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β‐Structures in Fibrous Proteins
b‐STRUCTURES IN FIBROUS PROTEINS By ANDREY V. KAJAVA,* JOHN M. SQUIRE,{ AND DAVID A. D. PARRY{ *Centre de Recherches de Biochimie Macromole´culaire, CNRS FRE‐2593, 1919 Route de Mende, 34293 Montpellier Cedex 5, France; { Biological Structure and Function Section, Biomedical Sciences Division, Imperial College London, London SW7 2AZ, United Kingdom; { Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand
I. II. III. IV. V.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Characteristics of Simple b‐Structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diversity of b‐Structural Fibrous Folds Revealed by Crystallographic Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Recent Advances in Structural Studies of Amyloid and Prion Fibrils . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract The b‐form of protein folding, one of the earliest protein structures to be defined, was originally observed in studies of silks. It was then seen in early studies of synthetic polypeptides and, of course, is now known to be present in a variety of guises as an essential component of globular protein structures. However, in the last decade or so it has become clear that the b‐conformation of chains is present not only in many of the amyloid structures associated with, for example, Alzheimer’s Disease, but also in the prion structures associated with the spongiform encephalopathies. Furthermore, X‐ray crystallography studies have revealed the high incidence of the b‐fibrous proteins among virulence factors of pathogenic bacteria and viruses. Here we describe the basic forms of the b‐fold, summarize the many different new forms of b‐structural fibrous arrangements that have been discovered, and review advances in structural studies of amyloid and prion fibrils. These and other issues are described in detail in later chapters.
I.
Introduction
Elucidation of the three‐dimensional structures of b‐structural fibrous proteins has attracted the interest of scientists for more than 50 years. In the early days, the objects of these studies were predominantly the naturally occurring fibrous assemblies obtained from b‐silk and stretched mammalian ADVANCES IN PROTEIN CHEMISTRY, Vol. 73 DOI: 10.1016/S0065-3233(06)73001-7
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Copyright 2006, Elsevier Inc. All rights reserved. 0065-3233/06 $35.00
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Fig. 1. The basic arrangements of b‐strands in hydrogen‐bonded b‐sheets (A) parallel chains, (B) antiparallel chains. Green spheres of different sizes denote side chain groups
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b‐keratin (Astbury and Street, 1931), but the crystalline structures formed by some synthetic polypeptides (Fraser and MacRae, 1973) were also investigated in detail. An important outcome of these studies was a description of the two basic b‐structural arrangements found in proteins: the parallel and antiparallel pleated b‐sheet structures (Fraser et al., 1969; Pauling and Corey, 1951; Fig. 1A and B). Most of these b‐sheet structures are nonplanar (i.e., twisted), as shown initially by Fraser et al. (1971) for feather keratin, but subsequently seen widely in virtually all crystalline globular proteins (Salemme and Weatherford, 1981). At the same time, significant progress was achieved in establishing the orientation of the b‐crystallites that composed the pleated sheet structures in b‐silk, b‐keratin, and the other fibrous polypeptide structures (Bradbury et al., 1960; Fraser and MacRae, 1973). More recently, research on fibrous b‐proteins has been stimulated by the observation that amyloids, prion fibrils, and a variety of denaturated globular proteins have cross‐b structures (Fig. 1C and D), in which the polypeptide chains are oriented perpendicular to the plane of the fibrils axis (Blake and Serpell, 1996; Caughey et al., 1991; Eanes and Glenner, 1968; Kirschner et al., 1986). The incidence of amyloid fibrils in important human diseases has attracted considerable efforts to solve their structures at the atomic level. Despite this, however, the structure of the amyloid fibril, and in particular the lateral packing of the b‐strands and their orientation (parallel vs antiparallel) within the b‐sheets, remains unknown. This failure may be attributed in part to the fact that methods of determining high‐resolution structure (protein crystallography and NMR spectroscopy) cannot be used because of the polymeric character and insolubility of the fibrils involved. Accordingly, X‐ray fiber diffraction, electron microscopy (EM), optical spectroscopy, and other biophysical approaches have been the principal sources of data underlying the models of b‐structural fibrils presented to date. Over the last 13 years, there have been major advances in the study of fibrous b‐proteins. In particular, this period has been marked by a rapid emergence of new structural information. First, a number of crystal structures having elongated b‐structural fibrous topologies have been resolved by X‐ray crystallography, thanks to improved expression and crystallization strategies. Second, several new experimental techniques, including solid‐ state NMR, scanning transmission EM mass measurements, and electron directed either toward (large spheres) or away from (small spheres) from the reader. Hydrogen bonds are shown by red dotted lines. Other colors follow the standard CPK scheme. (C) Chain folding back onto itself in a cross‐b sheet. (D) Stacking of several sheets as in (C); the spacing of the stacks, shown as 11 A˚, is actually very variable depending on the nature of the R groups.
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paramagnetic resonance spectroscopy of spin‐labeled derivatives, have been applied successfully and these have provided significant constraints on the structural models for b‐silk, amyloid, and prion fibrils. One of the aims in preparing this book has been to provide an overview of the progress made in the elucidation of the b‐fibrous proteins over the past decade.
II.
