Archives of Biochemistry and Biophysics 602 (2016) 116e126

Contents lists available at ScienceDirect

Archives of Biochemistry and Biophysics

journal homepage: www.elsevier.com/locate/yabbi

Crystallographic studies on protein misfolding: Domain swapping and

amyloid formation in the SH3 domain

mara-Artigas
Ana Ca
Department of Chemistry and Physics, University of Almera, Agrifood Campus of International Excellence (ceiA3), Research Centre for Agricultural and Food
Biotechnology (BITAL), Carretera de Sacramento s/n, Almera 04120, Spain

a r t i c l e i n f o a b s t r a c t

Article history: Oligomerization by 3D domain swapping is found in a variety of proteins of diverse size, fold and
Received 29 October 2015 function. In the early 1960s this phenomenon was postulated for the oligomers of ribonuclease A, but it
Received in revised form was not until the 1990s that X-ray diffraction provided the rst experimental evidence of this special
19 February 2016
manner of oligomerization. Nowadays, structural information has allowed the identication of these
Accepted 23 February 2016
Available online 26 February 2016
swapped oligomers in over one hundred proteins. Although the functional relevance of this phenomenon
is not clear, this alternative folding of protomers into intertwined oligomers has been related to amyloid
formation. Studies on proteins that develop 3D domain swapping might provide some clues on the early
Keywords:
3D domain swapping
stages of amyloid formation. The SH3 domain is a small modular domain that has been used as a model
Intertwined dimer to study the basis of protein folding. Among SH3 domains, the c-Src-SH3 domain emerges as a helpful
X-ray crystallography model to study 3D domain swapping and amyloid formation.
Protein folding 2016 Elsevier Inc. All rights reserved.
Amyloid
SH3 domain

1. Introduction difculties to obtain crystals. Other techniques that offer highly

Since Annsen's pioneering work on protein folding a number of
research works have studied how native structure acquisition is
inuenced by protein sequence and the milieu conditions [1].
Failure in the folding process of a protein may result in the devel-
opment of protein deposition diseases. Creutzfeldt-Jakob disease
[2] was the rst one to illustrate that a misfolded protein can be the
origin of some diseases. To date over 30 major human diseases have
been connected to amyloid formation; these include both geneti-
cally and non-genetically related diseases, some of which are
transmissible [3,4]. In addition, some amyloids have been classied
as functional amyloids and reported to play a role in diverse
physiological processes [5e8].
X-ray diffraction experiments conducted by Astbury and Dick-
inson in the 1930s rst showed the typical cross-b ber diffraction
pattern displayed by amyloid bers [9]. Nevertheless, structural
information on amyloids is still problematic because the two
structural techniques that provide high-resolution data, nuclear
magnetic resonance (NMR) and X-ray crystallography, are not
applicable to these aggregates owing to their large size and the
E-mail address: acamara@ual.es.
valuable structural information include high-resolution electron
microscopy, solid state NMR and other NMR techniques, Fourier
transform infrared spectroscopy, circular dichroism and electron
paramagnetic resonance [10]. Even though the information pro-
vided by these techniques has allowed researchers to obtain a
detailed view of several amyloid structures, the mechanism by
which amyloidogenic proteins become unfolded and initiate their
aggregation process is still unknown. The development of models
to explain this process is particularly challenging due to the dif-
culty in obtaining high-resolution structural information, and this
is where structural studies on three-dimensional (3D) domain-
swapped proteins can help.
3D domain swapping is a phenomenon that occurs when two or
more molecules of a partially unfolded protomer form homo-
dimers, or higher-order oligomers, by exchanging protein domains
or secondary structure elements. The formation of these oligomers
has been proposed as a possible mechanism underlying the
appearance of early aggregates of amyloid bril [11]. Some well-
characterized amyloid-forming globular proteins also form inter-
twined dimers: prion [12,13], cystatin [14e16] and b-2-
microglobulin [12]. Although to date only about one hundred
proteins have been reported to undergo 3D domain swapping, the
diversity of their sequences and secondary structures suggests that

http://dx.doi.org/10.1016/j.abb.2016.02.024
0003-9861/ 2016 Elsevier Inc. All rights reserved.

