Tài liệu Báo cáo khoa học: Mechanisms of amyloid fibril formation – focus on domain-swapping doc

REVIEW ARTICLE Mechanisms of amyloid fibril formation – focus on domain-swapping Eva Z ˇ erovnik 1 , Veronika Stoka 1 , Andreja Mirtic ˇ 2 , Gregor Gunc ˇ ar 3 , Joz ˇ e Grdadolnik 2,4 , Rosemary A. Staniforth 5 , Dus ˇ an Turk 1,6 and Vito Turk 1 1 Department of Biochemistry and Molecular and Structural Biology, Joz ˇ ef Stefan Institute, Ljubljana, Slovenia 2 Laboratory of Biomolecular Structure, National Institute of Chemistry, Ljubljana, Slovenia 3 Department of Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Slovenia 4 En-Fist Centre of Excellence, Ljubljana, Slovenia 5 Department of Molecular Biology and Biotechnology, University of Sheffield, UK 6 Center of Excellence for Integrated Approaches in Chemistry and Biology of Proteins, Ljubljana, Slovenia Introduction The ordered aggregation of proteins to amyloid fibrils is at the core of systemic diseases such as diabetes type II and immunoglobulin light-chain amyloidosis, and also prevalent in localized diseases, particularly in neurodegenerative disorders such as Alzheimer’s, Parkinson’s, Huntington’s disease, several other dementias, motor neuron disease, different ataxias and prion-related diseases [1–4]. Increasing evidence suggests that aberrant folding of the mutated protein and its aggregation might be the initial trigger of such diseases, followed by other consequences, such as Ca 2+ and metal ion imbalance, oxidative stress, and the overload of chaperone and ubiquitin proteasome systems [1,5,6]. The primary trigger in sporadic cases is Keywords domain-swapping; mechanisms of amyloid- fibril formation; protein aggregation; stefin B; toxic oligomers Correspondence E. Z ˇ erovnik, Department of Biochemistry and Molecular and Structural Biology, Joz ˇ ef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia Fax: + 386 1 477 3984 Tel: + 386 1 477 3753 E-mail: eva.zerovnik@ijs.si (Received 18 February 2011, revised 6 April 2011, accepted 28 April 2011) doi:10.1111/j.1742-4658.2011.08149.x Conformational diseases constitute a group of heterologous disorders in which a constituent host protein undergoes changes in conformation, lead- ing to aggregation and deposition. To understand the molecular mecha- nisms of the process of amyloid fibril formation, numerous in vitro and in vivo studies, including model and pathologically relevant proteins, have been performed. Understanding the molecular details of these processes is of major importance to understand neurodegenerative diseases and could contribute to more effective therapies. Many models have been proposed to describe the mechanism by which proteins undergo ordered aggregation into amyloid fibrils. We classify these as: (a) templating and nucleation; (b) linear, colloid-like assembly of spherical oligomers; and (c) domain-swap- ping. In this review, we stress the role of domain-swapping and discuss the role of proline switches. Abbreviations 1D, 1 dimensional; AFM, atomic force microscopy; CO, critical oligomers; DA, dipole assembly; DCF, double-concerted fibrillation; IDPs, intrinsically disordered proteins; MDC, monomer-directed conversion; NCC, nucleated conformational conversion; NDP, nucleation- dependent polymerization; NP, nucleated polymerization; OFF, off-pathway folding; TA, templated assembly; TEM, transmission electron microscopy; TFE, 2,2,2-trifluoroethanol. FEBS Journal 278 (2011) 2263–2282 ª 2011 The Authors Journal compilation ª 2011 FEBS 2263 still a matter of debate. Proteins and lipids become damaged by oxidative stress and by excessive metal interactions, which, in turn, could both promote pro- tein aggregation [7,8]. Aging by itself influences the performance of the ubiquitin–proteasome system [9] and autophagy [10], with a concomitant decline in pro- tein degradation capability. In addition, mitochondrial energy production becomes less efficient with age [11]. All these factors could contribute to the accumulation of protein aggregates. Understanding the rules governing protein folding should lead to a better understanding of protein ‘mis- folding’ (i.e. folding to an alternative, often multimeric state). The conversion to the cross-b structure observed in mature amyloid fibrils takes place starting from an intermediate conformation which, in the case of globu- lar proteins, forms after partial unfolding and, in natively unfolded proteins, after partial folding [12,13]. Dobson [14] proposed that any protein can be trans- formed into amyloid fibrils. Many disease-related and nonpathological proteins have been studied in an attempt to reveal the molecular mechanism of their aggregation into ordered, b-sheet rich amyloid fibrils. In this review, we focus on the possible mechanisms of amyloid-fibril formation and search for common grounds. We also discuss the interface between folding and aggregation. The field of protein aggregation into amyloid fibrils combines physicochemical and structural studies, cellular and animal models, and clinical studies. In addition to providing a basic understanding of the pro- cesses of protein folding and aggregation, such data help towards translational approaches in medicine. Structural and morphological data Pre-amyloid, oligomeric intermediates, at the cross- roads between protein folding and aggregation, possess some common structure, regardless of their amino acid sequence, because polyclonal antibodies raised against one can bind to most such oligomers of different amy- loid proteins [15]. It remains to be clarified whether the structure of the prefibrillar oligomers is indeed all b-sheet or whether the a-helical parts are the ones that