Characteristics of Simple b‐Structures
To set the scene for the detailed chapters in the rest of the book, we describe here the main features of the simple b‐structural components of proteins. The parallel and antiparallel b‐structures in Fig. 1A and B show certain characteristic features. There is a repeat along the chain direction (vertical in Fig. 1A and B) which consists of two‐amino acid residues and is often about 6.5‐ to 7‐A˚ long. The b‐sheet is not planar but pleated to permit the side chains (R groups) of the amino acids to project out from the plane of the backbone‐pleated sheet. In Fig. 1A and B, the backbone‐pleated sheets are in the plane of the page and the R groups are imagined projecting above and below this plane. The other relatively constant dimension in b‐structures is the repeat distance in the direction of the hydrogen bonding between adjacent chains. In the antiparallel b‐structures this distance is about 9.6 A˚, but this contains two chains so there is a marked repeat at half of this, about 4.8 A˚. In the silks (Dicko et al., this volume) and in stretched keratin, the chain axis is normally parallel to the fiber axis direction, as envisaged in Fig. 1A and B for a ‘‘vertical’’ fiber axis. However, early studies of synthetic polypeptides (Bradbury et al., 1960) showed that some structures existed where the chain axis was perpendicular to the direction of stroking or stretching when the polypeptide solutions were oriented before drying. This was termed the cross‐b structure (Fig. 1C); it was also found to exist in a number of denatured globular and fibrous proteins (see summary in Fraser and MacRae, 1973). The term ‘‘cross‐b structure’’ was originally used to imply an antiparallel arrangement of b‐strands lying perpendicular to the fibril axis. It is now used more generally, however, to describe any chain arrangement of b‐strands (parallel, antiparallel, or mixed) with a chain orientation perpendicular to the fibril axis. The in‐plane spacings in cross‐b structures are much the same as in Fig. 1A and B, except that the hydrogen‐bonded direction (9.6‐ and 4.8‐A˚ repeats) is now along the fiber axis and the in‐chain repeat of 7 A˚ is perpendicular to the fiber axis. For b‐crystallites in general (i.e., for those structural elements in which the chain directions lie either in a similar direction to the fibril axis or which lie approximately perpendicular to it), the repeat in the third
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dimension has proved to be quite variable. The hydrogen‐bonded sheets depicted in Fig. 1A–C can sometimes stack together as shown in Fig. 1D for a cross‐b antiparallel sheet. The exact separation of the sheets is much more variable than the other repeats and depends crucially on the nature of the side chains (R groups) of the amino acids in the sheets and how they pack together. This intersheet spacing has been observed to be as low as 5 or 6 A˚ in some simple synthetic polypeptides, and can vary between about 8 and 16 A˚ in some prion structures (see Inouye and Kirschner, this volume), although the crystallographic repeat in this direction can be multiples of this basic intersheet spacing depending on precisely how the sheets are arranged. In many amyloid structures this third direction, if it involves sheet packing (see later), often has a repeat around 10–11 A˚. There are several different techniques with which to categorize b‐structures into those with their chain axis oriented along the fiber axis or those which are cross‐b types, but among the most powerful is the method of fiber diffraction (usually with X‐rays, but sometimes using neutrons or electrons). Figure 2 illustrates schematically the differences that might be observed in the fiber diffraction pattern of well‐aligned samples depending on whether the chains are oriented along (A, B) or perpendicular to (C, D) the fiber axis. Once again the 7‐A˚ repeat along the chain (call it C for Chain) and the 9.6‐A˚ repeat (or half of this) in the hydrogen‐bonded direction (call it H for Hydrogen‐bonded) are relatively constant features, whereas the peaks associated with the intersheet distance (call it S for Sheet) can have a variety of positions and strengths. The C‐repeat along the chain is roughly the pitch of a 2/1 helix of amino acids where the subunit repeat is 3.5 A˚, so the 3.5‐A˚ repeat gives a meridional peak in the diffraction pattern from the parallel b‐structure (Fig. 2B), with the 7‐A˚ repeat showing up as off‐meridional intensity on the first layer‐line. The H and S directions are perpendicular to this, so for the axially aligned b‐structures they show up as intensities along the equator of the pattern. Once again, in the antiparallel sheet the 9.6‐A˚ H direction corresponds to the separation of two chains, so there is a strong pseudo‐repeat after 4.8 A˚ which often makes this peak much stronger than the 9.6‐A˚ peak (which in fact may not be observed). In the case of diffraction from the cross‐b structure, the repeat in the fiber axis direction is the hydrogen‐bonded H‐repeat of 9.6 or 4.8 A˚. The S‐repeat still shows up on the equator, but the C‐repeat along the chain axis has now switched from the meridian in Fig. 2C to the equator in Fig. 2D. Finally, a number of observed amyloid diffraction patterns, which are often quite disoriented, giving arced diffraction peaks, show simply the interchain spacing (H) at around 4.8 A˚ on the meridian and the intersheet spacing at around 10–11 A˚ on the equator. In Figs. 1C, D, and 2C, the
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Fig. 2. The differences that might be observed in the fiber diffraction patterns from oriented samples of antiparallel b‐structures depending on whether the chains are aligned along (A, B) or perpendicular to (C, D) the fiber axis. Color coding on (A, B) as in Fig. 1.
antiparallel chains are shown folding back on themselves so that a single sheet can be formed from just one chain. However, many well‐studied b‐structures, some natural and some synthetic, are aggregates of very short peptides, some of which are not long enough to fold back on themselves even once. In these cases, there is often ambiguity about whether adjacent chains are parallel or antiparallel and the intersheet stacking can be very variable. As discovered recently in structures solved by protein crystallography, there is in fact a great diversity of chain organizations in naturally occurring b‐structural folds, as summarized in the next section.
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Diversity of b‐Structural Fibrous Folds Revealed by Crystallographic Studies
During the past few years, a new set of b‐structural fibrous folds has emerged. One of these, the parallel b‐helix, was first described for bacterial pectate lyase in 1993 (Yoder et al., 1993). Since then more than a hundred crystal structures with similar or related fibrous morphologies have been solved. These have revealed a great diversity of b‐structural folds that can be categorized into at least five distinct groups (Fig. 3). They include b‐solenoids (Fig. 3A), triple‐stranded b‐solenoids (Fig. 3B), triangular cross‐b prisms (Fig. 3C), triple b‐spirals (Fig. 3D), and spiral b‐hairpin stacks (staircases; Fig. 3E). The crystal structures generally have axial dimensions that are comparable to their lateral ones and are, therefore, in a sense, not strictly fibrous. However, these structures are built of axially stacked repetitive structural blocks. This arrangement, in principle, allows ready elongation to form fibrils by the simple addition of recurrent blocks. The other common property of the fibrous b‐proteins is the repetitive character of their amino acid sequences. The majority of the known b‐fibrous proteins are located on the surfaces of either bacteria or viruses. A significant portion of these proteins forms homotrimers. The most frequently occurring b‐fibrous folds are based on solenoidal windings of the polypeptide chain. Each coil in the solenoid has an axial rise of about 4.8 A˚ and corresponding b‐strands in successive coils align to form parallel b‐sheets (Fig. 3A). The number of the known b‐solenoid proteins, which include b‐helices and b‐rolls, is now large enough to support their detailed analysis and classification. Kajava and Steven (this volume) present a systematic account of these structures distinguished by their handedness, twist, oligomerization state, and coil shape. This survey has also revealed some relationships between the amino acid sequences of b‐solenoids and their structures and functions. This has implications for structural prediction of other b‐solenoids and for elucidation of amyloid fibril structures. A recently discovered subset of triple‐stranded b‐helices from bacteriophage tail proteins (alternatively termed ‘‘triple‐stranded b‐solenoids’’) represents another distinct group of b‐fibrous folds (Fig. 3B). In these structures, three identical chains related by threefold rotational symmetry wind around a common axis. These chains form unusual parallel b‐sheets with no intra‐ and only intermolecular b‐structural hydrogen bonding. Kajava and Steven (this volume) survey the distinguishing structural features of the known triple‐stranded b‐solenoids, also documenting their notable diversity and differences in comparison to the single‐stranded b‐solenoids.