mara-Artigas / Archives of Biochemistry and Biophysics 602 (2016) 116e126
A. Ca 117

almost any protein may oligomerize via this mechanism [17]. The
swapped-oligomer appears as an alternative mechanism for pro-
tomer folding, possibly driven by a lower energy of self-associated
species compared with the single folded chain, or alternatively by
the faster formation of the oligomer and its later stabilization. Most
of the hydrophobic-core interactions of the swapped oligomer are
already present in the protomer, and the secondary structure ele-
ments are often conserved [18]. On the other hand, major changes
are present in the so-called hinge loops, which facilitate the pro-
tomer's opening.
Nowadays, the structures of several domain-swapping/amyloid-
forming proteins are available, and although the role of 3D domain
swapping is not clear, the structural information obtained from
these intertwined proteins may help to elucidate their plausible
functional role and, in addition, to clarify the complex mechanism
of amyloidogenesis. Furthermore, these oligomeric structures
deserve attention since some studies on deposition diseases have
attributed the toxicity to oligomeric assemblies of the protein,
rather than to the docking of mature brils [19e21].
1.1. Structural information on amyloids
High-resolution structural information on amyloids is limited,
but recent technological advances and the use of structural ap-
proaches in concert resulted in a more detailed picture of these
aggregates. Most of this information comes from the study of some
well-characterized amyloid-forming proteins, for example Sup35
prion proteins, Alzheimer's disease b-amyloid, Parkinson's disease
a-Synuclein, b2-Microglobulin [10]. The b-sheet structure is the
principal molecular structure of amyloid, which can appear in a
parallel or anti-parallel form. In the case of the parallel form, it can
be in-register or out of register. In addition, there are several ter-
tiary structures for these b-sheets: b-sandwich; b-solenoid, which
can form a b-helix or a b-roll [22]. Fig. 1 illustrates the structure of
Alzheimer's b-amyloid brils.
To date, solid-state NMR provides the few high-resolution
structures available for a complete amyloid bril, for example
those of the functional prion HET-s from the fungi Podospora
anserina [25] and the b-amyloid (1e42) bril [23,24,26]. Never-
theless, most of the crystallographic information about amyloid
aggregates is limited to short peptide sequences that are able to
form amyloid and crystallize at the same time. Eisenberg's group
solved the crystallographic structure of about one hundred amyloid
forming peptides derived from Sup35 [27,28], transthyretin [29], b-
amyloid [30] and insulin [31], among others. These structures
revealed shared characteristics, as is the case of the cross b-sheet
structures [32], the dry interface between sheets [27] and the steric
zipper [28]. They also show that the remarkable stability of these
bers is reached through near optimal values for main-chain
dihedral angles, along with an extensive packing of hydrophobic
side chains and salt bridge/hydrogen bond formation between the
peptides (Fig. 1).
The structural studies also revealed conformational diversity in
prion proteins [33] and other amyloid systems [28,34,35]. This
conformational heterogeneity has mostly been studied using sys-
tems where the amyloid has been obtained in vitro from peptides or
recombinant proteins. Chiti et al. proposed that amyloid bril for-
mation can occur when the native globular fold of a protein is desta-
bilized under conditions in which noncovalent interactions still remain
favorable [36]. In this way, manipulating the pH, temperature and
salt concentration gives the possibility of producing amyloids un-
der controlled conditions that might result in morphologically
different structures. In addition, it provides the opportunity to
investigate the molecular basis of amyloid formation in different
proteins.
1.2. Structural information on 3D domain-swapped proteins
Although domain swapping was rst proposed in the 1960s to
explain the behavior of a ribonuclease A dimer [37], no structural
evidence for these oligomers was obtained until the 1990s [38]. The
diphtheria toxin was the rst 3D domain-swapped dimer described

Fig. 1. b-sheet structure of b-amyloid. (A) The structure of Alzheimer's b-amyloid (Ab1-42) brils in a parallel in register b-sheets form (PDB entry 2BEG) [23] and (B) the structure
of Alzheimer's Iowa mutant b-amyloid (Ab1-40, D23N mutant) brils in an anti-parallel form (PDB entry 2LNQ) obtained by NMR techniques [24]. The left and right panels show two
different orientations of the b-amyloid bril, perpendicular and same direction that the bril propagation axis, respectively. Relevant residues are shown in stick: K28-D23 and K16-
E22 salt-bridge (shown in green dotted line) in the parallel and in the antiparallel form, respectively. In both forms the b-sheet structure is stabilized through p-p stacking
interaction between residues F19 and F20.
118
mara-Artigas / Archives of Biochemistry and Biophysics 602 (2016) 116e126
A. Ca

