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:
(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
An example of the propagated domain-swapping is
provided by human cystatin C. Amyloid fibril forma-
tion by human cystatin C has been studied [162,164]
and connected to the domain-swapping of this mole-
cule [120,165]. In an experiment where dimer forma-
tion was prevented by engineered disulfide bridges,
fibril formation was also prevented [165]. From these
studies, it is apparent that dimers play an important
role in fibril formation, although this does not imply
they should build the fibrils directly.
A further example of a domain-swapping mechanism
underlying amyloid fibril formation is provided by the
human stefins. In vitro studies have demonstrated that
stefin A, albeit under rather harsh preconditioning, is
able to form amyloid fibrils [64,125], as would be
expected if this process is generic [66]. In stefin A
monomer and dimer, there are more salt bridges inter-
connecting the a-helix and strand 2 to the rest of the
structure than in its stefin B homolog [59,125]. Conse-
quently, stefin A can only form amyloid fibrils in vitro
under very stringent conditions compared to the
almost physiological conditions needed for stefin B
[64,166]. Fibrillation of stefin A can be initiated by
heating the protein to predenaturing temperatures of
approximately 90 °C, which promotes domain-
swapped dimer formation, and by reducing the pH
below 2.5, which partially unwinds the dimer [121].

Staniforth et al. [122] originally proposed the propaga-
tion of the domain-swapped dimer of stefin A into
fibrils through the open ends on the N- and C-termini.
Similar to other cystatins, stefin B is prone to form
domain-swapped dimers (Fig. 2B). H ⁄ D exchange and
heteronuclear NMR studies on stefin B mature fibrils
do not appear to confirm the initial prediction [122]
based on the structure of stefin A dimers. Rather, they
suggest that the fibrils are themselves highly structured,
being made from a protected core of strands 2–5,
whereas the a-helix and strand 1 are unprotected. This
makes sense if the a-helix and strand 1 were flanking
from the spine of the fibril [163] (Fig. 2C). These
results imply that lower domain-swapped oligomers of
cystatins (dimers and tetramers) do not directly build
mature fibrils. It appears that the oligomers can
become fibril building blocks only after partial unfold-
ing of the a-helix from the body of the b-sheet
(Fig. 2C).
Possible role of domain-swapping in the ‘off-pathway
folding’ (OFF) model
The OFF model for amyloid formation was first
described by Pallitto and Murphy [167]. The general
model is described in more detail in Fig. 1F and is
included in Table 1. The off-pathway oligomers are the
dead end of an alternative folding pathway, which is
incapable of converting directly to fibrils and substan-
tially slowing their formation. As the protein concen-
tration increases, the off-pathway oligomers become
even longer lived, which is doubly detrimental in that

their lifetime increases when they become more abun-
dant [137,168].
An example of the OFF model, involving domain-
swapping, is provided by human stefin B (Fig. 1G).
The fibrillation of stefin B at room temperature and at
approximately pH 5 is characterized by an extensive
lag phase, in which granular aggregates have been
observed by TEM and AFM, appearing as micelle-like
arrangements of oligomers [124–127,169]. After the lag
phase, various morphologies have been detected during
the fibril growth phase, from annular to spherical, rod-
like and amorphous species [126,169]. Unlike at room
temperature, at temperatures above 35 °C, thioflavin T
fluorescence shows no visible lag phase [137]. The sub-
sequent growth phase shows an anomalous dependence
on protein concentration; at low concentrations, the
final value is reached faster than at higher concentra-
tions. This observation is explained in terms of an off-
pathway state with a rate-limiting escape rate [137].
However, there may be two (or more) pathways by
which this protein aggregates, depending on pH and
ionic strength [124,126,127].
Discussion
Although it is essential to study different conforma-
tional states populated by the amyloid precursor pro-
teins, it is a difficult task to draw links between states
occurring during folding and the ‘misfolded’ states
populated during amyloid formation. In some cases,
extensive study allows us to determine the pathways to
which different conformations belong. However, our

understanding of the importance of different parts of
the molecule and their flexibility in the process of amy-
loid fibril formation often results more from a struc-
tural analysis of the amyloid endpoint. As with many
other proteins [80], there is no clear link between the
fold, stability, unfolding or folding rates of stefins and
their propensity to form amyloid [59]. Studying the
effect of different factors (i.e. from pH, temperature to
concentration of TFE) on amyloid fibril formation,
and looking at how these combine to cause the specific
changes favoring amyloid over native folds, or even
alternative (off-pathway) oligomers, where the additiv-
ity of different effects may not be straightforward, is a
formidable task. Nevertheless, such studies do provide
us with a useful insight into the mechanism of amyloid
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 2273
formation. It often becomes apparent that changes in
specific parts of the protein molecule (e.g. either pro-
tonation of a number of side chains, binding of metals
to an unfolded state or mutations at specific sites) are
key to amyloid formation. One of the major players
appear to be prolines at critical loop positions, which
may act as gate-keepers to amyloid-preceding confor-
mation; this remains to be discussed. As the number of
studies grow, insight will be provided as to how spe-
cific (or indeed how random, directed or evolved) the
amyloid structure may be. The answer to the equation

is therefore not simply to determine the conditions that
are sufficiciently destabilizing to favor large conforma-
tional changes but, instead, those that are sufficiciently
stabilizing to produce a new structure. It is tempting
to imagine that these changes are controlled by nature,
just as they can be by the scientist.
In the case of globular proteins, the formation of
partially-unfolded intermediates populated from the
native state or accessible during refolding is the first
critical step of the pathway of amyloid fibril formation
[61,170,171]. Taking the example of stefin B, partial
unfolding is a prerequisite for both protofibril and
fibril formation. We have observed that protofibrils
tend to form from the structured molten globule
obtained at pH 3.3 and the mature fibrils from the
partially-unfolded monomer (native-like intermediate)
populated at pH 4.8, which transforms into domain-
swapped dimer [124,126,127]. We also have shown that
the aggregates formed from different partially-folded
intermediates differ in toxicity [172].
The intrinsically disordered proteins (IDPs) [173],
constitute a large fraction of naturally occurring amy-
loidogenic proteins [174]. In the case of IDPs (i.e.
natively unfolded proteins), the formation of partially
structured conformers occurs by partial folding, and
fibril formation is promoted by factors that induce
partial folding [12,13,175]. For example, in the case
of a-synuclein, either a decrease in pH or an increase
in temperature appears to induce partial folding, as
well as enhance the propensity of the protein to fibril-

