less than the potential energy benefit of 3442 kJ mol-1 23,24 derived by the formation of two extra stabilizing side chain interamide hydrogen bonds. This structure has no stereochemical clashes between atoms and is in a localized potential energy minimum. The calculated (Cerius2) diffraction pattern for the structure is shown in Figure 1c. This computer-generated diffraction pattern includes the appropriate intensity correction factors; the degree of arcing has been chosen to match that shown in Figure 1a. The fit between the X-ray patterns shown in Figures 1a and 1c is sufficiently good, in our judgment, to support the basic correctness of this β-sheet structure. Variations and Other Factors. (1) So far only apβ-sheets (antiparallel) have been considered. As discussed in the Introduction, parallel pβ-sheets are also possible. We therefore repeated the calculations using the pβ-sheets model, and the diffraction pattern is shown in Figure 1d. A comparison of parts c and d of Figure 1 highlights the fact that there is little difference between the two structures in terms of X-ray scattering, other than technically altering the Miller indices. In terms of hydrogen bonding interaction, the pβ-sheet structure is less satisfactory. This is illustrated in Figure 3d for the pβ-sheet; the central layer of backbone interchain hydrogen bonds is no longer close to linearity. (2) It needs to be remembered that the Fourier transform calculation is based on a lattice of polyQ, whereas the experimental X-ray diffraction pattern emanates from the D2Q15K2 molecule. The total potential scattering from the side chain atoms in the terminal D2 and K2 amino acids in the D2Q15K2 molecule is slightly under 10% and therefore cannot substantially alter the calculations used in the generation of Figure 1c. However, to double-check, we placed the four side chains of the D2 and K2 pairs into geometrically realistic conformations, commensurate with the polyQ sublattice, and recalculated the diffraction pattern for the whole D2Q15K2 molecule; only slight variations in relative intensity occurred. (3) A noticeable feature of the polyQ structure, compared with other β-sheet or cross-β-sheet structures (e.g., 7, 25), is the relative sharpness of the 020 (0.83 nm) intersheet diffraction signal. Usually, this diffraction signal is broad, reflecting the less well-defined sheet stacking (van der Waals interactions and room temperature mobility of side chains) and as a consequence relatively short correlation lengths in the b direction. However, in polyQ, the relatively compact intermeshing of sheets (see Figure 3), which emanates, in part, from the side chains being locked by the side chain interamide hydrogen bonds, generates long-range lattice order along the b direction. This compact intermeshing of sheets is also responsible for the relatively high calculated density (for the Qblock) of 1.54 g cm-3 as mention earlier. (4) The broadness of the 00l and hkl (l*0) diffraction signals, in particular the 002, suggests that the coherent scattering length (csize) in the chain c axis is limited. A priori, this is expected, since the molecule is only 19 peptides long with a crystalline lamellar thickness of 7.1 nm, as shown in Figure 4a. However, comparison of the observed line broadening (Figure 1a) of the 002 with hk0 diffraction signals and with the calculated diffraction pattern (top half of Figure
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Figure 3. (a,b) Computer-generated views, parallel to a axis, of a Qblock of our proposed structure; color coding as in Figure 2. (a) Highlights the intimate stacking of two β-sheets; all of the atoms in the lower sheet are in yellow to help illustrate the interdigitation of Q side chains. The intersheet stacking distance is 0.83 nm and the unit cell is shown as a black rectangle. (b) Ball and stick model of the same β-sheet structure as a polyQ crystal. The unit cell, represented by colored box, contains two sheet segments. The β-sheets in yellow are slipped along a axis, orthogonal to bc plane. (c) View of polyQ apβ-sheet orthogonal to ab plane illustrating the strings of hydrogen bonds; central yellow between the usual β-sheet backbone amides; light blue at top surface between Q side chain amides; pink: equivalent hydrogen-bonding string at lower surface. There are four strings of hydrogen bonds per diQ chain segment; one on each surface between side chain amides and two between backbone amides (one string hidden behind other in central yellow band). (d) Similar view for a polyQ pβ-sheet, illustrating that the hydrogen bonding scheme can be maintained, although those between the backbone amides (yellow) are less linear. (e) View of polyQ crystal structure parallel to chain c axis. This view illustrates how the locking of the Q side chains via hydrogen bonds creates the pronounced sheet surface ridges. The central sheet in the unit cell (rectangular box) is in yellow to enable the Q side chains at the β-sheet surfaces within the crystal to be easily identified.