from structural data [39]. In fact, it was the rst protein structure
determined in both the monomeric and domain-swapped dimer
forms (Fig. 2A) [40]. In this protein the segment swapped between
protomers was a structural domain, but 3D domain swapping ter-
minology has been applied to the subsequently discovered inter-
twined proteins, even in those cases where only a secondary
structure element (not regarded as a structural domain) is the
interchanged segment. One example is ribonuclease A, which ex-
changes the amino terminal a-helix and/or the carboxyl terminal b-
strand [41,42] (Fig. 2B).
Eisenberg and co-workers developed a terminology to describe
the 3D domain-swapping phenomenon [46,47]. They dened three
different classes of domain swapping taking into account the
availability of the structures of the swapped and un-swapped
forms: i) bona de domain swapping: the dimeric and monomeric
structures of the protein are known; ii) quasi domain swapping: the
structure has a homologous monomer; and iii) candidates for
domain swapping: no monomer structure is available. The poly-
peptide chain conformation adopted by the domain-swapped
oligomer is called open monomer, while the monomeric confor-
mation is called closed monomer. Each form shows different in-
terfaces: the closed interface or primary interface is the
intramolecular interface, which is the same in the oligomer as in
the monomer but is formed by different chains; the open interface
or secondary interface is unique to the oligomeric form [46,48,49].
An analysis of the bona de and quasi domain-swapped struc-
tures deposited at the Protein Data Bank (PDB) shows that more
than one hundred proteins oligomerize by this mechanism. Some
structures have been solved by means of NMR techniques (<10%),
but most of them have been solved by X-ray crystallography.
Recently, MacKinnon and Wodak [50] have performed an extensive
study on the interfaces of multi-domain homo-oligomeric proteins.
They found that intertwined oligomers are very common in
homomeric association, and they performed a classication taking
into account the interchanged segment of the protein. Interestingly,
these authors found that of all the structures included in this study
only three interchanged a protein segment that it is a structural
domain: diphtheria toxin [39], bB2-crystallin [51] and CIB1 [52].
The web database ProSwap (http://wodaklab.org/proswap/) com-
piles 3D domain-swapped structures up to 2011. Shameer et al. [53]
also developed a web page to compile all the 3D domain-swapped
proteins described up to 2010 (http://caps.ncbs.res.in/3dswap/).
Unfortunately, due to the complexity of the search, these web pages
have not been updated.
Another attempt to classify domain-swapped proteins was
performed by Huang et al. [17], who also performed a detailed
analysis of 500 structures at the PDB to identify their special fea-
tures. This structural analysis brought to light the diversity of the
domain-swapped structures. Among the bona de domain-
swapped proteins, sizes vary from the Eps8-SH3 domain (60
amino acid residues) to the diphtheria toxin (535 amino acid resi-
dues) for the smallest and largest proteins, respectively. There is no
preferred fold among the known 3D domain-swapped proteins:
some are all a, others all b, and most of them show both elements of
secondary structure in their fold. Sometimes the extent of the
secondary structure element interchanged is just a short segment
of the polypeptide chain or a single element of secondary structure
[41,42]. In other proteins the exchange of secondary structure is
complete, as for example the SH3 domain [54e57]. Most frequently,
domain swapping results in an intertwined dimer, but sometimes it
appears as an intertwined trimer or tetramer [58,59] and some
proteins exhibit multiple swapping modes [60,61] (Fig. 2B).
In order to be able to oligomerize by 3D domain swapping, a
protein must have a region of the polypeptide chain that can be
exchanged between monomers once the conformational change of
the hinge loop takes place. This loop must have suitable length and
exibility to allow the placement of the swapped region in the open
and closed monomer. For this reason, many studies on 3D domain

Fig. 2. Domain-swapped dimers. Cartoon representation of the intertwined dimers of the (A) diphtheria toxin (monomer, PDB code 1MDT [43]; intertwined dimer, PDB code 1DDT
[39]) and (B) RNase A (monomer, PDB code 2E3W [44]; N-terminal intertwined dimer, PDB code 1A2W [45]; C-terminal intertwined dimer, PDB code 1F0V [42]). Exchangeable
domains/secondary structure elements are shown in red. In the intertwined dimer, the complementary chain is shown in light blue and the exchangeable element in pink.