late [13]. Similar to intermediates formed from the
partial folding of globular proteins, the aggregate-
prone intermediates of IDPs can polymerize to form
fibrillar or amorphous aggregates, or soluble oligo-
mers.
The generalized picture that we describe below may
hold for the folded globular proteins more than for
IDPs. A common trait is the fact that the monomers
have to undergo a conformational change (i.e. partial
opening), which demands partially denaturing condi-
tions. The conformational change can happen more
easily on a template of another, already distorted,
monomer (MDC) or a nucleus acting as a template
(TA). In the case of stefin B, under mild solvent condi-
tions (from pH 5.5 to neutral), the partially-folded
monomers tend to form closed domain-swapped
dimers, tetramers, etc., up to octamers and even
dodecamers, which persist temporarily as off-pathway
oligomers. On the main pathway to fibrils, slightly
below pH 5, some of the oligomers (ring-like, annular
oligomers of 4–20 nm in diameter) [169] unwind and
rearrange to make short protofibrils. As discussed by
Wahlbom et al. [162], this transition can take place in
two ways: either the small oligomer rings gather one
above the other (meaning the protofibril would be hol-
low) or they open and rearrange to become a growing
filament, a number of which wind around each other.
Under more stringent denaturing solvent conditions
(of pH 3.3 or at a higher temperature at pH 7) that
destabilize the lower oligomers, oligomers higher than

dimers [126,127], and possibly tetramers [155], cannot
form. Therefore, partially-unfolded monomers and
dimers accumulate until they form the so-called ‘criti-
cal oligomers’, which comprise part of the insoluble
granular aggregate. When such a critical mass is
reached and the oligomeric spheres gain sufficiently
large dipole moments, they form linear chains in the
form of colloid particles to give protofibrils. These can
interact laterally, building up fibrils. Under suitable
solvent conditions, the protofibrils smooth out into fil-
aments, which wind around each other and form
mature fibrils, whereas, under some other solvent con-
ditions, they remain protofibrillar. It is also possible
that several fibril morphologies could exist side by
side.
In the NP, NDP and NCC models (Table 1), the
partially-unfolded intermediates, when present at a
critical concentration, slowly assemble into a nucleus,
within which the first conformational change takes
place. These oligomeric nuclei then rapidly grow into
globular oligomers, also termed ‘granules’ or ‘spher-
oids’ and, after reaching a ‘critical’ size, go on to form
chain-like protofibrils (CO and DA models), which
eventually form fibrils [97,176] or remain protofibrillar.
Some other models, such as the DCF model, predict
that a second concerted conformational change has to
take place within the globular oligomers, after which
they can chain up (i.e. as colloid particles) into
protofibrils [102,104].
We have noted that the amyloid fibril formation of

proteins that form domain-swapped oligomers (e.g.
cystatins and stefins) initially follow nucleation kinetics
with a prominent lag phase. However, when the high
barrier to partial unfolding is crossed (under different
solvent conditions or increased temperature), they
Domain-swapping and amyloid fibril formation E. Z
ˇ
erovnik et al.
2274 FEBS Journal 278 (2011) 2263–2282 ª 2011 The Authors Journal compilation ª 2011 FEBS
follow a linear growth of globular oligomers similar to
that of the DA or CO models.
Only a small change in reaction conditions, in which
different structural intermediates form, can turn the
mechanism from one path to another. Polyglutamine
repeat-containing proteins are a good example, for
which a domain-swapped model [177], polar zipper
[94], cross-linking [178] model and a nucleation-depen-
dent pathway [179] have all been proposed. Accord-
ingly, our model protein stefin B follows a different
pathway towards fibrils or protofibrils at different pHs
and temperatures (Table 1). Interestingly, several par-
allel mechanisms can apply even to a single protein,
leading to final fibril heterogeneity [102].
It is a great challenge to derive a common, generic
model of amyloid fibril formation that would define a
single preferred pathway for a given protein sequence,
covering most of the possible conditions. Is it likely
that there is only one generic mechanism of amyloid
fibril formation? Is there a common mechanism of pro-
tein folding? Or are there extreme cases of two-state

folding on one end and noncooperative transitions via
multiple intermediates on the other, with all the rest
inbetween? [180]. In folding, the energy landscape rep-
resentation [181] is used to show different scenarios,
with steep funnels or ragged surfaces, slowly descend-
ing into a final funnel. In certain cases of metastable
states, the funnels can end in two or three minima.
We propose that such metastable states preceding
fibril formation could well be domain-swapped dimers
and higher oligomers, preceding or gate-keeping the
amyloid fibril formation. High-energy barriers of the
order of 100 and 30 kcalÆmol
)1
occur in the domain-
swapped dimerization of stefin A [121] and in the tet-
ramerization of stefin B [123], respectively. These barri-
ers are equivalent to those corresponding to almost
complete unfolding and proline isomerization. Both
barriers occur in amyloid-fibril formation by this pro-
tein [137]. We further propose that the term ‘propa-
gated domain-swapping’ would encompass both
domain-swapping and loop-swapping. The domain-
swapping demands almost complete unfolding
(> 90 kcalÆmol
)1
) and loop-swapping, usually an
extensive conformational change involving proline cis–
trans isomerization. One such reaction would cost
28 kcalÆmol
)1

, and two, occurring within the nucleus,
would cost approximately 56 kcalÆmol
)1
[137].
The cis to trans isomerization of prolines is rate-lim-
iting for many processes from protein folding to
switching on ⁄ off of neurotransmitter ion channels
[182]. In many cases, proline isomerism also serves as
a gate-keeper to amyloid-fibril formation [130,137,
141,145,146].
In the context of neurodegenerative disorders, it was
shown that a prolyl isomerase facilitates the formation
of a-synuclein inclusions (i.e. the Lewy bodies of Par-
kinson’s disease) [183], and that the prolyl isomerase
Pin1 regulates amyloid precursor protein processing
and amyloid-b production in Alzheimer’s disease [184].
Taken together, there is a growing body of evidence
indicating that proline residues have crucial roles in
the processes of oligomerization and amyloid fibril for-
mation, suggesting mechanisms that could destabilize
the structure of toxic intermediates and thus prevent
their undesired activity [123].
Acknowledgements
The work was supported by programs P1-0140 (prote-
olysis and its regulation, led by B. Turk) and P1-0048
(Structural Biology, led by D. Turk) and by the project
J3-2258 (V. Stoka), all financed by the Slovenian
Research Agency (ARRS). Work in Sheffield is funded
by grants to RAS from the BBSRC (UK) (BB ⁄
C504035 ⁄ 1) and the Royal Society (516002.K5631).