cross the membranes. As revealed by atomic force microscopy (AFM), the structure of such annular olig- omers embedded in lipid bilayers resembles that of the well ordered bacterial toxins [15–17]. It still remains for us to capture and image the annular oligomers in their cellular environment where they are inserted in cellular membranes. We envisage that two-photon fluo- rescence correlation spectroscopy [18] may soon make this possible. However, the common structural details of the oligomers and their mode of toxic action remain unknown [4] and would profit from innovative research approaches. Mature amyloid fibrils are long and straight, usually comprising four to six filaments. They specifically bind certain dyes such as Congo red and thioflavin T, and they demonstrate a characteristic cross-b pattern on X-ray diffraction, reflecting distances between b-strands (4.7 A ˚ ) and distances between b-sheets (9–11 A ˚ ) [19,20]. High-resolution structural methods such as NMR and X-ray diffraction are of limited use for character- izing prefibrillar aggregates and amyloid fibrils, pri- marily as a result of their limitations in providing insight into the structure of heterogeneous species. However, they can be used to determine the structure of the precursor conformation, whereas, for the fibrils and oligomers, cryo-electron microscopy, transmission electron microscopy, small angle X-ray scattering and AFM are more suitable [21]. AFM and electron microscopy have revealed multiple morphological vari- ants of amyloid fibrils differing in the number of fila- ments and the helicity of their intertwining [22–24]. The structure of the mature fibrils has been deter- mined in a limited number of cases by either solid state NMR [25] or by H ⁄ D exchange quenched flow fol- lowed by heteronuclear NMR [26]. However, the struc- ture of the prefibrillar oligomers, which is more relevant to biomedically oriented research, remains rather elusive. Both Yu et al. [27] and Glabe [28] proposed that two kinds of b-structure are possible: the b-sheet that is observed in the mature fibrils and the a-pleated sheet [29], which could be the structure in the prefibrillar species, termed either globular oligo- mers (or ‘globulomers’), ‘granules’, ‘critical oligomers’ or ‘spheres’. The a-pleated sheet structure would give the globular oligomers higher dipole moments, which would lead to a linear, colloid-like growth of amyloid protofibrils. Glabe [28] suggested that, instead of selecting oligomers by size, they could be selected by the structural epitopes that become exposed. Trials with conformationally selective antibodies have shown that most of the prefibrillar species are bound by the selective A11 antibody, and only a few by OC anti- body, which also binds fibrils [28]. Comparison of amyloid aggregation and protein folding Under physiological conditions, protein folding takes place in the crowded milieu of the cell with a whole range of helper proteins [30]. These helpers include a series of molecular chaperones whose functions, Domain-swapping and amyloid fibril formation E. Z ˇ erovnik et al. 2264 FEBS Journal 278 (2011) 2263–2282 ª 2011 The Authors Journal compilation ª 2011 FEBS amongst others, are to prevent aggregation of incom- pletely folded polypeptide chains [31] and to disaggre- gate formed aggregates [32–34]. Protein folding involves a complex molecular recog- nition phenomenon that depends on the cooperative action of a large number of relatively weak, noncova- lent interactions involving thousands of atoms. Hydro- phobic [35,36], electrostatic [37–39] and van der Waals interactions [40,41]; peptide hydrogen bonds [42,43]; and peptide solvation [44,45] are major forces driving protein folding. The electrostatic interaction between polar C=O and NH groups in the peptide backbone depends strongly on the peptide backbone conforma- tion [37,38]. In the extended b-strand conformation, C=O and NH dipoles of adjacent peptide units are aligned antiparallel, whereas, in the a-helical confor- mation, they are parallel. The stability of both types of structure can be explained by the electrostatic screen- ing model [37,46]. This readily explains the distinct preferences of residues in native and denatured pro- teins [46] and in peptides [47,48]. In this model, it is assumed that the total free energy of an amino acid residue is determined predominantly by the local elec- trostatic energy of the backbone dipole moments (N-H, C=O) as a result of interaction with neighbor- ing peptide groups, and by the solvation free energy of the backbone dipole moments [37,49,50]. The u and w values of the ‘coil library’ of high-resolution protein structures, which represent residues outside the second- ary structure, adopt b, a R , a L and polyproline II back- bone conformations [51]. With regard to the electrostatic screening model [46,51], the b conformer is energetically more favorable than either of the two a conformers of a residue in the gas phase. The antipar- allel orientation of the backbone dipole moments stabilizes the b conformer, whereas the parallel orien- tation of dipole moments destabilizes the a R conformer. However, the parallel arrangement of dipole moments has advantages in polar solvents as a result of favorable interactions with the solvent. There- fore, the solvation of backbone atoms is much larger for a conformers than for b conformers. Interaction with solvent thus compensates for the destabilization of the a conformation as a result of peptide dipole moments. Alternation of the screening of backbone electrostatic interactions by side chains causes different conformational preferences of residues in aqueous solution. Moreover, the