1c or d) suggests that crystalline lamellar thickness is shorter still. An obvious conceptual mechanism to comply with this factor is for the D2Q15K2 molecule to fold into a hairpin conformation as illustrated in Figure 4b,c; in this case, the molecular length reduces to 3.6 nm and csize 3 nm. There are now effectively only eight repetitive crystallographic repeats (in the straight-stem portions of hairpin molecule) contributing to the 002 diffraction signal. As a consequence, the 002 (and neighboring higher layer line diffraction arcs) broaden, as shown in the bottom half of Figure 1c. This calculated broadening is a better match to the observed line broadening (Figure 1a). With straight stems of equal length,
to maximize the hydrogen bonding interaction, the fold involves three amino acids (two backbone amide unit), i.e., a γ-turn, similar to the reverse turn discovered in the series of chain-folded, sequence-designed genetically engineered polypeptides.15-20 The terminal D2 and K2 dimers would be adjacent to each other (see Figure 4b,c). We do not judge this latter facet to be absolutely essential, but it would encourage the hairpin formation in appropriate ionic conditions when both the negative and positive charges are active. It is likely, that a hairpin structure forms in solution prior to self-association and growth, especially so in the a direction (see Figure 4d).
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Figure 4. (a) View, orthogonal to sheet surface, of the nonfolded (a) and once-folded hairpin-like conformation (b-d) of the D2Q15K2 molecule self-assembling into an apβ-sheet. The backbone atoms are in yellow and side chain atoms colored as listed in Figure 2 caption. For the nonfolded conformation, a pβ-sheet equivalent is possible. For the hairpin-like once-folded conformation, there is only one way to dock the molecules together to form a cross-β sheet and that is illustrated in (c).
A once-folded, hairpin conformation for the D2Q15K2 molecule, and subsequent aggregation into β-sheets with increasing concentration, has also been suggested from data obtained using ultraviolet circular dichroism.26,27 Examination of the once-folded conformation of D2Q15K2 (Figure 4b,c) reveals that hairpin-like molecules can only dock together in one way to form a cross-β ribbon; that is, the dictates of the backbone amide and side chain amide hydrogen bonds require the self-assembly arrangement shown in Figure 4d. Thus, the straight stems in this cross-β ribbon are antiparallel and the folds are along one edge. It is generally the case in these situations that a specific docking arrangement ameliorates the rate of self-assembly and quality of the crystalline entity being generated. When the ribbons stack (in b direction) to from cross-β crystallites, the folds can decorate both surfaces. Comparison of Our Proposed Model with PreViously Published Structures of highQ Peptides or Proteins. The concept of polyQ peptides forming β-sheets and additionally hydrogen bonding via the side chain amide units emanates from at least the 1960s. For example, using infrared spectroscopy, optical rotary dispersion, and X-ray powder diffraction, Krull et al.21 suggested that the side chain amides hydrogen bonded together, although no detailed model was proposed. Their X-ray powder diffraction pattern has essentially the same distribution of intensities and d spacings as reported in ref 2. To our knowledge, the first detailed structural model, the so-called “polar zipper model”, was proposed by Perutz et al.26 in 1994. The structure proposed here is conceptually related to this and the other previously proposed models.21 Both models are based on β-sheets structures, and some form of hydrogen bonding between glutamine side chain amides is proposed.
Structure proposed here differs from the Perutz et al.26 1994 structure in the following most noticeable ways. (1) In our model, we propose continuous strings of hydrogen bonds between the side chain amide units (see Figure 3), whereas in the Perutz et al.26 model, shown in Figure 5a, only half of these hydrogen bonds are formed. (2) In our proposed model, the β-sheets are intimately stacked at 0.830 nm apart, whereas in the Perutz et al.26 model, the side groups have a different geometry, and an intersheet distance of 1.68 nm is reported as shown in Figure 5b. It is suggested that intersheet hydrogen bonds between the surface side chains maintain this intersheet distance. In our model building calculations of this structure, we find that the distances involved are too great (>0.4 nm) for direct intersheet hydrogen bonding to occur. General Discussion and Conclusions We judge that the recently proposed hollow nanotube structure for amyloid fibrils,2 based on the X-ray diffraction data from the D2Q15K2 oligopeptide crystals is incorrect. It is not our intention to scrutinize the diffraction analysis from which the model is derived. However, it might be helpful to others to point out some features of the paper [2] that caused us some initial confusion. The electron micrograph (Figure 5, Perutz et al.2) of fibrils is not of the self-assembled D2Q15K2 peptides but from a different polypeptide: the 22-peptide (Sup35 fragment) shown in Figure 2b. The amino acid sequence of this Sup35 molecule is H2N-DDNNQQNYQQYSQNGNQQQGKKCOOH, the longest Qblock being a tripeptide. Thus, the helical tube or twisted ribbon appearance of this selfassembled molecule is not directly related to the D2Q15K2 molecule or to the X-ray diffraction pattern shown in Figure 1a.28 That this Sup35 fragment does not crystallize like the
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ameliorates the intimate stacking of the sheets, resulting in a compact crystalline structure. Amyloid fibrillar structures are relatively insoluble; thus, even in general terms, thin-wall water-filled nanotubes2 would seem to be inappropriate structural entities to resist enzymatic degradation. On the other hand, the robust cross-β Q crystallites that are proposed here would appear to be a more logical molecular architecture for creating insoluble protein fibrils. They derive their robustness from a set of concomitant molecular features: additional strings of hydrogen bonds locking the glutamine side chains into prominent ridges on the cross-β ribbon surfaces, which in turn foster intimate stacking. In proteins in general, if Qblocks are present, there is a basis for nucleation and localized self-assembly that has the potential to encourage other segments of the protein chain into β-sheet conformation. In the wheat gluten proteins for example, there are many Qblocks incorporated into the protein chain. Perhaps, therefore, it is not too surprising to find that such a macromolecule is rather insoluable.29 Acknowledgment. We thank Professor Sir Aaron Klug and Dr. John Finch for listening to our interpretations of the diffraction physics involved, scrutinizing our basic model structure, and encouraging us to submit it for publication. We also thank the Engineering and Physical Sciences Research Council (EPSRC) for support including a postdoctoral fellowship to P.S.. P.S. also acknowledges financial support from The Norwegian Research Council under Centre for Biopolymer Engineering at NOBIPOL, NTNU (Grant No. 145945/130). We also acknowledge Dr. L. C. Serpell for many useful discussions. Figure 5. Views of the polyQ zipper structure proposed by Perutz et al. (26). (a) Single apβ-sheet; note that 50% of the potential intrasheet, interamide hydrogen bonds are not made. (b) Successive sheets are placed 1.66 nm apart. The gap between intersheet atoms is g0.4 nm.