We thank Professor R. H. Pain for editing the English
and making useful suggestions.
References
1 Irvine GB, El-Agnaf OM, Shankar GM & Walsh DM
(2008) Protein aggregation in the brain: the molecular
basis for Alzheimer’s and Parkinson’s diseases.
Mol Med 14 , 451–464.
2 Morris AM, Watzky MA & Finke RG (2009) Protein
aggregation kinetics, mechanism, and curve-fitting: a
review of the literature. Biochim Biophys Acta 1794,
375–397.
3 Ross CA & Poirier MA (2004) Protein aggregation
and neurodegenerative disease. Nat Med 10(Suppl),
S10–S17.
4 Uversky VN (2010) Mysterious oligomerization of the
amyloidogenic proteins. FEBS J 277, 2940–2953.
5 Almeida CG, Takahashi RH & Gouras GK (2006)
Beta-amyloid accumulation impairs multivesicular
body sorting by inhibiting the ubiquitin-proteasome
system. J Neurosci 26, 4277–4288.
6 Ross CA & Pickart CM (2004) The ubiquitin-protea-
some pathway in Parkinson’s disease and other
neurodegenerative diseases. Trends Cell Biol 14,
703–711.
7 Bush AI & Tanzi RE (2008) Therapeutics for
Alzheimer’s disease based on the metal hypothesis.
Neurotherapeutics 5, 421–432.
8 Duce JA & Bush AI (2010) Biological metals and
Alzheimer’s disease: implications for therapeutics and
diagnostics. Prog Neurobiol 92, 1–18.

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 2275
9 Vernace VA, Schmidt-Glenewinkel T & Figueiredo-
Pereira ME (2007) Aging and regulated protein
degradation: who has the UPPer hand? Aging Cell 6,
599–606.
10 Wong E & Cuervo AM (2010) Autophagy gone awry
in neurodegenerative diseases. Nat Neurosci 13, 805–
811.
11 Hipkiss AR (2010) Mitochondrial dysfunction, proteo-
toxicity, and aging: causes or effects, and the possible
impact of NAD
+
-controlled protein glycation.
Adv Clin Chem 50, 123–150.
12 Uversky VN & Fink AL (2005) Pathways to Amyloid
Fibril Formation: Partially Folded Intermediates in
the Fibril Formation of Natively Unfolded Proteins.
Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
13 Uversky VN, Li J & Fink AL (2001) Evidence for a
partially folded intermediate in alpha-synuclein fibril
formation. J Biol Chem 276, 10737–10744.
14 Dobson CM (2002) Protein-misfolding diseases: getting
out of shape. Nature 418, 729–730.
15 Kayed R, Head E, Thompson JL, McIntire TM,
Milton SC, Cotman CW & Glabe CG (2003) Common
structure of soluble amyloid oligomers implies common
mechanism of pathogenesis. Science 300, 486–489.

16 Jang H, Zheng J & Nussinov R (2007) Models of
beta-amyloid ion channels in the membrane suggest
that channel formation in the bilayer is a dynamic
process. Biophys J 93, 1938–1949.
17 Lashuel HA & Lansbury PT (2006) Are amyloid dis-
eases caused by protein aggregates that mimic bacterial
pore-forming toxins? Q Rev Biophys 39, 167–201.
18 Sahoo B, Balaji J, Nag S, Kaushalya SK & Maiti S
(2008) Protein aggregation probed by two-photon fluo-
rescence correlation spectroscopy of native tryptophan.
J Chem Phys 129, 075103.
19 Sipe JD (2005) The b -Pleated Sheet Conformation and
Protein Folding: A Brief History. Wiley-VCH Verlag
GmbH & Co. KGaA, Weinheim.
20 Sunde M & Blake CCF (1998) From the globular to the
fibrous state: protein structure and structural conversion
in amyloid formation. Q Rev Biophys 31, 1–39.
21 Langkilde AE & Vestergaard B (2009) Methods for
structural characterization of prefibrillar intermediates
and amyloid fibrils. FEBS Lett 583, 2600–2609.
22 Goldsbury C, Frey P, Olivieri V, Aebi U & Muller SA
(2005) Multiple assembly pathways underlie amyloid-
beta fibril polymorphisms. J Mol Biol 352, 282–298.
23 Kad NM, Thomson NH, Smith DP, Smith DA &
Radford SE (2001) beta(2)-microglobulin and its deam-
idated variant, N17D form amyloid fibrils with a range
of morphologies in vitro. J Mol Biol 313, 559–571.
24 Petkova AT, Leapman RD, Guo Z, Yau WM, Matt-
son MP & Tycko R (2005) Self-propagating, molecu-
lar-level polymorphism in Alzheimer’s beta-amyloid

fibrils. Science 307, 262–265.
25 Tycko R (2006) Molecular structure of amyloid fibrils:
insights from solid-state NMR. Q Rev Biophys 39, 1–55.
26 Hoshino M, Katou H, Hagihara Y, Hasegawa K,
Naiki H & Goto Y (2002) Mapping the core of the
beta(2)-microglobulin amyloid fibril by H ⁄ D exchange.
Nat Struct Biol 9, 332–336.
27 Yu L, Edalji R, Harlan JE, Holzman TF, Lopez AP,
Labkovsky B, Hillen H, Barghorn S, Ebert U,
Richardson PL et al. (2009) Structural characterization
of a soluble amyloid beta-peptide oligomer. Biochemis-
try 48, 1870–1877.
28 Glabe CG (2008) Structural classification of toxic
amyloid oligomers. J Biol Chem 283, 29639–29643.
29 Armen RS, DeMarco ML, Alonso DO & Daggett V
(2004) Pauling and Corey’s alpha-pleated sheet struc-
ture may define the prefibrillar amyloidogenic interme-
diate in amyloid disease. Proc Natl Acad Sci USA 101,
11622–11627.
30 Gething MJ & Sambrook J (1992) Protein folding in
the cell. Nature 33, 355.
31 Ellis RJ & Hartl FU (1999) Principles of folding in the
cellular environment. Curr Opin Struct Biol 9, 102.
32 Arimon M, Grimminger V, Sanz F & Lashuel HA
(2008) Hsp104 targets multiple intermediates on the
amyloid pathway and suppresses the seeding capacity
of Abeta fibrils and protofibrils. J Mol Biol 384,
1157–1173.
33 Glover JR & Lum R (2009) Remodeling of
protein aggregates by Hsp104. Protein Pept Lett 16,

587–597.
34 Vashist S, Cushman M & Shorter J (2010) Applying
Hsp104 to protein-misfolding disorders. Biochem Cell
Biol 88, 1–13.
35 Dill KA (1990) Dominant forces in protein folding.
Biochemistry 29, 7133–7155.
36 Kauzmann W (1959) Factors in interpretation of
protein denaturation. Adv Protein Chem 14, 1–63.
37 Avbelj F & Moult J (1995) Role of electrostatic screen-
ing in determining protein main chain conformational
preferences. Biochemistry 34, 755–764.
38 Brant DA & Flory PJ (1965) The role of dipole inter-
actionsin determining polypeptide conformation. JAm
Chem Soc 87, 663–664.
39 Perutz MF (1978) Electrostatic effects in proteins.
Science 201, 1187–1191.
40 Klapper MH (1971) On the nature of the protein
interior. Biochim Biophys Acta 229, 557–566.
41 Richards FM (1977) Areas, volumes, packing and
protein structure. Annu Rev Biophys Bioeng 6, 151–176.
42 Schellman JA (1955) The stability of hydrogen-bonded
peptide structures in aqueous solution. CR Trav Lab
Carlsberg Ser Chim 29, 230–259.
43 Stickle DF, Presta LG, Dill KA & Rose GD (1992)
Hydrogen bonding in globular proteins. J Mol Biol
226, 1143–1159.
Domain-swapping and amyloid fibril formation E. Z
ˇ
erovnik et al.
2276 FEBS Journal 278 (2011) 2263–2282 ª 2011 The Authors Journal compilation ª 2011 FEBS