additional modulation of screening by changing the local environment and inter- and ⁄ or intramolecular interactions may have a signifi- cant influence on the preferential conformations of a single amino acid residue. Therefore, even small varia- tions in pH, temperature and ionic strength may have sufficient potential to induce changes in the conforma- tional propensities of amino acid residues to form sec- ondary structure, as well as their ability to aggregate. Computer simulations of protein aggregation indi- cate that the hydrophobic effect plays an important role in promoting the aggregation process [52]. Molec- ular dynamics simulations of small peptides show that b-sheet aggregates are stabilized by backbone hydro- gen bonds, as well as by specific side-chain interac- tions, such as hydrophobic stacking of polar side chains and formation of salt bridges [53,54]. Coulom- bic interactions also play an important role in protein aggregation [54–57]. Synthetic amyloidogenic peptides polymerize into fibrils only when the net charge is ± 1 [54], whereas a neutral or higher effective charge pre- vents fibril formation. These results were explained on the assumption that nonspecific, amorphous aggrega- tion and fibril formation represent competing events. When the structure of the side chains permits, poly- peptides in the b-pleated sheet conformation can self- assemble into 1D, crystal-like structures involving a very large number of b-sheets. The capacity of unlim- ited interchain hydrogen bonding in the absence of structural restraints is considered to drive the assembly of susceptible proteins into amyloid fibrils [19]. The structure of amyloid fibrils reflects the aggregation of strands of b-pleated sheet polypeptides into a long cross-b assembly, with the strands oriented perpendicu- lar to the fibril axis. The dominant forces driving the association of b-sheet formations are dipole–dipole interactions and the dehydration propensity of pre- formed intrasheet hydrogen bonds [58]. Factors influencing the propensity to aggregate The degree of conformational stability of the protein native state plays an important but not always decisive [59,60] role in the process of aggregation. A partially- unfolded conformation favors specific intermolecular interactions, including electrostatic attraction, hydro- gen bonding and hydrophobic contacts, which result in oligomerization and fibrillation [14,61–64]. In general, amyloid formation in vitro can be achieved by destabi- lizing the native state of the protein under conditions in which noncovalent interactions still remain favor- able [65–67]. However, a local conformational change before aggregation is not a necessary step in the fibril formation of every protein. For some proteins, it was shown that the native structure is preserved in the fibrils [68,69]. Even all-a [70] or mixed a ⁄ b proteins can transform into amyloid fibrils. It has also been observed that the ability of a protein to undergo an E. Z ˇ erovnik et al. Domain-swapping and amyloid fibril formation FEBS Journal 278 (2011) 2263–2282 ª 2011 The Authors Journal compilation ª 2011 FEBS 2265 a to b conformational change is facilitated by amino acid regions that adopt an a-helical conformation within the native structure, at the same time as having a higher statistical propensity for the b-structure [71]. Mutations and changes in environmental conditions both affect the aggregation reaction [72–76]. A protein may assemble into amyloid fibrils with multiple distinct morphologies in response to a change in amino acid sequence [74] or upon a change in aggregation condi- tions [23,24,76], as well as under the same growth con- dition [22,77,78]. A study of b-lactoglobulin has shown that charge repulsion makes amyloid fibrils more regu- lar, whereas a lower charge, caused by a pH change in the direction of the pI and⁄ or screening electrostatic interactions by salt, results in shorter fibrillar rods that pack into spheres [56]. Analysis of naturally occurring b-sheet proteins and mixed a ⁄ b proteins has identified a number of struc- tural motifs that interrupt self-assembly of the edge strands into the intermolecular b-pleated sheet. For example, charged side chains within the hydrophobic region of the edge strand and proline residues both limit interactions with other b-pleated sheet edge strands [79]. It has been suggested that the edge strands have evolved as guards against uncontrolled propagation of the b-pleated sheet conformation that would otherwise interfere with productive protein fold- ing [79]. Partial proteolysis often results in amyloidogenic fragments. Algorithms have been developed to predict the location of amyloidogenic fragments in the poly- peptide sequence [80–82]. In globular proteins, such amyloidogenic parts are usually surrounded by resi- dues that have a low aggregation propensity, the so- called ‘amyloid-breakers’ [82], and inhibit amyloid propagation. The software used to calculate the propensity of a protein to aggregate is based on either sequence or structural data, thus taking into consideration the known data, including intrinsic and external factors [83–85]. The universe of proteins capable of forming amyloid-like fibrils has been named the ‘amylome’ [86]. The major determinants qualifying a protein to belong to the amylome can be summarized as: (a) the forma- tion of a ‘steric zipper’ consisting of two self-comple- mentary b-sheets that form the spine of an amyloid fibril and (b) sufficient ‘conformational freedom’ of the self-complementary segment to interact with other molecules. Although self-complementary segments are found in almost all proteins, the size of