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A
B
b -Solenoid
C
Triple-stranded b -solenoid
D
Cross-b -prism E
Triple b -spiral
Spiral b -hairpin staircase
Fig. 3. Representative structures of five principal groups of b‐structural fibrous folds that have recently been established by X‐ray crystallographic studies. Arrows denote b‐strands. b‐Strands that belong to the same chains have the same color.
The abundance, location, stability, and folding of the triple‐stranded b‐helices are also reviewed in a chapter by Mitraki, Papanikolopoulou, and van Raaij, which is dedicated to triple b‐stranded fibrous folds in the viral fibers. Mitraki and colleagues also overview the other distinctive family of b‐fibrous folds, called the triple b‐spirals (Fig. 3D). The b‐spiral folds are more complicated than the solenoidal fold, with long central b‐strands that hold the trimer together through interchain hydrogen bonds, and
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interactions of the apolar side chains and short peripheral b‐strands to stabilize the structure. The distinctive structural property of this trimeric fold is that its longest core b‐strands run parallel to the fiber axis in contrast to most of the other b‐fibrous folds representing variations of the cross‐b structure. Mitraki and coauthors also describe the structures of b‐structural globular domains identified in the viral fibers and demonstrate their crucial role in the correct folding of the adjacent b‐fibrous folds such as triple‐stranded b‐helices and b‐spirals. Finally, the chapter by Mitraki et al. illustrates some crystallographic strategies that may lead to the discovery of even more new b‐fibrous modules from the trimeric fibers of viruses. For completeness of classification, it is pertinent to mention two other families of the b‐fibrous folds. One of these is a homotrimeric structure that resembles a triangular prism with equivalent sides formed by antiparallel cross‐b sheets (Fig. 3C). The internal side of these b‐sheets is composed of apolar side chains, while the opposite side consists primarily of polar side chains. This fold was found in the crystal structure of autotransporter protein Hia from Haemophilus influenzae (Yeo et al., 2004) and in the tailspike protein of Salmonella typhimurium phage P22 (Schuler et al., 2000). The other distinctive b‐fibrous fold was discovered among the surface proteins of pathogenic Gram‐positive bacteria (Fernandez‐Tornero et al., 2001; Ho et al., 2005) or their bacteriophages (Hermoso et al., 2003). This is a single‐stranded b‐fibrous fold with b‐hairpins as repetitive structural units (Fig. 3E). The b‐hairpins extend perpendicularly from the axis and the relation between adjacent hairpins can be approximated by a threefold screw‐axis transformation characterized by a 90–120 unit rotation and an axial translation of about 10 A˚, thereby creating a left‐handed superhelix. This fold resembles a spiral staircase with b‐hairpins as the steps. The ‘‘spiral b‐hairpin staircase’’ fold was found in choline‐binding domains in the pneumococcal virulence factor LytA (Fernandez‐Tornero et al., 2001) and endolysin from pneumococcal bacteriophage Cp‐1 (Hermoso et al., 2003) as well as in carbohydrate‐binding domain of toxin A from Clostridium difficile (Ho et al., 2005). It is worth mentioning that, in contrast to the other b‐fibrous folds, the fibrils generated by the spiral b‐hairpin staircase fold can readily curve due to the absence of hydrogen bonding between adjacent b‐hairpins. Thus, the classical pleated b‐sheet structures have now been supplemented by several new b‐structural fibrous folds that have been established by X‐ray crystallographic studies. As a consequence, today, the b‐fibrous folds represent a more diverse class of fibrous structures than those defined by the a‐ or collagen‐helices.
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Recent Advances in Structural Studies of Amyloid and Prion Fibrils
Over the past decade, significant progress has been made in understanding the structural arrangements of prion and amyloid fibrils. This can be attributed to the establishment of several b‐solenoid folds that are consistent with the available constraints imposed by the structure of amyloid fibrils. Furthermore, new experimental techniques such as solid‐ state NMR, scanning transmission EM mass measurements, and electron paramagnetic resonance spectroscopy of spin‐labeled derivatives have provided a number of new constraints for modeling amyloid and prion structures. An important achievement of this work was the establishment of parallel and in‐register arrangement of b‐strands in several amyloid fibrils. These included b‐amyloid, a‐synuclein, human amylin, and yeast Ure2p (Benzinger et al., 1998; Chan et al., 2005; Der‐Sarkissian et al., 2003; Jayasinghe and Langen, 2004). Based on this and other experimental data, several new structural models for amyloid and prion fibrils with parallel in‐register stacking of b‐strands have been formulated (Govaerts et al., 2004; Guo et al., 2004; Kajava et al., 2004, 2005; Petkova et al., 2002; Ritter et al., 2005; Wang et al., 2005; Fig. 4). These results have effectively put an end to the dominance of those models characterized by antiparallel b‐sheet arrangements. It has also revealed that the parallel b‐sheets in these prion and amyloid fibrils differ from the antiparallel ones observed in the fibrils formed by short (7–10 residue) fragments of the same peptides (Balbach et al., 2000; Griffiths et al., 1995). These data clearly indicate the possibility that the unconstrained short peptides may not have the same structure/ properties as they do in the context of a full‐length peptide. Fibrils of the first mammalian prion protein discovered, namely PrP (Prusiner, 1991), proved to be difficult to study as a consequence of poor in vitro reproduction of what were homogeneous well‐ordered fibrils in vivo (Baskakov and Bocharova, 2005). Nevertheless, a considerable body of data has been collected on the structure and formation of these fibrils. In this volume, Kirschner and Inouye review current knowledge of PrP prion pathologies and summarize X‐ray fiber and powder diffraction studies on the N‐terminal fragments of prion proteins. They also compare structures of PrP peptide assemblies with those of the PrP‐related polyalanine and polyglutamine peptides. Recently, prions have been found in fungal systems (Wickner, 1994) and this has advanced the field considerably, due to the improved experimental tractability of these prions. Baxa and colleagues (this volume) have focused on the structures of the fungal prion fibrils and in so doing have both summarized current experimental constraints and appraised the various models proposed. The authors have concluded
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A
B
b-Helical models
C
b-Superpleated model
Fig. 4. New structural models for amyloid and prion filaments with the parallel and in‐register arrangement of b‐strands in the b‐sheets. b‐Strands are denoted by arrows. The filaments are formed by hydrogen‐bonded stacks of repetitive units. Axial projections of single repetitive units corresponding to each model are shown on the top. Lateral views of the overall structures are on the bottom. (A) The core of a b‐helical model of the b‐amyloid protofilament (Petkova et al., 2002). Two such protofilaments coil around one another to form a b‐amyloid fibril. (B) The core of a b‐helical model of the HET‐s prion fibril (Ritter et al., 2005). The repetitive unit consists of two b‐helical coils. (C) The core of a superpleated b‐structural model suggested for yeast prion Ure2p protofilaments and other amyloids (Kajava et al., 2004).