44 Makhatadze GI & Privalov PL (1993) Contribution of
hydration to protein folding. I. The enthalpy of hydra-
tion. J Mol Biol 232, 639–659.
45 Wolfenden R (1978) Interaction of the peptide bond
with solvent water: a vapor Phase Analysis. Biochemis-
try 17, 201–204.
46 Avbelj F & Fele L (1998) Role of main-chain electro-
statics, hydrophobic effect and side-chain conforma-
tional entropy in determining the secondary structure
of proteins. J Mol Biol 279, 665–684.
47 Avbelj F, Golic
ˇ
Grdadolnik S, Grdadolnik J & Bald-
wine LR (2006) Intrinsic backbone preferences are fully
present in blocked amino acids. Proc Natl Acad Sci
USA 103, 1272–1277.
48 Grdadolnik J, Grdadolnik SG & Avbelj F (2008)
Determination of conformational preferences of dipep-
tides using vibrational spectroscopy. J Phys Chem B
112, 2712–2718.
49 Avbelj F (2000) Amino acid conformational prefer-
ences and solvation of polar backbone atoms in pep-
tides and proteins. J Mol Biol 300, 1335–1359.
50 Avbelj F, Luo P & Baldwin RL (2000) Energetics of
the interaction between water and the helical peptide
group and its role in determining helix propensities.
Proc Natl Acad Sci USA 97, 10786–10791.
51 Avbelj F & Baldwin RL (2003) Role of backbone sol-
vation and electrostatics in generating preferred peptide
backbone conformations: distributions of phi. Proc

Natl Acad Sci USA 100, 5742–5747.
52 Wu C, Lei XH & Duan Y (2004) Formation of par-
tially ordered oligomers of amyloidogenic hexapeptide
(NFGAIL) in aqueous solution observed in molecular
dynamics simulations. Biophys J 87, 3000–3009.
53 Gsponer J, Haberthur U & Caflisch A (2003) The role
of side-chain interactions in the early steps of aggrega-
tion: molecular dynamics simulations of an amyloid-
forming peptide from the yeast prion Sup35. Proc Natl
Acad Sci USA 100, 5154–5159.
54 Lopez de la Paz M, Goldie K, Zurdo J, Lacroix E,
Dobson CM, Hoenger A & Serrano L (2002) De novo
designed peptide-based amyloid fibrils. Proc Natl Acad
Sci USA 99, 16052–16057.
55 Aggeli A, Nyrkova IA, Bell M, Harding R, Carrick L,
McLeish TCB, Semenov AN & Boden N (2001) Hier-
archical self-assembly of chiral rod-like molecules as a
model for peptide b-sheet tapes, ribbons, fibrils, and
fibers. Proc Natl Acad Sci USA 98, 11857–11862.
56 Krebs MR, Devlin GL & Donald AM (2009) Amyloid
fibril-like structure underlies the aggregate structure
across the pH range for beta-lactoglobulin. Biophys J
96, 5013–5019.
57 Zhang S & Rich A (1997) Direct conversion of an
oligopeptide from a b-sheet to an a-helix: a model
for amyloid formation. Proc Natl Acad Sci USA 94,
23–28.
58 Ferna
´
ndez A (2005) What factor drives the fibrillogenic

association of b-sheets? FEBS Lett 579, 6635–6640.
59 Kenig M, Jenko-Kokalj S, Tusek-Znidaric M, Pompe-
Novak M, Guncar G, Turk D, Waltho JP, Staniforth
RA, Avbelj F & Zerovnik E (2006) Folding and amy-
loid-fibril formation for a series of human stefins’ chi-
meras: any correlation? Proteins
62, 918–927.
60 Rabzelj S, Turk V & Zerovnik E (2005) In vitro study
of stability and amyloid-fibril formation of two
mutants of human stefin B (cystatin B) occurring in
patients with EPM1. Protein Sci 14, 2713–2722.
61 Kelly JW (1998) The alternative conformations of amy-
loidogenic proteins and their multi-step assembly path-
ways. Curr Opin Struct Biol 8, 101–106.
62 Lansbury PT (1999) Evolution of amyloid: what nor-
mal protein folding may tell us about fibrillogenesis
and disease. Proc Natl Acad Sci USA 96, 3342–3344.
63 Rochet JC & Lansbury PT (2000) Amyloid fibrillogen-
esis: themes and variations. Curr Opin Struct Biol 10,
60–68.
64 Zerovnik E (2002) Amyloid-fibril formation. Proposed
mechanisms and relevance to conformational disease.
Eur J Biochem 269, 3362–3371.
65 Daizo H & Dobson CM (2002) A kinetic study of b-
lactoglobulin amyloid fibril formation promoted by
urea. Protein Sci 11, 2417–2426.
66 Guijarro JI, Sunde M, Jones JA, Campbell ID & Dob-
son CM (1998) Amyloid fibril formation by an SH3
domain. Proc Natl Acad Sci USA 95, 4224–4228.
67 Ramirez-Alvarado M, Merkel JS & Regan L (2000) A

systematic exploration of the influence of the protein
stability on amyloid fibril formation in vitro. Proc Natl
Acad Sci USA 97, 8979–8984.
68 Bousset L, Thomson NH, Radford SE & Melki R
(2002) The yeast prion Ure2p retains its native a con-
formation upon assembly into protein fibrils. EMBO J
21, 2903–2911.
69 Laurine E, Gregoire C, Faendrich M, Engemann S,
Marchal S, Thion L, Mohr M, Monsarrat B, Michel
B, Dobson CM et al. (2003) Lithostathine quadruple-
helical filaments form proteinase K-resistant deposits in
Creutzfeldt-Jakob disease. J Biol Chem 278, 51770–
51778.
70 Fandrich M, Fletcher MA & Dobson CM (2001)
Amyloid fibrils from muscle myoglobin. Nature 410,
165–166.
71 Kallberg Y, Gustafsson M, Persson B, Thyberg J &
Johansson J (2001) Prediction of amyloid fibril-forming
proteins. J Biol Chem 276, 12945–12950.
72 Bitan G, Vollers SS & Teplow DB (2003) Elucidation
of primary structure elements controlling early amyloid
beta-protein oligomerization. J Biol Chem 278, 34882–
34889.
73 Collins SR, Douglass A, Vale RD & Weissman JS
(2004) Mechanism of prion propagation: amyloid
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 2277
growth occurs by monomer addition. PLoS Biol 2,