the amylome is limited, suggesting that chaperoning effects have evolved to prevent self-complementary segments from interacting with each other [86]. Mechanisms of amyloid fibril formation The models reported before the year 2000 have been described in older reviews [63,64,87] and some excellent reviews have been written subsequently [2,4,88–90]. On the basis of the main features of the models, we have classified them into three groups (Table 1): (a) templat- ing and nucleation; (b) linear, colloid-like assembly of spherical oligomers; and (c) domain-swapping. For some of the case proteins relevant to the focus of this review on domain-swapping, descriptions of the mechanisms are provided, whereas, for most of the other cases, the original publications are cited. On the basis of our research on cystatins, which are capable of domain-swapping, and on a literature survey of a number of other amyloidogenic proteins that initially form dimers, we emphasize domain-swapping as a pos- sible mechanism underlying amyloid fibril formation (see below). We also describe several factors that are Table 1. Models for the mechanism of amyloid fibril formation. Templating (A) and nucleation (B) Examples a A TA model [91] (Fig. 1A) Prion A MDC model [92] (Fig. 1B) Prion, stefin B at pH 7 (from monomer) B NP model [97] Amyloid-b peptide B NDP model [99] (Fig. 1C) Amyloid-b peptide B NCC model [98] Yeast prion protein Sup35 C ‘Polar zipper’ model [93–96] Huntingtin, ataxin-3 Linear colloid-like assembly of spherical oligomers examples A Model of CO [104] Yeast phosphoglycerate kinase B DA model [107] (Fig. 1D) Tau 40 protein C DCF mechanism [88,108] (Fig. 1E) a-Synuclein D Isodesmic (linear) polymerization [104,185] b 2 -Microglobulin stefin B at pH 3 (from globular oligomer) Domain swapping [150,160] Examples A Propagated domain-swapping [120,186] Cystatin C B Off-pathway model [137] with domain-swapped oligomers [123] and propagated domain-swapping (Fig. 1G) Stefin B at pH 5 (from dimers) B Off-pathway model [137] with domain-swapped oligomers [121,122,163] and likely propagated domain-swapping Stefin A a All human proteins, with a representative case example. Domain-swapping and amyloid fibril formation E. Z ˇ erovnik et al. 2266 FEBS Journal 278 (2011) 2263–2282 ª 2011 The Authors Journal compilation ª 2011 FEBS decisive for folding, misfolding, domain-swapping and amyloid fibril formation. Templating and nucleation models Templating models comprise the templated assembly (TA) and the monomer-directed conversion (MDC) models. These models were originally proposed for the prion protein transformations [91,92]. The TA and DSC models are presented in Fig. 1A,B. The ‘Polar zipper’ model proposed by Perutz et al. [93] can also be classified as a templating model. This model applies to amyloid forming proteins whose b-sheets are stabilized by hydrogen bonds between polar side chains, such as those between glutamine and asparagine [94,95]. Molecular modeling has shown that such polar residues link b-strands together into b-sheets by a network of hydrogen bonds between the main-chain amides and the polar side chains. The glu- tamine- and asparagine-rich regions are commonly found in the N-termini of both mammalian and yeast prion proteins [96] and several other proteins with polyglutamine expansions such as huntingtin and ataxin-3. The nucleation-based models [97–99] comprise the nucleated polymerization (NP) model [97], the nucle- ated conformational conversion (NCC) model [98] and the nucleation-dependent polymerization (NDP) model [99]; for a review, see Kelly [87]. An example of the NP model is that used by Loma- kin et al. [100] to describe fibril formation by the amy- loid-b peptide. The model predicts that the lag phase, which disappears upon seeding, decreases exponentially as the protein concentration increases; however in a recent, very reproducible study of the kinetics of Ab assembly, this was found not to be the case [101]. The NP model predicts micelle formation above a critical protein concentration, where fibrils nucleate on heter- ologous seeds. In this model, fibrils grow by irrevers- ible binding of monomers to the fibril ends. The NDP model (Fig. 1C) predicts that the lag phase arises from the fact that the dissociation rate is initially greater than the association rate. This is reversed after a critical nucleus size is reached. In this model, the lag phase is also predicted to show a high concentration dependence and to disappear on seeding [102]. The NCC model of Serio et al. [98] is applicable when little or no concentration dependence is observed for both the nucleation and assembly rates. In this model, a steady rate is ensured by an almost constant concentration of the assembly competent oligomers [98,103]. In the NCC model, the rate-determining step is a conformational change that occurs in the nucleus of preformed oligomers, rather than oligomer growth itself. The concentration of soluble oligomers does not increase with higher soluble protein concentration as a result of the formation of assembly-ineligible com- plexes. An example of NCC mechanism of amyloid assembly is provided by the yeast prion protein Sup35 [103]. Linear colloid-like assembly of spherical oligomers Model of ‘critical oligomers’ (CO) In the kinetics of yeast phosphoglycerate kinase fibril- lation studied by Modler et al. [104], two steps were observed during the formation of amyloid. ‘CO’ were formed in the first step, whereas, in the second step, a linear growth of oligomers into protofibrils was observed. The kinetics of both steps were found to be irreversible. Phosphoglycerate kinase was converted into protofibrils, starting