mara-Artigas / Archives of Biochemistry and Biophysics 602 (2016) 116e126
A. Ca
swapping have focused on the hinge loop by performing muta-
genesis of some critical residues [60] or shortening/lengthening it
[49]. The manipulation of the hinge loop might result in changes in
the behavior of these domain-swapped oligomers as it affects its
exibility [62] or the propensity of the residues to stabilize the b-
turn [63,64]. Sometimes the manipulation of a loop in a monomeric
protein results in the formation of a de novo domain-swapped
oligomer. As an example, the articial insertion of a long gluta-
mine repeats in the inhibitory loop of the chymotrypsin inhibitor 2,
a monomeric protein, resulting in an intertwined dimer [65].
Other experimental and computational evidence demonstrated
that the hinge loop is not the only factor responsible for oligo-
merization [66]. Point mutations in the hydrophobic core of the
protein and in amino acid residues stabilizing the secondary
interface also affect domain swapping [55,67,68].
1.3. Functional role of 3D domain swapping
For many years, domain swapping has been considered an
in vitro artefact [69]. The high protein concentrations used in the
crystallization setups and the presence of additives can promote 3D
domain swapping by introducing unfavorable protein-solvent in-
teractions, but similar conditions might also take place in vivo. As
the number of 3D domain-swapped structures available increases,
there is growing evidence of the biological relevance of some of
these oligomers. Moreover, there are cases where only the inter-
twined oligomer of the protein is known, and it is the functional
form [70e73].
It has been suggested that domain swapping plays a role in the
evolution of oligomeric proteins [74,75]. These proteins are present
in all organisms and show structural and functional advantages
compared to monomeric ones: oligomeric proteins are commonly
more stable and, from a functional point of view, oligomerization
can bear some control mechanism that improves the accessibility
and specicity of active sites. Thus, domain swapping has been
described to participate in the appearance of new binding sites,
such as in the coagulation factor IX binding protein, where a Mg2
ion is bound to the domain-swapped dimer [76]. These ions have
been shown to maintain the native conformation and in vivo
function of factor IX intertwined Gla domain during blood coagu-
lation [77]. Another example is the human zinc metalloenzyme
glyoxalase I, where the active site zinc ion interacts with both
chains in the intertwined dimer [78]. This protein is homologous
with the bleomycin resistance protein, which has been proposed to
arise from gene duplication and is only known in its intertwined
form [79,80].
Another putative function of intertwined proteins is the for-
mation of allosteric sites in enzymes, for example the type II re-
striction endonuclease SgrAI, which forms a tetramer by swapping
the amino-terminal 24 amino acid residues [81]. The formation of
an allosteric site was also proposed for the domain-swapped bovine
seminal ribonuclease [82]. In this case, the two chains forming the
intertwined dimer exchange their amino-terminal a-helix and a
new nucleotide-binding site is generated in a cavity at the interface
between the two subunits. Another example is the survival protein
SurE from Salmonella typhimurium, whose dimers are formed by
the swapping of the carboxyl-terminal a-helix and a loop with two
b-strands [83]. These examples indicate that oligomerization
through 3D domain swapping may play a role in the modulation of
the catalytic activity of some enzymes.
Structural evidence also put forward the involvement of 3D
domain swapping in the formation of supra-macromolecular ag-
gregates such as those appearing in viral capsid structures. Ivanov
et al. [84] reported the crystal structure of a distinct domain-
swapped variant of the HIV-1 CA-CTD dimer stabilized by a single
119
amino acid deletion and suggested that domain swapping of the
major homology region (MHR) segment of adjacent Gag molecules
might play a role in viral assembly. In contrast, a biophysical
analysis of the MHR motif indicates that domain-swapped dimers
are not present in the nal assembly of the HIV capsid and this
reaction might be an evolutionary remnant of the biologically
relevant dimerization of some oligomeric proteins, such as the
homologous mammalian SCAN domain [85,86]. Nevertheless, the
role of domain swapping in capsid assembly is reinforced by the
crystal structure of the icosahedral rice yellow mottle virus and
biochemical analysis, which justies an increase in the stability of
this capsid as compared to other homologous protein capsids that
do not show this phenomenon [87].
Without doubt, the most widely claimed function of domain-
swapped oligomers is their plausible participation in the rst
steps of the development of higher-order oligomers that lead to the
formation of amyloids. The rst experimental evidence was
brought to light by the intertwined dimer of cystatin C, which was a
known amyloidogenic protein [88,89]. At the same time, a model
for amyloid formation by a polar zipper with 3D domain swapping
was proposed for ribonuclease A and other proteins [42].
At present, many of the known domain-swapped proteins have
been described to form amyloid aggregates. Although there is some
controversy regarding the relationship between 3D domain
swapping and amyloid formation [90], the structural information
available on different swapped proteins is of great value to analyze
the rst steps of oligomer formation. Unveiling the mechanism of
the rst steps of this misfolding phenomenon could help to
determine why a specic protein changes its folding preferences
and how oligomerization can account for the additional stabiliza-
tion factors. In this way, structural information on 3D domain-
swapped proteins may provide a key to build models of protein
misfolding. Certainly, additional thermodynamic and kinetic in-
formation on the folding process can help to develop these models.
There are a few 3D domain-swapped proteins forming intertwined
oligomers and amyloids that have been extensively studied from
the thermodynamic and kinetic points of view. One of these pro-
teins is the SH3 domain of the c-Src tyrosine kinase. Recently, our
group has demonstrated that this protein forms amyloid brils in a
pH-dependent manner that it is related to the nature of the residue
at position 128 [55].
1.4. 3D domain swapping and amyloid formation in SH3 domains
The SH3 domain is a small modular domain (~60-residue long)
present in a large number of proteins. It is an all-b protein where
the b-strands form an open b-barrel structure. Comparison of
different SH3 domains reveals that the hydrophobic core of these
domains is mainly conserved, but the sequence and conformation
of their loops show considerable variability (Fig. 3). This small
modular domain is also one of the most widely present in all ge-
nomes, and it is found in different kinds of proteins that develop
key functions in the cell [91].
To date, only four SH3 domains have been described to form
domain-swapped dimers: Eps8 [54], p47phox [92], the C-terminal
SH3 domain of CRKL [93] and c-Src [57,94]. However, the diverse
nature of these SH3 domains suggests that other SH3 domains
might also show 3D domain swapping. On the other hand, amyloid
formation has been experimentally described in ve SH3 domains:
c-Yes [95], Fyn [96], c-Src [55], PI3K [97] and a-spectrin [98]. c-Yes,
Fyn and c-Src SH3 domains are part of tyrosine kinases that belong
to the Src family and show a high degree of sequence homology
(Fig. 3). Among all the SH3 domains, the c-Src-SH3 is the only one
that has been described to form both amyloid and 3D domain-
swapped dimers. Table 1 compiles the above information and
120
mara-Artigas / Archives of Biochemistry and Biophysics 602 (2016) 116e126
A. Ca