1582–1590.
74 Hess S, Lindquist SL & Scheibel T (2007) Alternative
assembly pathways of the amyloidogenic yeast prion
determinant Sup35-NM. EMBO Rep 8, 1196–1201.
75 Kumar S & Udgaonkar JB (2009) Conformational con-
version may precede or follow aggregate elongation on
alternative pathways of amyloid protofibril formation.
J Mol Biol 385, 1266–1276.
76 Thakur AK, Jayaraman M, Mishra R, Thakur M,
Chellgren VM, Byeon IJ, Anjum DH, Kodali R,
Creamer TP, Conway JF et al. (2009) Polyglutamine
disruption of the Huntingtin exon 1 N terminus trig-
gers a complex aggregation mechanism. Nat Struct
Mol Biol 16, 380–389.
77 Heise H, Hoyer W, Becker S, Andronesi OC, Riedel D
& Baldus M (2005) Molecular-level secondary struc-
ture, polymorphism, and dynamics of full-length alpha-
synuclein fibrils studied by solid-state NMR. Proc Natl
Acad Sci USA 102, 15871–15876.
78 Kad NM, Myers SL, Smith DP, Smith DA, Radford
SE & Thomson NH (2003) Hierarchical assembly of
beta(2)-microglobulin amyloid in vitro revealed by
atomic force microscopy. J Mol Biol 330, 785–797.
79 Richardson JS & Richardson DC (2002) Natural
b-sheet proteins use negative design to avoid edge-to-
edge aggregation. Proc Natl Acad Sci USA 99, 2754–
2759.
80 Chiti F, Stefani M, Taddei N, Ramponi G & Dobson
CM (2003) Rationalization of the effects of mutations
on peptide and protein aggregation rates. Nature 424,

805–808.
81 Pawar AP, Dubay KF, Zurdo J, Chiti F, Vendruscolo
M & Dobson CM (2005) Prediction of ‘aggregation-
prone’ and ‘aggregation-susceptible’ regions in proteins
associated with neurodegenerative diseases. J Mol Biol
350, 379–392.
82 Ventura S, Zurdo J, Narayanan S, Parreno M, Man-
gues R, Reif B, Chiti F, Giannoni E, Dobson CM,
Aviles FX et al. (2004) Short amino acid stretches can
mediate amyloid formation in globular proteins: the
Src homology 3 (SH3) case. Proc Natl Acad Sci USA
101, 7258–7263.
83 Conchillo-Sole O, de Groot NS, Aviles FX, Vendrell J,
Daura X & Ventura S (2007) AGGRESCAN: a server
for the prediction and evaluation of ‘hot spots’ of
aggregation in polypeptides. BMC Bioinformatics 8, 65.
84 Fernandez-Escamilla AM, Rousseau F, Schymkowitz J
& Serrano L (2004) Prediction of sequence-dependent
and mutational effects on the aggregation of peptides
and proteins. Nat Biotechnol 22, 1302–1306.
85 Thompson MJ, Sievers SA, Karanicolas J, Ivanova
MI, Baker D & Eisenberg D (2006) The 3D profile
method for identifying fibril-forming segments of pro-
teins. Proc Natl Acad Sci USA 103, 4074–4078.
86 Goldschmidt L, Teng PK, Riek R & Eisenberg D
(2010) Identifying the amylome, proteins capable of
forming amyloid-like fibrils. Proc Natl Acad Sci USA
107, 3487–3492.
87 Kelly JW (2000) Mechanisms of amyloidogenesis. Nat
Struct Biol 7, 824–826.

88 Bhak G, Choe YJ & Paik SR (2009) Mechanism of
amyloidogenesis: nucleation-dependent fibrillation
versus double-concerted fibrillation. BMB Rep 42,
541–551.
89 Hamada D, Tanaka T, Tartaglia GG, Pawar A,
Vendruscolo M, Kawamura M, Tamura A, Tanaka N
& Dobson CM (2009) Competition between folding,
native-state dimerisation and amyloid aggregation in
beta-lactoglobulin. J Mol Biol 386, 878–890.
90 Merlini G & Bellotti V (2003) Molecular mechanisms
of amyloidosis. N Engl J Med 349, 583–596.
91 Griffith JS (1967) Self-replication and scrapie. Nature
215, 1043–1044.
92 Prusiner SB (1982) Novel proteinaceous infectious
particles cause scrapie. Science 216, 136–144.
93 Perutz MF, Staden R, Moens L & De Baere I (1993)
Polar zippers. Curr Biol 3, 249–253.
94 Perutz M (1994) Polar zippers: their role in human
disease. Protein Sci 3, 1629–1637.
95 Perutz MF, Johnson T, Suzuki M & Finch JT (1994)
Glutamine repeats as polar zippers – their possible
role in inherited neurodegenerative diseases. Proc Natl
Acad Sci USA 91, 5355–5358.
96 Michelitsch MD & Weissman JS (2000) A census of
glutamine ⁄ asparagine-rich regions: implications for
their conserved function and the prediction of novel
prions. Proc Natl Acad Sci USA 97, 11910–11915.
97 Jarrett JT & Lansbury PT Jr (1993) Seeding ‘one-
dimensional crystallization’ of amyloid: a pathogenic
mechanism in Alzheimer’s disease and scrapie? Cell 73,

1055–1058.
98 Serio TR, Cashikar AG, Kowal AS, Sawicki GJ, Mos-
lehi JJ, Serpell L, Arnsdorf MF & Lindquist SL (2000)
Nucleated conformational conversion and the replica-
tion of conformational information by a prion determi-
nant. Science 289, 1317–1321.
99 Wood SJ, Wypych J, Steavenson S, Louis JC, Citron
M & Biere AL (1999) alpha-synuclein fibrillogenesis is
nucleation-dependent. Implications for the pathogene-
sis of Parkinson’s disease. J Biol Chem 274, 19509–
19512.
100 Lomakin A, Teplow DB, Kirschner DA & Benedek
GB (1997) Kinetic theory of fibrillogenesis of amy-
loid beta-protein. Proc Natl Acad Sci USA 94, 7942–
7947.
101 Hellstrand E, Boland B, Walsh DM & Linse S (2010)
Amyloid beta-protein aggregation produces highly
reproducible kinetic data and occurs by a two-phase
process. ACS Chem Neurosci 1, 13–18.
Domain-swapping and amyloid fibril formation E. Z
ˇ
erovnik et al.
2278 FEBS Journal 278 (2011) 2263–2282 ª 2011 The Authors Journal compilation ª 2011 FEBS
102 Kumar S & Udgaonkar JB (2010) Mechanisms of amy-
loid fibril formation by proteins. Curr Sci India 98,
639–656.
103 DePace AH, Santoso A, Hillner P & Weissman JS
(1998) A critical role for amino-terminal gluta-
mine ⁄ asparagine repeats in the formation and propaga-
tion of a yeast prion. Cell 93, 1241–1252.