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that two parallel b‐structural folds, the superpleated b‐structure (Kajava et al., 2004) and a specific b‐helical formulation (Ritter et al., 2005), are the most valid candidates to explain the known experimental data on Ure2p and HET‐s prion filaments. The polymorphism of the yeast prion fibrils and its possible structural basis have also been discussed in several chapters of this volume. Polymorphism appears to be a common property of prion and amyloid fibrils. The same amyloidogenic peptide can form different fibril structures depending on slight changes in the fibrillogenesis conditions. This adds complexity to what is already a difficult problem in analyzing the structure of amyloid fibrils. Kreplak and Aebi (this volume) have surveyed the polymorphism of fibrils formed by b‐amyloid peptides, calcitonin, human amylin, and other proteins as observed by EM. They have linked this phenomenon with differences in the structure of small intermediate oligomers that initiate fibrillogeneses. Approaches that allow a study of such oligomers and provide information on the relation of the intermediates with the toxicity of the amyloid peptides have also been discussed. Nelson and Eisenberg (this volume) have provided a detailed review of the current structural models of prion and amyloid fibrils. Despite the fact that atomic‐level structures of amyloid‐like fibrils have yet to be determined, many models of these fibrils have been proposed. Nelson and Eisenberg categorize these models into three classes: (i) Refolding models, in which the protein has different structures in the native and fibrillar states; (ii) Gain‐of‐Interaction models, which propose a largely native‐like structure for proteins in the fibril; and (iii) Natively Disordered models, which are formed by peptides whose native state is not structured. It was shown that the cores of several models contain a packing of the b‐strands similar to that in the so‐called cross‐b spine structure. This has recently been determined at atomic resolution using X‐ray diffraction of the crystal formed from a seven‐residue peptide from the yeast prion Sup35 (Nelson et al., 2005).
V. Conclusions The contributions to this volume demonstrate that structural studies of fibrous b‐proteins, as well as prion and amyloid fibrils, have advanced rapidly thanks in large part to improved experimental techniques and better theoretical analysis of the ever‐increasing structural data. It is also possible to learn from studies of naturally occurring silks (Dicko et al., this volume) how variations in the conditions of production of silk threads from the same protein can produce a variety of b‐structures with very distinct
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properties. Further progress in studies of b‐structures is likely to lead to the discovery of new b‐fibrous folds, to the determination of more precise structural models for amyloid‐like fibrils, and to a better understanding of the factors that determine why they fold as they do. Since diseases like Alzheimer’s are likely to affect a gradually increasing proportion of the population as life expectancy increases, this is not an unimportant task. The observed abundance of the b‐fibrous folds among virulence factors of bacteria and viruses also indicates that the fibrous b‐proteins will be an attractive target for future structural studies, especially in the context of emerging infectious threats.
References Astbury, W. T., and Street, A. (1931). X‐ray studies of the structures of hair, wool and related fibres. I. General. Trans. R. Soc. Lond. A230, 75–101. Balbach, J. J., Ishii, Y., Antzutkin, O. N., Leapman, R. D., Rizzo, N. W., Dyda, F., Reed, J., and Tycko, R. (2000). Amyloid fibril formation by A beta 16–22, a seven‐residue fragment of the Alzheimer’s beta‐amyloid peptide, and structural characterization by solid state NMR. Biochemistry 39, 13748–13759. Baskakov, I. V., and Bocharova, O. V. (2005). In vitro conversion of mammalian prion protein into amyloid fibrils displays unusual features. Biochemistry 44, 2339–2348. Benzinger, T. L., Gregory, D. M., Burkoth, T. S., Miller‐Auer, H., Lynn, D. G., Botto, R. E., and Meredith, S. C. (1998). Propagating structure of Alzheimer’s beta‐ amyloid(10–35) is parallel beta‐sheet with residues in exact register. Proc. Natl. Acad. Sci. USA 95, 13407–13412. Blake, C., and Serpell, L. (1996). Synchrotron X‐ray studies suggest that the core of the transthyretin amyloid fibril is a continuous beta‐sheet helix. Structure 4, 989–998. Bradbury, E. M., Brown, L., Downie, A. R., Elliott, A., Fraser, R. D. B., Hanby, W. E., and Macdonald, T. R. R. (1960). The ‘‘cross‐beta’’ structure in polypeptides of low molecular weight. J. Mol. Biol. 2, 276–281. Caughey, B. W., Dong, A., Bhat, K. S., Ernst, D., Hayes, S. F., and Caughey, W. S. (1991). Secondary structure analysis of the scrapie‐associated protein PrP 27‐30 in water by infrared spectroscopy. Biochemistry 30, 7672–7680. Chan, J. C., Oyler, N. A., Yau, W. M., and Tycko, R. (2005). Parallel beta‐sheets and polar zippers in amyloid fibrils formed by residues 10‐39 of the yeast prion protein Ure2p. Biochemistry 44, 10669–10680. Der‐Sarkissian, A., Jao, C. C., Chen, J., and Langen, R. (2003). Structural organization of alpha‐synuclein fibrils studied by site‐directed spin labeling. J. Biol. Chem. 278, 37530–37535. Eanes, E. D., and Glenner, G. G. (1968). X‐ray diffraction studies on amyloid filaments. J. Histochem. Cytochem. 16, 673–677. Fernandez‐Tornero, C., Lopez, R., Garcia, E., Gimenez‐Gallego, G., and Romero, A. (2001). A novel solenoid fold in the cell wall anchoring domain of the pneumococcal virulence factor LytA. Nat. Struct. Biol. 8, 1020–1024. Fraser, R. D. B., and MacRae, T. P. (1973). ‘‘Conformation in Fibrous Proteins and Related Synthetic Polypeptides.’’ Academic Press, London, New York.