Fig. 3. Sequence alignment of the domain swapping and amyloid-forming SH3 domains and overall fold. (A) Sequence alignment of the intertwined and amyloid-forming SH3
domains. Sequence numbering corresponds to the c-Src-SH3 domain and its secondary structure is shown in the upper part. (B) Overall fold of the c-Src-SH3 domain, where the
names of the loop regions and the different structural elements of the protein are indicated. In addition, residues involved in the folding nucleation of the c-Src-SH3 are shown in
magenta.

relevant references.
1.5. 3D domain swapping in SH3 domain
The rst 3D domain-swapped SH3 domain described was Eps8
(Eps8-SH3), an oncoprotein that participates in v-Src-induced
cellular transformation. The crystallographic structure of this SH3
domain was solved in the triclinic space group P1, and the asym-
metric unit was made up of two chains of the domain which formed
an intertwined dimer. In this domain-swapped structure, the n-Src
loop acts as hinge loop and the canonical SH3 fold is formed by
interchanging the C-terminal b-strands b3-b5 between the two
chains (Fig. 4A) [54]. Some years later, Kishan et al. [99] also solved
the structure of the monomeric form of this SH3 domain and the
availability of the structures of both forms allowed them to study
the factors that favor the formation of the domain-swapped dimer.
The main difference was that each crystal form was obtained at a
different pH: the monomeric and dimeric forms were obtained at
pH 4.0 and 7.0, respectively. Analysis of the potential salt bridges/
hydrogen bonds between acidic and basic residues suggested a role

Table 1
3D domain-swapping and amyloid formation in the SH3 domains.