104 Modler AJ, Gast K, Lutsch G & Damaschun G (2003)
Assembly of amyloid protofibrils via critical oligo-
mers – a novel pathway of amyloid formation. J Mol
Biol 325, 135–148.
105 Damaschun G, Damaschun H, Gast K & Zirwer D
(1999) Proteins can adopt totally different folded con-
formations. J Mol Biol 291, 715–725.
106 Damaschun G, Damaschun H, Fabian H, Gast K,
Krober R, Wieske M & Zirwer D (2000) Conversion
of yeast phosphoglycerate kinase into amyloid-like
structure. Proteins 39, 204–211.
107 Xu SH, Bevis B & Arnsdorf MF (2001) The assembly
of amyloidogenic yeast Sup35 as assessed by scanning
(atomic) force microscopy: an analogy to linear colloi-
dal aggregation? Biophys J 81, 446–454.
108 Bhak G, Lee JH, Hahn JS & Paik SR (2009) Granular
assembly of alpha-synuclein leading to the accelerated
amyloid fibril formation with shear stress. PLoS ONE
4, e4177.
109 Kordis D & Turk V (2009) Phylogenomic analysis of
the cystatin superfamily in eukaryotes and prokaryotes.
BMC Evol Biol 9, 266.
110 Turk V, Stoka V & Turk D (2008) Cystatins: biochem-
ical and structural properties, and medical relevance.
Front Biosci 13, 5406–5420.
111 Palsdottir A, Abrahamson M, Thorsteinsson L,
Arnason A, Olafsson I, Grubb A & Jensson O (1988)
Mutation in cystatin C gene causes hereditary brain
haemorrhage. Lancet 2, 603–604.
112 Tizon B, Sahoo S, Yu H, Gauthier S, Kumar AR,

Mohan P, Figliola M, Pawlik M, Grubb A, Uchiyama
Y et al. (2010) Induction of autophagy by cystatin C:
a mechanism that protects murine primary cortical
neurons and neuronal cell lines. PLoS ONE 5, e9819.
113 Benussi L, Ghidoni R, Galimberti D, Boccardi M,
Fenoglio C, Scarpini E, Frisoni GB & Binetti G (2010)
The CST3 B haplotype is associated with frontotempo-
ral lobar degeneration. Eur J Neurol 17, 143–146.
114 Benussi L, Ghidoni R, Steinhoff T, Alberici A, Villa
A, Mazzoli F, Nicosia F, Barbiero L, Broglio L,
Feudatari E et al. (2003) Alzheimer disease-associated
cystatin C variant undergoes impaired secretion.
Neurobiol Dis 13, 15–21.
115 Ii K, Ito H, Kominami E & Hirano A (1993) Abnor-
mal distribution of cathepsin proteinases and endoge-
nous inhibitors (cystatins) in the hippocampus of
patients with Alzheimer’s disease, parkinsonism-demen-
tia complex on Guam, and senile dementia and in the
aged. Virchows Arch A Pathol Anat Histopathol 423,
185–194.
116 Lalioti MD, Mirotsou M, Buresi C, Peitsch MC,
Rossier C, Ouazzani R, Baldy-Moulinier M, Bottani
A, Malafosse A & Antonarakis SE (1997) Identifica-
tion of mutations in cystatin B, the gene responsible
for the Unverricht-Lundborg type of progressive
myoclonus epilepsy (EPM1). Am J Hum Genet 60,
342–351.
117 Pennacchio LA, Lehesjoki AE, Stone NE, Willour VL,
Virtaneva K, Miao J, D’Amato E, Ramirez L, Faham
M, Koskiniemi M et al. (1996) Mutations in the gene

encoding cystatin B in progressive myoclonus epilepsy
(EPM1). Science 271, 1731–1734.
118 Pennacchio LA, Bouley DM, Higgins KM, Scott MP,
Noebels JL & Myers RM (1998) Progressive ataxia,
myoclonic epilepsy and cerebellar apoptosis in cystatin
B-deficient mice. Nat Genet 20, 251–258.
119 Lehtinen MK, Tegelberg S, Schipper H, Su H, Zukor
H, Manninen O, Kopra O, Joensuu T, Hakala P,
Bonni A et al. (2009) Cystatin B deficiency sensitizes
neurons to oxidative stress in progressive myoclonus
epilepsy, EPM1. J Neurosci 29, 5910–5915.
120 Janowski R, Kozak M, Jankowska E, Grzonka Z,
Grubb A, Abrahamson M & Jaskolski M (2001)
Human cystatin C, an amyloidogenic protein, dimerizes
through three-dimensional domain swapping.
Nat Struct Biol 8, 316–320.
121 Jerala R & Zerovnik E (1999) Accessing the global
minimum conformation of stefin A dimer by annealing
under partially denaturing conditions. J Mol Biol 291,
1079–1089.
122 Staniforth RA, Giannini S, Higgins LD, Conroy MJ,
Hounslow AM, Jerala R, Craven CJ & Waltho JP
(2001) Three-dimensional domain swapping in the
folded and molten-globule states of cystatins, an
amyloid-forming structural superfamily. EMBO J 20,
4774–4781.
123 Jenko Kokalj S, Guncar G, Stern I, Morgan G,
Rabzelj S, Kenig M, Staniforth RA, Waltho JP,
Zerovnik E & Turk D (2007) Essential role of proline
isomerization in stefin B tetramer formation. J Mol

Biol 366, 1569–1579.
124 Zerovnik E, Turk V & Waltho JP (2002) Amyloid fibril
formation by human stefin B: influence of the initial
pH-induced intermediate state. Biochem Soc Trans 30,
543–547.
125 Jenko S, Skarabot M, Kenig M, Guncar G, Musevic I,
Turk D & Zerovnik E (2004) Different propensity to
form amyloid fibrils by two homologous proteins-
Human stefins A and B: searching for an explanation.
Proteins 55, 417–425.
126 Zerovnik E, Skarabot M, Skerget K, Giannini S, Stoka
V, Jenko-Kokalj S & Staniforth RA (2007) Amyloid
fibril formation by human stefin B: influence of pH
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 2279
and TFE on fibril growth and morphology. Amyloid
14, 237–247.
127 Zerovnik E, Pompe-Novak M, Skarabot M, Ravnikar
M, Musevic I & Turk V (2002) Human stefin B readily
forms amyloid fibrils in vitro. Biochim Biophys Acta
1594, 1–5.
128 Staniforth RA, Dean JL, Zhong Q, Zerovnik E, Clarke
AR & Waltho JP (2000) The major transition state in
folding need not involve the immobilization of side
chains. Proc Natl Acad Sci USA 97, 5790–5795.
129 Zerovnik E, Virden R, Jerala R, Kroon-Zitko L, Turk
V & Waltho JP (1999) Differences in the effects of
TFE on the folding pathways of human stefins A and