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Fraser, R. D. B., MacRae, T. P., Parry, D. A. D., and Suzuki, E. (1969). Structure of beta‐ keratin. Polymer 10, 810–826. Fraser, R. D. B., MacRae, T. P., Parry, D. A. D., and Suzuki, E. (1971). The structure of feather keratin. Polymer 12, 35–56. Govaerts, C., Wille, H., Prusiner, S. B., and Cohen, F. E. (2004). Evidence for assembly of prions with left‐handed beta‐helices into trimers. Proc. Natl. Acad. Sci. USA 101, 8342–8347. Griffiths, J. M., Ashburn, T. T., Auger, M., Costa, P. R., Griffin, R. G., and Lansbury, P. T. (1995). Rotational resonance solid‐state NMR elucidates a structural model of pancreatic amyloid. J. Am. Chem. Soc. 117, 3539–3546. Guo, J. T., Wetzel, R., and Xu, Y. (2004). Molecular modeling of the core of Abeta amyloid fibrils. Proteins 57, 357–364. Hermoso, J. A., Monterroso, B., Albert, A., Galan, B., Ahrazem, O., Garcia, P., Martinez‐ Ripoll, M., Garcia, J. L., and Menendez, M. (2003). Structural basis for selective recognition of pneumococcal cell wall by modular endolysin from phage Cp‐1. Structure 11, 1239–1249. Ho, J. G., Greco, A., Rupnik, M., and Ng, K. K. (2005). Crystal structure of receptor‐ binding C‐terminal repeats from Clostridium difficile toxin A. Proc. Natl. Acad. Sci. USA 102, 18373–18378. Jayasinghe, S. A., and Langen, R. (2004). Identifying structural features of fibrillar islet amyloid polypeptide using site‐directed spin labeling. J. Biol. Chem. 279, 48420–48425. Kajava, A. V., Baxa, U., Wickner, R. B., and Steven, A. C. (2004). A model for Ure2p prion filaments and other amyloids: The parallel superpleated beta‐structure. Proc. Natl. Acad. Sci. USA 101, 7885–7890. Kajava, A. V., Aebi, U., and Steven, A. C. (2005). The parallel superpleated beta‐structure as a model for amyloid fibrils of human amylin. J. Mol. Biol. 348, 247–252. Kirschner, D. A., Abraham, C., and Selkoe, D. J. (1986). X‐ray diffraction from intraneuronal paired helical filaments and extraneuronal amyloid fibers in Alzheimer disease indicates cross‐beta conformation. Proc. Natl. Acad. Sci. USA 83, 503–507. Nelson, R., Sawaya, M. R., Balbirnie, M., Madsen, A. O., Riekel, C., Grothe, R., and Eisenberg, D. (2005). Structure of the cross‐beta spine of amyloid‐like fibrils. Nature 435, 773–778. Pauling, L., and Corey, R. B. (1951). Configurations of polypeptide chains with favored orientations around single bonds: Two new pleated sheets. Proc. Natl. Acad. Sci. USA 37, 729–740. Petkova, A. T., Ishii, Y., Balbach, J. J., Antzutkin, O. N., Leapman, R. D., Delaglio, F., and Tycko, R. (2002). A structural model for Alzheimer’s beta‐amyloid fibrils based on experimental constraints from solid state NMR. Proc. Natl. Acad. Sci. USA 99, 16742–16747. Prusiner, S. B. (1991). Molecular biology of prion diseases. Science 252, 1515–1522. Ritter, C., Maddelein, M. L., Siemer, A. B., Luhrs, T., Ernst, M., Meier, B. H., Saupe, S. J., and Riek, R. (2005). Correlation of structural elements and infectivity of the HET‐s prion. Nature 435, 844–848. Salemme, F. R., and Weatherford, D. W. (1981). Conformational and geometrical properties of beta‐sheets in proteins. I. Parallel beta‐sheets. J. Mol. Biol. 146, 101–117. Schuler, B., Furst, F., Osterroth, F., Steinbacher, S., Huber, R., and Seckler, R. (2000). Plasticity and steric strain in a parallel beta‐helix: Rational mutations in the P22 tailspike protein. Proteins 39, 89–101.
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Wang, J., Gulich, S., Bradford, C., Ramirez‐Alvarado, M., and Regan, L. (2005). A twisted four‐sheeted model for an amyloid fibril. Structure 13, 1279–1288. Wickner, R. B. (1994). [URE3] as an altered URE2 protein: Evidence for a prion analog in Saccharomyces cerevisiae. Science 264, 566–569. Yeo, H. J., Cotter, S. E., Laarmann, S., Juehne, T., St Geme, J. W., III, and Waksman, G. (2004). Structural basis for host recognition by the Haemophilus influenzae Hia autotransporter. EMBO J 23, 1245–1256. Yoder, M. D., Keen, N. T., and Jurnak, F. (1993). New domain motif: The structure of pectate lyase C, a secreted plant virulence factor. Science 260, 1503–1507.
I. II. III. IV. V.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Characteristics of Simple b‐Structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diversity of b‐Structural Fibrous Folds Revealed by Crystallographic Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Recent Advances in Structural Studies of Amyloid and Prion Fibrils . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract The b‐form of protein folding, one of the earliest protein structures to be defined, was originally observed in studies of silks. It was then seen in early studies of synthetic polypeptides and, of course, is now known to be present in a variety of guises as an essential component of globular protein structures. However, in the last decade or so it has become clear that the b‐conformation of chains is present not only in many of the amyloid structures associated with, for example, Alzheimer’s Disease, but also in the prion structures associated with the spongiform encephalopathies. Furthermore, X‐ray crystallography studies have revealed the high incidence of the b‐fibrous proteins among virulence factors of pathogenic bacteria and viruses. Here we describe the basic forms of the b‐fold, summarize the many different new forms of b‐structural fibrous arrangements that have been discovered, and review advances in structural studies of amyloid and prion fibrils. These and other issues are described in detail in later chapters.
I.