with a partially-unfolded intermediate [105,106]. According to this model [104], the acquisition of a b-sheet structure and fibril growth are coupled events subsequent to a generalized diffu- sion-collision process. Dipole assembly (DA) model Xu et al. [107] proposed a similar two-step model, which they termed the ‘DA’ model. In the first step, nucleation units (i.e. globular oligomers resembling ‘spheres’ or ‘granules’) form in a process driven by the surface chemical potential. The oligomeric and spheri- cal nucleation units reach a uniform size as a result of the electrostatic repulsion between these species and the monomers. Xu et al. [107] proposed that nucle- ation units aggregate linearly as a result of their intrin- sic dipole moment. Their growth is governed by charge-dipole and dipole–dipole interactions (Fig. 1D). Double-concerted fibrillation (DCF) model Bhak et al. [88,108] proposed the ‘DCF’ model as an alternative to the prevailing nucleation-dependent fibrillation models [97–99]. In this model (Fig. 1E), amyloid fibril formation also occurs in two steps: (a) association of the monomers into oligomeric units (globular oligomers; also termed ‘granules’ or ‘spher- oids’) and (b) linear growth of the oligomeric units into protofibrils in the absence of a template [108]. According to this model, the major driving force for fibril formation is a structural rearrangement within the oligomeric granules achieved by shear stress. E. Z ˇ erovnik et al. Domain-swapping and amyloid fibril formation FEBS Journal 278 (2011) 2263–2282 ª 2011 The Authors Journal compilation ª 2011 FEBS 2267 k 1 k −OP k 2 k I k G DimerMonomer Oligomer Fibril Rearrangement Protofibril Off-path oligomer A B C D E F G Domain-swapping and amyloid fibril formation E. Z ˇ erovnik et al. 2268 FEBS Journal 278 (2011) 2263–2282 ª 2011 The Authors Journal compilation ª 2011 FEBS Domain-swapping as a mechanism of amyloid-fibril formation Here, we feel we need to explain more of our main model proteins: cystatins and stefins. Given their example, we illustrate the principle of domain-swap- ping and how this can underlie the process of amyloid- fibril formation. Cystatins and stefins: an example of domain-swapping proteins forming amyloids Cystatins and stefins are a large family of cysteine pro- teinase inhibitors, examples of which have been linked to amyloid diseases and degenerative conditions. These small globular proteins (11–13 kDa), albeit evolution- ary distinct [109], are structurally and functionally analogous and those studied so far show evidence of 3D domain-swapping both in vitro and in vivo. Human cystatin C is a member of the cystatin II family of cysteine cathepsin inhibitors [110] but may have additional functions. It is a well known amyloi- dogenic protein whose mutations cause hereditary cyst- atin C amyloid angiopathy [111]. Recently, it was reported that cystatin C induces autophagy [112] in a cathepsin-independent manner and, in this way, con- tributes to neuroprotection. It is also known that the cystatin C A ⁄ A allele, which leads to impaired secre- tion of the protein and intracellular accumulation, influences negatively the outcome of late-onset Alzhei- mer’s disease and frontotemporal lobar degeneration [113,114]. Human stefins are representative of the cystatin I family of the cysteine protease inhibitors [110]. Human stefins A and B (sometimes referred to as cystatins A and B), together with some cathepsins, were identified in the core of amyloid plaques of various origins [115]. Human stefin B (i.e. cystatin B gene) mutations cause progressive myoclonus epilepsy of type 1-EPM1 [116,117], with signs of cerebellar neurodegeneration [118] and oxidative stress [119]. The structures of cystatin domain-swapped dimers have been solved, both by X-ray crystallography (human cystatin C) [120] and by heteronuclear NMR (human stefin A and chicken cystatin) [121,122]. The domain-swapped dimer of stefin A (Fig. 2A) is made of strand 1, the a-helix and strand 2 from one mono- mer, and strands 3–5 from the other monomer [120,122]. Similar to other cystatins, stefin B is prone to form domain-swapped dimers (Fig. 2B). The 3D structure of its tetramer [123] is composed of two domain-swapped dimer units. The two domain- swapped dimers interact through loop-swapping, also termed ‘hand-shaking’ [123]. Folding mechanisms and oligomer formation by domain-swapping Folding studies are usually focused on unraveling the conformational changes occurring within the mono- meric protein under conditions often referred to as ‘physiological’, generally comprising pH 7.0 and room temperature. It is clear that different folding conditions must be examined when the focus switches to what is occurring in the early steps of amyloid-fibril formation. For many systems, including the stefins [124–126], amyloid-fibrils form at nonphysiological pHs and in the presence of further additives, such as metal ions or Fig. 1. Schematic representations of the chosen mechanisms. (A) The TA model [98]. In the TA model, in a rapid pre-equilibrium step, the soluble state (S) molecules that are initially in a random coil conformation bind to a pre-assembled (A) state nucleus. This binding induces the rate-determining structural change from the random coil to the b-pleated sheet structure as the molecule is added to the growing end of the fibril [91]. (B) The MDC model [98]. In the MDC model, a pre-existing monomer in the A-state conformation, analogous to the conforma- tion adopted in the fibrils, binds to the soluble S-state monomer and converts it to an A-state dimer [92] in a rate-determining step. The dimer then dissociates, and the constituent A-state monomers