B. Proteins 36, 205–216.
130 Miller KH, Karr JR & Marqusee S (2010) A hinge
region cis-proline in ribonuclease A acts as a confor-
mational gatekeeper for C-terminal domain swapping.
J Mol Biol 400, 567–578.
131 Ferreira ST & De Felice FG (2001) PABMB Lecture.
Protein dynamics, folding and misfolding: from basic
physical chemistry to human conformational diseases.
FEBS Lett 498, 129–134.
132 Liu Y & Eisenberg D (2002) 3D domain swapping: as
domains continue to swap. Protein Sci 11, 1285–1299.
133 Knaus KJ, Morillas M, Swietnicki W, Malone M,
Surewicz WK & Yee VC (2001) Crystal structure of
the human prion protein reveals a mechanism for olig-
omerization. Nat Struct Biol 8, 770–774.
134 Serag AA, Altenbach C, Gingery M, Hubbell WL &
Yeates TO (2001) Identification of a subunit interface
in transthyretin amyloid fibrils: evidence for self-assem-
bly from oligomeric building blocks. Biochemistry 40,
9089–9096.
135 Sabate R, Gallardo M & Estelrich J (2005) Tempera-
ture dependence of the nucleation constant rate in beta
amyloid fibrillogenesis. Int J Biol Macromol 35, 9–13.
136 Auer S, Dobson CM & Vendruscolo M (2007) Charac-
terization of the nucleation barriers for protein aggre-
gation and amyloid formation. HFSP J 1, 137–146.
137 Skerget K, Vilfan A, Pompe-Novak M, Turk V, Wal-
tho JP, Turk D & Zerovnik E (2009) The mechanism
of amyloid-fibril formation by stefin B: temperature
and protein concentration dependence of the rates.

Proteins 74, 425–436.
138 Nilsson M, Wang X, Rodziewicz-Motowidlo S, Janow-
ski R, Lindstrom V, Onnerfjord P, Westermark G,
Grzonka Z, Jaskolski M & Grubb A (2004) Prevention
of domain swapping inhibits dimerization and amyloid
fibril formation of cystatin C: use of engineered disul-
fide bridges, antibodies, and carboxymethylpapain to
stabilize the monomeric form of cystatin C. J Biol
Chem 279, 24236–24245.
139 Laganowsky A & Eisenberg D (2010) Non-3D domain
swapped crystal structure of truncated zebrafish al-
phaA crystallin. Protein Sci 19, 1978–1984.
140 Gotte G, Vottariello F & Libonati M (2003) Thermal
aggregation of ribonuclease A. A contribution to the
understanding of the role of 3D domain swapping in
protein aggregation. J Biol Chem 278, 10763–10769.
141 Rousseau F, Schymkowitz JW, Wilkinson HR &
Itzhaki LS (2001) Three-dimensional domain swapping
in p13suc1 occurs in the unfolded state and is con-
trolled by conserved proline residues. Proc Natl Acad
Sci USA 98, 5596–5601.
142 Stubbs MT, Laber B, Bode W, Huber R, Jerala R,
Lenarcic B & Turk V (1990) The refined 2.4 A X-ray
crystal structure of recombinant human stefin B in
complex with the cysteine proteinase papain: a novel
type of proteinase inhibitor interaction. EMBO J 9,
1939–1947.
143 Smajlovic A, Berbic S, Schiene-Fischer C, Tusek-Znid-
aric M, Taler A, Jenko-Kokalj S, Turk D & Zerovnik
E (2009) Essential role of Pro 74 in stefin B amyloid-

fibril formation: dual action of cyclophilin A on the
process. FEBS Lett 583, 1114–1120.
144 Eakin CM, Attenello FJ, Morgan CJ & Miranker AD
(2004) Oligomeric assembly of native-like precursors
precedes amyloid formation by beta-2 microglobulin.
Biochemistry 43, 7808–7815.
145 Eakin CM, Berman AJ & Miranker AD (2006) A
native to amyloidogenic transition regulated by a back-
bone trigger. Nat Struct Mol Biol 13, 202–208.
146 Jahn TR, Parker MJ, Homans SW & Radford SE
(2006) Amyloid formation under physiological condi-
tions proceeds via a native-like folding intermediate.
Nat Struct Mol Biol 13, 195–201.
147 Blaho DV & Miranker AD (2009) Delineating the con-
formational elements responsible for Cu(2+)-induced
oligomerization of beta-2 microglobulin. Biochemistry
48, 6610–6617.
148 Calabrese MF, Eakin CM, Wang JM & Miranker AD
(2008) A regulatable switch mediates self-association in
an immunoglobulin fold. Nat Struct Mol Biol 15, 965–
971.
149 Zerovnik E, Skerget K, Tusek-Znidaric M, Loeschner
C, Brazier MW & Brown DR (2006) High affinity
copper binding by stefin B (cystatin B) and its role in
the inhibition of amyloid fibrillation. FEBS J 273,
4250–4263.
150 Bennett MJ, Schlunegger MP & Eisenberg D (1995)
3D domain swapping: a mechanism for oligomer
assembly. Protein Sci 4, 2455–2468.
151 Liu Y, Gotte G, Libonati M & Eisenberg D (2001) A

domain-swapped RNase A dimer with implications for
amyloid formation. Nat Struct Biol 8, 211–214.
152 Janowski R, Abrahamson M, Grubb A & Jaskolski M
(2004) Domain swapping in N-truncated human cysta-
tin C. J Mol Biol 341, 151–160.
153 Janowski R, Kozak M, Abrahamson M, Grubb A &
Jaskolski M (2005) 3D domain-swapped human
Domain-swapping and amyloid fibril formation E. Z
ˇ
erovnik et al.
2280 FEBS Journal 278 (2011) 2263–2282 ª 2011 The Authors Journal compilation ª 2011 FEBS
cystatin C with amyloidlike intermolecular beta-sheets.
Proteins 61, 570–578.
154 Zerovnik E, Staniforth RA & Turk D (2010) Amyloid
fibril formation by human stefins: structure, mechanism
& putative functions. Biochimie 92, 1597–1607.
155 Sanders A, Jeremy Craven C, Higgins LD, Giannini S,
Conroy MJ, Hounslow AM, Waltho JP & Staniforth
RA (2004) Cystatin forms a tetramer through
structural rearrangement of domain-swapped
dimers prior to amyloidogenesis. J Mol Biol 336, 165–
178.
156 Nordlund A & Oliveberg M (2006) Folding of Cu ⁄ Zn
superoxide dismutase suggests structural hotspots for
gain of neurotoxic function in ALS: parallels to precur-
sors in amyloid disease. Proc Natl Acad Sci USA 103,
10218–10223.
157 Newcomer ME (2002) Protein folding and three-dimen-
sional domain swapping: a strained relationship? Curr
Opin Struct Biol 12, 48–53.