Introduction
Elucidation of the three‐dimensional structures of b‐structural fibrous proteins has attracted the interest of scientists for more than 50 years. In the early days, the objects of these studies were predominantly the naturally occurring fibrous assemblies obtained from b‐silk and stretched mammalian ADVANCES IN PROTEIN CHEMISTRY, Vol. 73 DOI: 10.1016/S0065-3233(06)73001-7
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Copyright 2006, Elsevier Inc. All rights reserved. 0065-3233/06 $35.00
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Fig. 1. The basic arrangements of b‐strands in hydrogen‐bonded b‐sheets (A) parallel chains, (B) antiparallel chains. Green spheres of different sizes denote side chain groups
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b‐keratin (Astbury and Street, 1931), but the crystalline structures formed by some synthetic polypeptides (Fraser and MacRae, 1973) were also investigated in detail. An important outcome of these studies was a description of the two basic b‐structural arrangements found in proteins: the parallel and antiparallel pleated b‐sheet structures (Fraser et al., 1969; Pauling and Corey, 1951; Fig. 1A and B). Most of these b‐sheet structures are nonplanar (i.e., twisted), as shown initially by Fraser et al. (1971) for feather keratin, but subsequently seen widely in virtually all crystalline globular proteins (Salemme and Weatherford, 1981). At the same time, significant progress was achieved in establishing the orientation of the b‐crystallites that composed the pleated sheet structures in b‐silk, b‐keratin, and the other fibrous polypeptide structures (Bradbury et al., 1960; Fraser and MacRae, 1973). More recently, research on fibrous b‐proteins has been stimulated by the observation that amyloids, prion fibrils, and a variety of denaturated globular proteins have cross‐b structures (Fig. 1C and D), in which the polypeptide chains are oriented perpendicular to the plane of the fibrils axis (Blake and Serpell, 1996; Caughey et al., 1991; Eanes and Glenner, 1968; Kirschner et al., 1986). The incidence of amyloid fibrils in important human diseases has attracted considerable efforts to solve their structures at the atomic level. Despite this, however, the structure of the amyloid fibril, and in particular the lateral packing of the b‐strands and their orientation (parallel vs antiparallel) within the b‐sheets, remains unknown. This failure may be attributed in part to the fact that methods of determining high‐resolution structure (protein crystallography and NMR spectroscopy) cannot be used because of the polymeric character and insolubility of the fibrils involved. Accordingly, X‐ray fiber diffraction, electron microscopy (EM), optical spectroscopy, and other biophysical approaches have been the principal sources of data underlying the models of b‐structural fibrils presented to date. Over the last 13 years, there have been major advances in the study of fibrous b‐proteins. In particular, this period has been marked by a rapid emergence of new structural information. First, a number of crystal structures having elongated b‐structural fibrous topologies have been resolved by X‐ray crystallography, thanks to improved expression and crystallization strategies. Second, several new experimental techniques, including solid‐ state NMR, scanning transmission EM mass measurements, and electron directed either toward (large spheres) or away from (small spheres) from the reader. Hydrogen bonds are shown by red dotted lines. Other colors follow the standard CPK scheme. (C) Chain folding back onto itself in a cross‐b sheet. (D) Stacking of several sheets as in (C); the spacing of the stacks, shown as 11 A˚, is actually very variable depending on the nature of the R groups.
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paramagnetic resonance spectroscopy of spin‐labeled derivatives, have been applied successfully and these have provided significant constraints on the structural models for b‐silk, amyloid, and prion fibrils. One of the aims in preparing this book has been to provide an overview of the progress made in the elucidation of the b‐fibrous proteins over the past decade.
II.
Characteristics of Simple b‐Structures
To set the scene for the detailed chapters in the rest of the book, we describe here the main features of the simple b‐structural components of proteins. The parallel and antiparallel b‐structures in Fig. 1A and B show certain characteristic features. There is a repeat along the chain direction (vertical in Fig. 1A and B) which consists of two‐amino acid residues and is often about 6.5‐ to 7‐A˚ long. The b‐sheet is not planar but pleated to permit the side chains (R groups) of the amino acids to project out from the plane of the backbone‐pleated sheet. In Fig. 1A and B, the backbone‐pleated sheets are in the plane of the page and the R groups are imagined projecting above and below this plane. The other relatively constant dimension in b‐structures is the repeat distance in the direction of the hydrogen bonding between adjacent chains. In the antiparallel b‐structures this distance is about 9.6 A˚, but this contains two chains so there is a marked repeat at half of this, about 4.8 A˚. In the silks (Dicko et al., this volume) and in stretched keratin, the chain axis is normally parallel to the fiber axis direction, as envisaged in Fig. 1A and B for a ‘‘vertical’’ fiber axis. However, early studies of synthetic polypeptides (Bradbury et al., 1960) showed that some structures existed where the chain axis was perpendicular to the direction of stroking or stretching when the polypeptide solutions were oriented before drying. This was termed the cross‐b structure (Fig. 1C); it was also found to exist in a number of denatured globular and fibrous proteins (see summary in Fraser and MacRae, 1973). The term ‘‘cross‐b structure’’ was originally used to imply an antiparallel arrangement of b‐strands lying perpendicular to the fibril axis. It is now used more generally, however, to describe any chain arrangement of b‐strands (parallel, antiparallel, or mixed) with a chain orientation perpendicular to the fibril axis. The in‐plane spacings in cross‐b structures are much the same as in Fig. 1A and B, except that the hydrogen‐bonded direction (9.6‐ and 4.8‐A˚ repeats) is now along the fiber axis and the in‐chain repeat of 7 A˚ is perpendicular to the fiber axis. For b‐crystallites in general (i.e., for those structural elements in which the chain directions lie either in a similar direction to the fibril axis or which lie approximately perpendicular to it), the repeat in the third
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dimension has proved to be quite variable. The hydrogen‐bonded sheets depicted in Fig. 1A–C can sometimes stack together as shown in Fig. 1D for a cross‐b antiparallel sheet. The exact separation of the sheets is much more variable than the other repeats and depends crucially on the nature of the side chains (R groups) of the amino acids in the sheets and how they pack together. This intersheet spacing has been observed to be as low as 5 or 6 A˚ in some simple synthetic polypeptides, and can vary between about 8 and 16 A˚ in some prion structures (see Inouye and Kirschner, this volume), although the crystallographic repeat in this direction can be multiples of this basic intersheet spacing depending on precisely how the sheets are arranged. In many amyloid structures this third direction, if it involves sheet packing (see later), often has a repeat around 10–11 A˚. There are several different techniques with which to categorize b‐structures into those with their chain axis oriented along the fiber axis or those which are cross‐b types, but among the most powerful is the method of fiber diffraction (usually with X‐rays, but sometimes using neutrons or electrons). Figure 2 illustrates schematically the differences that might be observed in the fiber diffraction pattern of well‐aligned samples depending on whether the chains are oriented along (A, B) or perpendicular to (C, D) the fiber axis. Once again the 7‐A˚ repeat along the chain (call it C for Chain) and the 9.6‐A˚ repeat (or half of this) in the hydrogen‐bonded direction (call it H for Hydrogen‐bonded) are relatively constant features, whereas the peaks associated with the intersheet distance (call it S for Sheet) can have a variety of positions and strengths. The C‐repeat along the chain is roughly the pitch of a 2/1 helix of amino acids where the subunit repeat is 3.5 A˚, so the 3.5‐A˚ repeat gives a meridional peak in the diffraction pattern from the parallel b‐structure (Fig. 2B), with the 7‐A˚ repeat showing up as off‐meridional intensity on the first layer‐line. The H and S directions are perpendicular to this, so for the axially aligned b‐structures they show up as intensities along the equator of the pattern. Once again, in the antiparallel sheet the 9.6‐A˚ H direction corresponds to the separation of two chains, so there is a strong pseudo‐repeat after 4.8 A˚ which often makes this peak much stronger than the 9.6‐A˚ peak (which in fact may not be observed). In the case of diffraction from the cross‐b structure, the repeat in the fiber axis direction is the hydrogen‐bonded H‐repeat of 9.6 or 4.8 A˚. The S‐repeat still shows up on the equator, but the C‐repeat along the chain axis has now switched from the meridian in Fig. 2C to the equator in Fig. 2D. Finally, a number of observed amyloid diffraction patterns, which are often quite disoriented, giving arced diffraction peaks, show simply the interchain spacing (H) at around 4.8 A˚ on the meridian and the intersheet spacing at around 10–11 A˚ on the equator. In Figs. 1C, D, and 2C, the
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Fig. 2. The differences that might be observed in the fiber diffraction patterns from oriented samples of antiparallel b‐structures depending on whether the chains are aligned along (A, B) or perpendicular to (C, D) the fiber axis. Color coding on (A, B) as in Fig. 1.