add to the growing end of the fibril. (C) The NDP model [88]. We consider that the final structure labeled as ‘amyloid’ represents protofibrils rather than fibrils. The NDP model also predicts a lag phase that arises from the fact that the dissociation rate is initially greater than the association rate. (D) The DA model [107]. In the first step, nucleation units (globular oligomers) form in a process driven by the surface chemical potential. In the second step, the nucleation units aggregate linearly as a result of their intrinsic dipole moment [107]. (E) The DCF model [88]. We consider that the final structure labeled as ‘amyloid’ represents protofibrils rather than fibrils. In this step, the interactive surfaces of the monomers shift from intra-oligomeric to interoligomeric. With the application of shear stress or organic solvents, oligomeric granules become distorted [108,187] and fibril growth takes place almost instanta- neously. (F) The general OFF model [167]. In this model, denatured monomers M u are refolded into either stable monomer M or dimer D (the latter could be domain-swapped) or a less stable dimeric intermediate I (which again could be a partially-unfolded domain-swapped dimer). The initial steps are practically irreversible, and are followed by cooperative assembly of the fibril prone dimeric intermediates, I, into a nucleus, N, from which thin filaments, f, originate. Filaments grow linearly by repeated addition of I, and fibrils, F, form by lateral associa- tion of the filaments. F also elongate by end-to-end association [167]. (G) Off-pathway oligomers model, branching at domain-swapped dimer, as derived for stefin B [137]. Andrej Vilfan (Joz ˇ ef Stefan Institute, Ljubljana) prepared the artwork. The growth phase shows an anom- alous dependence on protein concentration, which is explained by off-pathway oligomer formation with a rate-limiting escape rate [137]. E. Z ˇ erovnik et al. Domain-swapping and amyloid fibril formation FEBS Journal 278 (2011) 2263–2282 ª 2011 The Authors Journal compilation ª 2011 FEBS 2269 organic solvents that are proposed to mimic the effects of biological surfaces. The most extensively studied example of a cystatin amyloid is that of stefin B, which is triggered by mildly acidic conditions and a low con- centration of TFE [127]. It is notable that stefin B forms long unbranched amyloid fibrils from a native- like intermediate [124,125]. These conditions often cor- respond to conditions that favor oligomeric states [123,124,126]. Proteins in which folding intermediates are popu- lated, such as cystatin C and stefin B [128,129], are more likely to form oligomers of the domain-swapped type than those folding in a two-state (N-U) manner. A number of conformational changes to the cystatin molecule (as a representative of globular proteins) undergoing oligomerization and, by extension, amyloid formation will be considered below, including the role of 3D domain-swapping and proline isomerization. The energetics of domain-swapping Intramolecular and intermolecular forces do not differ. The only parameters favoring the monomeric state are thus entropic. However, the edge strands usually pro- tect a monomer from direct interaction with another monomer [79], whereas the internal strands do not possess such built-in protection. Under denaturing conditions, the internal strands become exposed and they can shift from intra- to intermolecular arrange- ments. There also is considerable backbone strain in the loop between strands 2 and 3 in the monomer structure of stefin A [122] because this is required for its proteinase inhibitory activity. The driving force for dimerization may thus be the alleviation of this strain as loop 1 extends on formation of the dimer [122]. Whether kinetic or thermodynamic factors govern the oligomer formation remains to be clarified [130]. In certain proteins, metastable states can exist site by site because the kinetic barriers are too high to allow the energetic minimum to be reached in a rea- sonable time [131]. However, when barriers are crossed (e.g. by raising the temperature or pressure, by lower- ing the pH or adding denaturant), the thermodynami- cally most stable state [i.e. the lower oligomer (dimer), then higher oligomers and, finally, fibrils] can be attained. Because the temperature dependences of fibrillation and domain-swapping are the same (i.e. activation energy of approximately 100 kcalÆmol )1 ), it was con- cluded that domain-swapping may be the rate-deter- mining step [132]. Domain-swapping demands almost complete unfolding before the two chains can rearrange and swap strands [132]. Domain-swapped dimers have been observed for both the mammalian prion protein [133] and the cystatins [120–122], and, for a number of amyloidogenic proteins, it is observed that the process of fibrillogenesis starts with dimerization [134]. The height of the first barrier to fibrillation observed for the stefins is distinct from that measured in the case of a synuclein [13] and also HET prion [135], where a smaller activation energy of 22 kcalÆmol )1 was observed. The value of 100 kcalÆmol )1 is close to the energies needed for unfolding, whereas the value of 25 kcalÆmol )1 is characteristic for Pro cis–trans isomeri- zation. Because native a-synuclein is not folded, whereas stefin B is a globular protein, different interme- diates may be rate-determining for fibrillation. Theoret- ical studies [136] point to a role for hydrophobicity in the nucleation barriers. Fig. 2. Involvement of domain-swapping in amyloid fibril formation of cystatins. (A) Stefin A monomer (Protein Data Bank code: 1dvc) and domain-swapped dimer as found in the structure