158 Ogihara NL, Ghirlanda G, Bryson JW, Gingery M,
DeGrado WF & Eisenberg D (2001) Design of three-
dimensional domain-swapped dimers and fibrous oligo-
mers. Proc Natl Acad Sci USA 98, 1404–1409.
159 Rousseau F, Schymkowitz J & Itzhaki LS (2007)
Implications of 3D domain swapping for protein fold-
ing, misfolding and function. In Protein Dimerization
(and Oligomerization) in Biology (Matthews J, ed.),
Landes Bioscience, Austin, TX, USA.
160 Guo Z & Eisenberg D (2006) Runaway domain
swapping in amyloid-like fibrils of T7 endonuclease I.
Proc Natl Acad Sci USA 103, 8042–8047.
161 Liu C, Sawaya MR & Eisenberg D (2011) Beta-micro-
globulin forms three-dimensional domain-swapped
amyloid fibrils with disulfide linkages. Nat Struct Mol
Biol 18, 49–55.
162 Wahlbom M, Wang X, Lindstrom V, Carlemalm E,
Jaskolski M & Grubb A (2007) Fibrillogenic oligomers
of human cystatin C are formed by propagated domain
swapping. J Biol Chem 282, 18318–18326.
163 Morgan GJ, Giannini S, Hounslow AM, Craven CJ,
Zerovnik E, Turk V, Waltho JP & Staniforth RA
(2008) Exclusion of the native alpha-helix from the
amyloid fibrils of a mixed alpha ⁄ beta protein. J Mol
Biol 375, 487–498.
164 Nilsson MR & Raleigh DP (1999) Analysis of amylin
cleavage products provides new insights into the amy-
loidogenic region of human amylin. J Mol Biol 294,
1375–1385.
165 Kolodziejczyk R, Michalska K, Hernandez-Santoyo A,

Wahlbom M, Grubb A & Jaskolski M (2010) Crystal
structure of human cystatin C stabilized against amy-
loid formation. Febs J 277, 1726–1737.
166 Zerovnik E, Zavasnik-Bergant V, Kopitar-Jerala N,
Pompe-Novak M, Skarabot M, Goldie K, Ravnikar
M, Musevic I & Turk V (2002) Amyloid fibril forma-
tion by human stefin B in vitro: immunogold labelling
and comparison to stefin A. Biol Chem 383, 859–863.
167 Pallitto MM & Murphy RM (2001) A mathematical
model of the kinetics of beta-amyloid fibril growth
from the denatured state. Biophys J 81, 1805–1822.
168 Powers ET & Powers DL (2008) Mechanisms of pro-
tein fibril formation: nucleated polymerization with
competing off-pathway aggregation. Biophys J 94, 379–
391.
169 Ceru S, Kokalj SJ, Rabzelj S, Skarabot M, Gutierrez-
Aguirre I, Kopitar-Jerala N, Anderluh G, Turk D,
Turk V & Zerovnik E (2008) Size and morphology of
toxic oligomers of amyloidogenic proteins: a case study
of human stefin B. Amyloid 15, 147–159.
170 Horwich A (2002) Protein aggregation in disease: a role
for folding intermediates forming specific multimeric
interactions. J Clin Invest 110, 1221–1232.
171 Wetzel R (1996) For protein misassembly, it’s the ‘I’
decade. Cell 86, 699–702.
172 Ceru S & Zerovnik E (2008) Similar toxicity of the
oligomeric molten globule state and the prefibrillar
oligomers. FEBS Lett 582, 203–209.
173 Uversky VN (2002) Natively unfolded proteins: a point
where biology waits for physics. Protein Sci 11, 739–756.

174 Uversky VN (2009) Intrinsic disorder in proteins asso-
ciated with neurodegenerative diseases. Front Biosci 14,
5188–5238.
175 Uversky VN & Fink AL (2004) Conformational con-
straints for amyloid fibrillation: the importance of
being unfolded. Biochim Biophys Acta 1698, 131–153.
176 Harper JD & Lansbury PT Jr (1997) Models of amy-
loid seeding in Alzheimer’s disease and scrapie: mecha-
nistic truths and physiological consequences of the
time-dependent solubility of amyloid proteins. Annu
Rev Biochem 66, 385–407.
177 Ross CA, Wood JD, Schilling G, Peters MF, Nucifora
FC Jr, Cooper JK, Sharp AH, Margolis RL & Bor-
chelt DR (1999) Polyglutamine pathogenesis. Philos
Trans R Soc Lond B Biol Sci 354, 1005–1011.
178 Dahlgren PR, Karymov MA, Bankston J, Holden T,
Thumfort P, Ingram VM & Lyubchenko YL (2005)
Atomic force microscopy analysis of the Huntington
protein nanofibril formation. Nanomedicine 1, 52–57.
179 Scherzinger E, Sittler A, Schweiger K, Heiser V, Lurz
R, Hasenbank R, Bates GP, Lehrach H & Wanker EE
(1999) Self-assembly of polyglutamine-containing hun-
tingtin fragments into amyloid-like fibrils: implications
for Huntington’s disease pathology. Proc Natl Acad
Sci USA 96, 4604–4609.
180 Pellarin R & Caflisch A (2006) Interpreting the aggre-
gation kinetics of amyloid peptides. J Mol Biol 360,
882–892.
181 Chan HS & Dill KA (1998) Protein folding in the land-
scape perspective: chevron plots and non-Arrhenius

kinetics. Proteins 30, 2–33.
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 2281
182 Lummis SC, Beene DL, Lee LW, Lester HA, Broad-
hurst RW & Dougherty DA (2005) Cis-trans isomeriza-
tion at a proline opens the pore of a neurotransmitter-
gated ion channel. Nature 438, 248–252.
183 Ryo A, Togo T, Nakai T, Hirai A, Nishi M,
Yamaguchi A, Suzuki K, Hirayasu Y, Kobayashi H,
Perrem K et al. (2006) Prolyl-isomerase Pin1 accumu-
lates in lewy bodies of parkinson disease and facilitates
formation of alpha-synuclein inclusions. J Biol Chem
281, 4117–4125.
184 Pastorino L, Sun A, Lu PJ, Zhou XZ, Balastik M,
Finn G, Wulf G, Lim J, Li SH, Li X et al. (2006) The
prolyl isomerase Pin1 regulates amyloid precursor
protein processing and amyloid-beta production.
Nature 440, 528–534.
185 Carrotta R, Manno M, Bulone D, Martorana V & San
Biagio PL (2005) Protofibril formation of amyloid
beta-protein at low pH via a non-cooperative elonga-
tion mechanism. J Biol Chem 280, 30001–30008.
186 Jaskolski M (2001) 3D domain swapping, protein olig-
omerization, and amyloid formation. Acta Biochim Pol
48, 807–827.
187 Lee JH, Bhak G, Lee SG & Paik SR (2008) Instanta-
neous amyloid fibril formation of alpha-synuclein from
the oligomeric granular structures in the presence of

hexane. Biophys J 95, L16–L18.
Domain-swapping and amyloid fibril formation E. Z
ˇ
erovnik et al.
2282 FEBS Journal 278 (2011) 2263–2282 ª 2011 The Authors Journal compilation ª 2011 FEBS