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
Trang 1Mechanisms of amyloid fibril formation – focus on
domain-swapping
Eva Zˇerovnik1, Veronika Stoka1, Andreja Mirticˇ2, Gregor Guncˇar3, Jozˇe Grdadolnik2,4,
Rosemary A Staniforth5, Dusˇan Turk1,6and Vito Turk1
1 Department of Biochemistry and Molecular and Structural Biology, Jozˇef 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
Ca2+ 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 Zˇerovnik, Department of Biochemistry
and Molecular and Structural Biology, Jozˇef
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.
Trang 2still 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 ‘globulooligo-mers’), ‘granules’, ‘critical oligooligo-mers’
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,
Trang 3amongst others, are to prevent aggregation of
incom-pletely folded polypeptide chains [31] and to
disaggre-gate formed aggredisaggre-gates [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 conformaconforma-tion,
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, aR, aLand 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 aR
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 formaaggrega-tion 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
Trang 4a 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 MDC model [92] (Fig 1B) Prion, stefin B at pH 7
(from monomer)
B NDP model [99] (Fig 1C) Amyloid-b peptide
protein Sup35
C ‘Polar zipper’ model [93–96] Huntingtin, ataxin-3 Linear colloid-like assembly of spherical oligomers examples
kinase
B DA model [107] (Fig 1D) Tau 40 protein
[88,108] (Fig 1E)
a-Synuclein
D Isodesmic (linear) polymerization [104,185]
b2-Microglobulin stefin B at
pH 3 (from globular oligomer)
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.
Trang 5decisive 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
Trang 6k1 k−OPk2 kI kG
Dimer
Rearrangement
Protofibril Off-path oligomer
A
B
C
D
E
F
G
Trang 7Domain-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 accumulasecre-tion,
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 associaassocia-tion [167] (G) Off-pathway oligomers model, branching at domain-swapped dimer, as derived for stefin B [137] Andrej Vilfan (Jozˇef 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].
Trang 8organic 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 addlower-ing 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)1is 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].
Trang 9Thus, 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 formadimeriza-tion [138]
Role of proline cis–trans isomerization as a
gate-keeper against oligomerization
Studies on stefin B and b2-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 b2 -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 isomerizaforma-tion is facilitated by complexation with Cu2+, which is an important metal influencing amyloid formation in the brain [145,147,148] Interestingly, stefin B also binds
Cu2+ in an oligomer-dependent manner [149], indicat-ing similar underlyindicat-ing 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
Trang 10high- 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 conformaisomeriza-tional 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 transitransi-tion 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 designgrow-ing 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 b2-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