of the tetramer (Protein Data Bank code: 1N9J); (B) stefin B monomer (Protein Data Bank code: 1stf) and domain-swapped dimer (Protein Data Bank code: 2oct); and (C) proposed mechanism of the building up of amyloid fibrils obtained on the basis of stefin B H ⁄ D exchange and heteronuclear NMR. Adapted from Morgan et al. [163]. Domain-swapping and amyloid fibril formation E. Z ˇ erovnik et al. 2270 FEBS Journal 278 (2011) 2263–2282 ª 2011 The Authors Journal compilation ª 2011 FEBS Thus, we have shown that domain-swapping of ste- fins demands almost complete unfolding, with a high activation energy of approximately 100 kcalÆmol )1 pre- ceding stefin A domain-swapped dimerization [121]. It has been shown for RNAse A that dimerization is not always energy demanding, as indicated by the presence of a variety of different domain-swapped and non- swapped dimers [130]. However, for stefins, a high activation energy (as observed for domain-swapped dimerization) is also a prerequisite for the initiation of amyloid fibril growth [137] which, together with a prominent role of the dimers accumulating in the lag phase [126,127], supports the hypothesis that the domain-swapped dimers are directly or indirectly involved in the amyloid fibril formation of stefins. This is consistent with the case of the homologous cystatin C, where the prevention of domain-swapped dimeriza- tion also prevents amyloid fibril formation [138]. Role of proline cis–trans isomerization as a gate-keeper against oligomerization Studies on stefin B and b 2 -microglobulin have shown a link between oligomerization and cis to trans proline isomerization. The critical prolines are usually positioned in the loops that have to extend in the domain-swapping process, as also was the case with aA crystallin [139]. RNAse A forms a C-terminal domain-swapped dimer in which the b-strand consisting of residues 114– 124 (among them Pro114) is exchanged. Dimerization of RNAse A occurs under extreme conditions of acid, organic solvents or temperatures [140]. This is reminis- cent of stefin A domain-swapping [121] and implies a high-energy barrier. The crystal structures of the RNAse A monomer and C-terminal dimer reveal that Pro114 is trans in the dimer and cis in the monomer [130]. Another example is provided by domain-swapping in p13suc1, which occurs in the unfolded state and is controlled by conserved proline residues [141]. The monomer–dimer equilibrium is controlled by two con- served prolines in the hinge loop that connects the exchanging domains. They exploit the backbone strain to specifically direct dimer formation, at the same time as preventing higher-order oligomerization. Further- more, an excellent correlation between domain-swap- ping and aggregation has been observed, which again suggests a common mechanism. In the structure of the monomeric stefin B in com- plex with papain [142], the Pro103I is found to be trans, whereas, in the tetrameric structure, the homolo- gous residue Pro74 is cis. Hence, in the stefin B tetra- mer, the proline residue in the loops undergoing the exchange [123] has to isomerize from trans to cis. Accordingly, in amyloid fibril formation of the wild- type stefin B, the Pro74 cis isomeric state was found to be critically important. Its mutation to Ser prolonged the lag phase by up to ten-fold at room temperature and almost stopped fibril growth [143]. Furthermore, it was shown that the prolyl peptidyl cis–trans isomerase, cyclophilin A, profoundly delayed the fibrillation rate of the wild-type protein [143]. The potentially impor- tant role of proline isomerization in stefin B oligomeri- zation and fibril formation is also reflected in the activation energy of approximately 27 kcalÆmol )1 for the fibril elongation phase [137], which is in the range of proline isomerization reactions. Pro32 is cis in the native structure of b 2 -microglobu- lin. For this protein, cis to trans isomerism acts as the ‘gate-keeper’ for the transition to an intermediate con- formation serving as a direct precursor of fibril forma- tion [144–146]. The Pro32 trans to cis isomerization is facilitated by complexation with Cu 2+ , which is an important metal influencing amyloid formation in the brain [145,147,148]. Interestingly, stefin B also binds Cu 2+ in an oligomer-dependent manner [149], indicat- ing similar underlying processes. Domain versus loop-swapping In the process of 3D domain-swapping, as originally proposed by Bennett et al. [150] and Liu et al. [151], two protein chains of partially open monomers exchange the whole parts of their chains from the hinge loop to the termini, and fold back to two mono- meric domains. The extended surface of the ‘hinge loop’ is the only region of the protein that adopts a different conformation in the domain-swapped dimer from that in the monomer [120,122]. By contrast, in the process of loop-swapping, as seen in the tetramer of stefin B, which is a dimer of domain-swapped dimers [123], swapping of additional internal parts of the chain occurs from residues 72–80. It is therefore possible that an analogous mechanism of domain exchange is also present in the higher-order oligomers. In the ‘hand-shake’ of the loops observed by stefin B tetramer [123], the loop position from residues Ser72 to Leu80 is enabled by Pro74 and Pro79. The adopted loop position differs in the tetramer from that in the monomer and domain-swapped dimer. The monomer and domain-swapped dimers of stefins A and B are illustrated in Fig. 2. Pro74 is widely conserved in stefins and cystatins, and is found in trans isomeric state in all of the reported structures [120,122,142,152,153]. Only in the E. Z ˇ erovnik et al. Domain-swapping and