D2Q15K2 peptide is not surprising since the non Q amino acids would disrupt the Qblock hydrogen bonding scheme and the large tyrosine (Y) units would not allow precise stacking of the sheets at the 0.830 nm intersheet distance. Based on the X-ray diffraction match between parts a and c of Figure 1, we believe that the structure of the D2Q15K2 peptide is close to that shown in Figures 3 and 4. Our molecular models show that all strong and linear hydrogen bonds are parallel to the ac plane and no intersheet hydrogen bonding is important in formation of the structure. The hydrogen bonding scheme (Figure 3c) is attractive and realistic; however, X-ray diffraction alone cannot prove this feature even if we had access to the original X-ray data. This aspect is best left to spectroscopic studies for further scrutiny. On the basis of the evidence available, it would appear that the D2Q15K2 peptides form sufficiently long-lived, selfsustaining hairpin-like, primary β-sheet entities that selfassemble along the hydrogen bond direction (a axis) by continuous hydrogen bonded strings; these “continuous zippers” occur via inter backbone amides and inter side chain amides. Thus, we judge that the D2Q15K2 hairpins are the basic nucleating and building units. The consequences of locking in and reducing the flexibility of the side chains
References and Notes (1) Michalik, A.; Van Broeckhoven, C. Hum. Mol. Genet. 2003, 12, 173186. (2) Perutz, M. F.; Finch, J. T.; Berriman, J.; Lesk, A. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5591-5595. (3) Elam, J. S.; Taylor, A. B.; Strange, R.; Antonyuk, S.; Doucette, P. A.; Rodriguez, J. A.; Hasnain, S. S.; Hayward, L. J.; Valentine, J. S.; Yeates, T. O.; Hart, P. J. Nat. Struct. Biol. 2003,10, 461-467 (4) Pauling, L., Corey, R. B. Proc. Natl. Acad. Sci. U.S.A. 1953, 39, 253-256. (5) Marsh, R. E., Corey, R. B.; Pauling, L. Biochim. Biophys. Acta 1955, 16, 1-34. (6) Marsh, R. E.; Corey, R. B.; Pauling, L. Acta Crystallogr. 1955, 8, 710-715. (7) Fraser, R. D. B.; MacRae, T. P. Conformation in Fibrous Proteins and Related Synthetic Polypeptides; Academic Press: New York, 1973 (8) Rudall, K. M. In Symposium on Fibrous Proteins, University of Leeds; J. Soc. Dyers Colourists 1946, 15-23. (9) Rudall, K. M. In Progress in Biophysics and Biophysical Chemistry, 1st ed.; Butler, J. A. V., Randall, J. T., Eds.; Butterworth-Springer: London, 1950; pp 39-72. (10) Rudall, K. M. In AdVances in Protein Chemistry, VII; Anson, M. L., Bailey, K., Edsall, J. T., Eds.; Academic Press: New York, 1952; pp 253-290. (11) Rudall, K. M. Lectures on the Scientific Basis of Medicine, V, British Postgraduate Medical Federation; University of London, Athlone Press: London, 1957; pp 217-230. (12) Eanes, E. D.; Glenner, G. G. J. Histochem. Cytochem. 1968, 16, 673677 (13) Sunde, M.; Serpell, L. C.; Bartlam, M.; Fraser, P. E.; Pepys, M. B.; Blake, C. C. F. J. Mol. Biol. 1997, 273, 729-739. (14) Sikorski, P.; Atkins, E.; Serpell, L. C. Structure 2003, 11, 915-926 (15) Krejchi, M. T.; Atkins, E. D. T.; Waddon, A. J.; Fournier, M. J.; Mason, T. L.; Tirrell, D. A. Science 1994, 265, 1427-1432.
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