antiparallel chains are shown folding back on themselves so that a single sheet can be formed from just one chain. However, many well‐studied b‐structures, some natural and some synthetic, are aggregates of very short peptides, some of which are not long enough to fold back on themselves even once. In these cases, there is often ambiguity about whether adjacent chains are parallel or antiparallel and the intersheet stacking can be very variable. As discovered recently in structures solved by protein crystallography, there is in fact a great diversity of chain organizations in naturally occurring b‐structural folds, as summarized in the next section.
FIBROUS b‐STRUCTURES
III.
7
Diversity of b‐Structural Fibrous Folds Revealed by Crystallographic Studies
During the past few years, a new set of b‐structural fibrous folds has emerged. One of these, the parallel b‐helix, was first described for bacterial pectate lyase in 1993 (Yoder et al., 1993). Since then more than a hundred crystal structures with similar or related fibrous morphologies have been solved. These have revealed a great diversity of b‐structural folds that can be categorized into at least five distinct groups (Fig. 3). They include b‐solenoids (Fig. 3A), triple‐stranded b‐solenoids (Fig. 3B), triangular cross‐b prisms (Fig. 3C), triple b‐spirals (Fig. 3D), and spiral b‐hairpin stacks (staircases; Fig. 3E). The crystal structures generally have axial dimensions that are comparable to their lateral ones and are, therefore, in a sense, not strictly fibrous. However, these structures are built of axially stacked repetitive structural blocks. This arrangement, in principle, allows ready elongation to form fibrils by the simple addition of recurrent blocks. The other common property of the fibrous b‐proteins is the repetitive character of their amino acid sequences. The majority of the known b‐fibrous proteins are located on the surfaces of either bacteria or viruses. A significant portion of these proteins forms homotrimers. The most frequently occurring b‐fibrous folds are based on solenoidal windings of the polypeptide chain. Each coil in the solenoid has an axial rise of about 4.8 A˚ and corresponding b‐strands in successive coils align to form parallel b‐sheets (Fig. 3A). The number of the known b‐solenoid proteins, which include b‐helices and b‐rolls, is now large enough to support their detailed analysis and classification. Kajava and Steven (this volume) present a systematic account of these structures distinguished by their handedness, twist, oligomerization state, and coil shape. This survey has also revealed some relationships between the amino acid sequences of b‐solenoids and their structures and functions. This has implications for structural prediction of other b‐solenoids and for elucidation of amyloid fibril structures. A recently discovered subset of triple‐stranded b‐helices from bacteriophage tail proteins (alternatively termed ‘‘triple‐stranded b‐solenoids’’) represents another distinct group of b‐fibrous folds (Fig. 3B). In these structures, three identical chains related by threefold rotational symmetry wind around a common axis. These chains form unusual parallel b‐sheets with no intra‐ and only intermolecular b‐structural hydrogen bonding. Kajava and Steven (this volume) survey the distinguishing structural features of the known triple‐stranded b‐solenoids, also documenting their notable diversity and differences in comparison to the single‐stranded b‐solenoids.
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A
B
b -Solenoid
C
Triple-stranded b -solenoid
D
Cross-b -prism E
Triple b -spiral
Spiral b -hairpin staircase
Fig. 3. Representative structures of five principal groups of b‐structural fibrous folds that have recently been established by X‐ray crystallographic studies. Arrows denote b‐strands. b‐Strands that belong to the same chains have the same color.
The abundance, location, stability, and folding of the triple‐stranded b‐helices are also reviewed in a chapter by Mitraki, Papanikolopoulou, and van Raaij, which is dedicated to triple b‐stranded fibrous folds in the viral fibers. Mitraki and colleagues also overview the other distinctive family of b‐fibrous folds, called the triple b‐spirals (Fig. 3D). The b‐spiral folds are more complicated than the solenoidal fold, with long central b‐strands that hold the trimer together through interchain hydrogen bonds, and
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interactions of the apolar side chains and short peripheral b‐strands to stabilize the structure. The distinctive structural property of this trimeric fold is that its longest core b‐strands run parallel to the fiber axis in contrast to most of the other b‐fibrous folds representing variations of the cross‐b structure. Mitraki and coauthors also describe the structures of b‐structural globular domains identified in the viral fibers and demonstrate their crucial role in the correct folding of the adjacent b‐fibrous folds such as triple‐stranded b‐helices and b‐spirals. Finally, the chapter by Mitraki et al. illustrates some crystallographic strategies that may lead to the discovery of even more new b‐fibrous modules from the trimeric fibers of viruses. For completeness of classification, it is pertinent to mention two other families of the b‐fibrous folds. One of these is a homotrimeric structure that resembles a triangular prism with equivalent sides formed by antiparallel cross‐b sheets (Fig. 3C). The internal side of these b‐sheets is composed of apolar side chains, while the opposite side consists primarily of polar side chains. This fold was found in the crystal structure of autotransporter protein Hia from Haemophilus influenzae (Yeo et al., 2004) and in the tailspike protein of Salmonella typhimurium phage P22 (Schuler et al., 2000). The other distinctive b‐fibrous fold was discovered among the surface proteins of pathogenic Gram‐positive bacteria (Fernandez‐Tornero et al., 2001; Ho et al., 2005) or their bacteriophages (Hermoso et al., 2003). This is a single‐stranded b‐fibrous fold with b‐hairpins as repetitive structural units (Fig. 3E). The b‐hairpins extend perpendicularly from the axis and the relation between adjacent hairpins can be approximated by a threefold screw‐axis transformation characterized by a 90–120 unit rotation and an axial translation of about 10 A˚, thereby creating a left‐handed superhelix. This fold resembles a spiral staircase with b‐hairpins as the steps. The ‘‘spiral b‐hairpin staircase’’ fold was found in choline‐binding domains in the pneumococcal virulence factor LytA (Fernandez‐Tornero et al., 2001) and endolysin from pneumococcal bacteriophage Cp‐1 (Hermoso et al., 2003) as well as in carbohydrate‐binding domain of toxin A from Clostridium difficile (Ho et al., 2005). It is worth mentioning that, in contrast to the other b‐fibrous folds, the fibrils generated by the spiral b‐hairpin staircase fold can readily curve due to the absence of hydrogen bonding between adjacent b‐hairpins. Thus, the classical pleated b‐sheet structures have now been supplemented by several new b‐structural fibrous folds that have been established by X‐ray crystallographic studies. As a consequence, today, the b‐fibrous folds represent a more diverse class of fibrous structures than those defined by the a‐ or collagen‐helices.