amyloid fibril formation FEBS Journal 278 (2011) 2263–2282 ª 2011 The Authors Journal compilation ª 2011 FEBS 2271 high- resolution structure of the stefin B tetramer is it in the cis isomeric state [123]. The dimer to tetramer transition is associated with a rotation of domains, which appears mandatory for the 90° repositioning of the exchanged loops. From the superposition of stefin B monomers and stefin A and cystatin C domain- swapped dimers onto the tetramer structure, it is evi- dent that the Ser72-Leu80 loops and the N-terminal trunks have to adopt different conformation in the tet- ramer to prevent clashes [154]. The adopted conforma- tion of the Ser72-Leu80 loop and the N-terminal trunk is made possible only by the proline in the cis confor- mation. Indirectly, we have confirmed that proline isomeriza- tion is at the root of the slow conformational change coupled to tetramerization by measuring the tempera- ture dependence of the kinetics [123]. The value for the activation energy of 28 ± 3 kcalÆmol )1 observed for the P79S mutant tetramer formation is consistent with the contribution of one proline isomerization event, most likely the conversion of Pro74 from trans to cis. In the case of recombinant stefin B, in which both P74 and P79 are present, the activation energy is higher (i.e. 36 kcalÆmol )1 ), suggesting that Pro79 also contrib- utes to the loop rigidity, and its conformation would be strictly trans. These findings are consistent with those of Sanders et al. [155]. On the basis of thermodynamic and kinetic data, they concluded that oligomerization of the chicken cystatin occurred in the pre-exponential phase of the fibril growth. They describe that cystatin first undergoes a bimolecular transition to a domain- swapped dimer via a predominantly unfolded transi- tion state, followed by a unimolecular transition to a tetramer via a predominantly folded transition state [155]. Models for amyloid fibril formation based on domain-swapping ‘Run-away’ and ‘propagated domain-swapping’ models The domain-swapped oligomer can act either as a seed for fibril elongation (propagated domain-swapping) or as an end product (off-pathway domain-swapped dimers, tetramers) [156]. The process of domain-swap- ping is rate-limiting for the initiation of amyloid fibril formation, as reflected by a high energetic barrier [121,150]. In principle, any protein is capable of oligo- merization by 3D domain-swapping [157]. Ogihara et al. [158] designed a sequence of RNAse A that underwent a reciprocated swap and another that ended in a propagated swap (Table 1). Under partially denaturing conditions, the protein molecule partially opens and, when stabilizing condi- tions are restored, the partially-unfolded monomers can swap domains. When the exchange of secondary structure elements is not reciprocated but propagated along multiple polypeptide chains, this can result in higher-order assemblies [159]. Guo and Eisenberg [160] proposed the term ‘run-away domain-swapping’ mech- anism for such a process of continuous domain-swap- ping. In their study of T7 endonuclease, Guo and Eisen- berg [160] define ‘run-away domain-swapping’ as a mechanism in which each protein molecule swaps a domain into the neighboring molecule along the grow- ing fibril. By designing disulfide bonds that form only at the domain-swapped dimer interface, they were able to show that the resulting covalently-linked fibrils con- tained domain-swapped dimers. If these were locked in a close-ended dimeric form by making internal disul- fide bonds, they were unable to form fibrils. A study by Liu et al. [161] indicates that the b-sheet spine in amyloid fibrils of b 2 -microglobulin could be made from amyloidogenic peptide sequences of the hinge regions of domain-swapped dimers, which also build the prefibrillar, curvelinear oligomers. For the example of aA crystallin, Laganowsky and Eisenberg [139] have shown even more plasticity in the way that the N- or C- terminal parts can swap from one molecule to another. Wahlbom et al. [162] used the term ‘propagated domain-swapping’ to describe a similar process of con- tinuous domain-swapping in the formation of cystatin C prefibrillar oligomers and fibrils. They showed annu- lar oligomers with an outer diameter of 13 nm at the beginning of fibril formation, which transformed to mature fibrils of 10 nm in width. From their study, it is not shown clear at which state the disulfide bond stabilizes the domain-swap. On the basis of the H ⁄ D exchange study of Morgan et al. [163], we suggest that, in the case of stefins, and in addition to initial domain-swapping to produce the domain-swapped dimer, there could be further exchange of loops. We propose that such additional loop-swapping could occur between the loop extending from the only a-helix to strand 2 of one domain- swapped dimer with another acting as one ‘click’, and between loops from strands 4–5 as another ‘click’, in a similar process to that taking place in the tetramer. Alternatively, whole a-helices and N-terminals could swap. Clearly, a 3D structure of a higher oligomer in the range of 12–16 mers is mandatory to provide insight into such exchange events. Domain-swapping and amyloid fibril formation E. Z ˇ erovnik et al. 2272 FEBS Journal 278 (2011) 2263–2282 ª 2011 The Authors Journal compilation ª 2011 FEBS [...]... 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REVIEW ARTICLE Mechanisms of amyloid fibril formation – focus on domain-swapping Eva Z ˇ erovnik 1 , Veronika Stoka 1 , Andreja Mirtic ˇ 2 ,. double-concerted fibrillation; IDPs, intrinsically disordered proteins; MDC, monomer-directed conversion; NCC, nucleated conformational conversion; NDP,