10 IV.
KAJAVA ET AL.
Recent Advances in Structural Studies of Amyloid and Prion Fibrils
Over the past decade, significant progress has been made in understanding the structural arrangements of prion and amyloid fibrils. This can be attributed to the establishment of several b‐solenoid folds that are consistent with the available constraints imposed by the structure of amyloid fibrils. Furthermore, new experimental techniques such as solid‐ state NMR, scanning transmission EM mass measurements, and electron paramagnetic resonance spectroscopy of spin‐labeled derivatives have provided a number of new constraints for modeling amyloid and prion structures. An important achievement of this work was the establishment of parallel and in‐register arrangement of b‐strands in several amyloid fibrils. These included b‐amyloid, a‐synuclein, human amylin, and yeast Ure2p (Benzinger et al., 1998; Chan et al., 2005; Der‐Sarkissian et al., 2003; Jayasinghe and Langen, 2004). Based on this and other experimental data, several new structural models for amyloid and prion fibrils with parallel in‐register stacking of b‐strands have been formulated (Govaerts et al., 2004; Guo et al., 2004; Kajava et al., 2004, 2005; Petkova et al., 2002; Ritter et al., 2005; Wang et al., 2005; Fig. 4). These results have effectively put an end to the dominance of those models characterized by antiparallel b‐sheet arrangements. It has also revealed that the parallel b‐sheets in these prion and amyloid fibrils differ from the antiparallel ones observed in the fibrils formed by short (7–10 residue) fragments of the same peptides (Balbach et al., 2000; Griffiths et al., 1995). These data clearly indicate the possibility that the unconstrained short peptides may not have the same structure/ properties as they do in the context of a full‐length peptide. Fibrils of the first mammalian prion protein discovered, namely PrP (Prusiner, 1991), proved to be difficult to study as a consequence of poor in vitro reproduction of what were homogeneous well‐ordered fibrils in vivo (Baskakov and Bocharova, 2005). Nevertheless, a considerable body of data has been collected on the structure and formation of these fibrils. In this volume, Kirschner and Inouye review current knowledge of PrP prion pathologies and summarize X‐ray fiber and powder diffraction studies on the N‐terminal fragments of prion proteins. They also compare structures of PrP peptide assemblies with those of the PrP‐related polyalanine and polyglutamine peptides. Recently, prions have been found in fungal systems (Wickner, 1994) and this has advanced the field considerably, due to the improved experimental tractability of these prions. Baxa and colleagues (this volume) have focused on the structures of the fungal prion fibrils and in so doing have both summarized current experimental constraints and appraised the various models proposed. The authors have concluded
11
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A
B
b-Helical models
C
b-Superpleated model
Fig. 4. New structural models for amyloid and prion filaments with the parallel and in‐register arrangement of b‐strands in the b‐sheets. b‐Strands are denoted by arrows. The filaments are formed by hydrogen‐bonded stacks of repetitive units. Axial projections of single repetitive units corresponding to each model are shown on the top. Lateral views of the overall structures are on the bottom. (A) The core of a b‐helical model of the b‐amyloid protofilament (Petkova et al., 2002). Two such protofilaments coil around one another to form a b‐amyloid fibril. (B) The core of a b‐helical model of the HET‐s prion fibril (Ritter et al., 2005). The repetitive unit consists of two b‐helical coils. (C) The core of a superpleated b‐structural model suggested for yeast prion Ure2p protofilaments and other amyloids (Kajava et al., 2004).
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that two parallel b‐structural folds, the superpleated b‐structure (Kajava et al., 2004) and a specific b‐helical formulation (Ritter et al., 2005), are the most valid candidates to explain the known experimental data on Ure2p and HET‐s prion filaments. The polymorphism of the yeast prion fibrils and its possible structural basis have also been discussed in several chapters of this volume. Polymorphism appears to be a common property of prion and amyloid fibrils. The same amyloidogenic peptide can form different fibril structures depending on slight changes in the fibrillogenesis conditions. This adds complexity to what is already a difficult problem in analyzing the structure of amyloid fibrils. Kreplak and Aebi (this volume) have surveyed the polymorphism of fibrils formed by b‐amyloid peptides, calcitonin, human amylin, and other proteins as observed by EM. They have linked this phenomenon with differences in the structure of small intermediate oligomers that initiate fibrillogeneses. Approaches that allow a study of such oligomers and provide information on the relation of the intermediates with the toxicity of the amyloid peptides have also been discussed. Nelson and Eisenberg (this volume) have provided a detailed review of the current structural models of prion and amyloid fibrils. Despite the fact that atomic‐level structures of amyloid‐like fibrils have yet to be determined, many models of these fibrils have been proposed. Nelson and Eisenberg categorize these models into three classes: (i) Refolding models, in which the protein has different structures in the native and fibrillar states; (ii) Gain‐of‐Interaction models, which propose a largely native‐like structure for proteins in the fibril; and (iii) Natively Disordered models, which are formed by peptides whose native state is not structured. It was shown that the cores of several models contain a packing of the b‐strands similar to that in the so‐called cross‐b spine structure. This has recently been determined at atomic resolution using X‐ray diffraction of the crystal formed from a seven‐residue peptide from the yeast prion Sup35 (Nelson et al., 2005).
V. Conclusions The contributions to this volume demonstrate that structural studies of fibrous b‐proteins, as well as prion and amyloid fibrils, have advanced rapidly thanks in large part to improved experimental techniques and better theoretical analysis of the ever‐increasing structural data. It is also possible to learn from studies of naturally occurring silks (Dicko et al., this volume) how variations in the conditions of production of silk threads from the same protein can produce a variety of b‐structures with very distinct
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properties. Further progress in studies of b‐structures is likely to lead to the discovery of new b‐fibrous folds, to the determination of more precise structural models for amyloid‐like fibrils, and to a better understanding of the factors that determine why they fold as they do. Since diseases like Alzheimer’s are likely to affect a gradually increasing proportion of the population as life expectancy increases, this is not an unimportant task. The observed abundance of the b‐fibrous folds among virulence factors of bacteria and viruses also indicates that the fibrous b‐proteins will be an attractive target for future structural studies, especially in the context of emerging infectious threats.
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