SH3 domain PDB 3D domain-swapping pH Reference Amyloid formation pH Reference

Eps8 1I07 Dimer WT 7.0 [51,96] e

1I0C Monomer WT 4.0 [96] e
N-ter p47phox 1NG2 Dimer WT 5.0 [89,97] e
1UEC Dimer WT 5.4 [89,97] e
1WLP Monomer WT 6.5 [99] e
C-ter CRKL 2BZY Dimer WT 5.6 [90] e
2BZY Monomer WT 6.5 [90] e
PI3K 3I5S e [156] WT 2.0 [94]
a-spectrin 1SHG e [157] WT 3.2 [118]
1QKX e [111] N47A 3.0 [95]
N47A double mutants 3.2 [117]
Best2, D48G(2Y) 3.2 [125]
c-Yes 2HDA e [92] WT 3.0
Fyn e e A39V/N53P/V55L/D(57e60) 7.0 [93]
c-Src 4JZ3 Dimer WT 5.0 [52] WT 5.0 [52]
4JZ4 Monomer WT 7.0 [52]
4OMN Dimer Q128E 5.0 [52] Q128E 7.0 [52]
4OMO Monomer Q128E 7.0 [52]
4OMP Dimer Q128K 5.0 [52] e
4OML Dimer Q128R 5.0 [52] e

mara-Artigas / Archives of Biochemistry and Biophysics 602 (2016) 116e126
A. Ca 121

of these interactions in the formation of the intertwined dimer.
Additionally, the formation of the dimer resulted in the burial of a
surface area of 4000 2, and Kishan et al. also suggested that the
stacking of aromatic residues W40 and F52 plays a role in the
stabilization of the intertwined dimer.
Owing to its small size, the SH3 domain is an ideal model to
study protein folding by molecular dynamics. Yang et al. [66]
applied the symmetrized Go -type potential model to the Eps8-
SH3 domain to determine how domain-swapped proteins un-
dergo a transition from the monomeric to the swapped confor-
mation. To establish whether the local properties of the hinge loop
were responsible for the domain swapping, these authors increased
the local exibility of the n-Src loop and other loops. Under con-
ditions of high exibility of two loops more trap states were found,
with the experimentally observed domain-swapped dimer at the
highest representation. They concluded that under conditions fa-
voring intermolecular interactions, swapping is a means of
reducing energy frustration and is determined by the native-state
topology rather than local signals at the hinge region.
Indeed, other domain-swapped SH3 domains have different
hinge loops. The structure of the tandem SH3 domains of the
p47phox, a cytosolic regulatory component of NADPH oxidase, was
solved by Groemping et al. [100] as an intertwined dimer of the N-
terminal SH3 domain (SH3-A, residues 159e213), where the distal
loop acts as hinge loop (Fig. 4B). However, in the Crk-like (CRKL) C-
terminal SH3 domain it is the RT loop that acts as hinge loop and
strand b1 is interchanged between neighbouring monomers
(Fig. 4C) [93]. In these SH3 domains, as in the Eps8-SH3, the
monomeric and dimeric species are in equilibrium in solution and
each form seems to be favored at different pH values. In this way,
the monomeric and dimeric form crystal structures of the CRKL C-
terminal SH3 domain were obtained at slightly different pH (6.5
and 5.6, respectively) and ionic strength [93]. The intertwined
dimer of p47phox seems to arise from the quite slow conversion
from monomeric to dimeric form under the crystallization condi-
tions [92,100,101]. The structure of the monomeric form was solved
by NMR at pH 6.5 in complex with a proline rich motif peptide
[102]. The pH and the presence of the proline rich motif peptide
bound to the N-terminal SH3 domain of the p47phox might stabilize
the monomeric form, as occurs in the c-Src-SH3 domain
[55,103,104].
1.6. Amyloid formation in SH3 domains
The phosphatidylinositol 3-kinase (PI3K) SH3 domain was the
rst one described to form amyloid [97]. This amyloid is obtained at
acidic pH (2.0) and moderate temperatures (25e37  C) and it is one
of the best characterized from the structural point of view. Studies
performed at different pH showed that amyloid bril formation
proceeds through different types of intermediates. NMR experi-
ments showed the presence of partly folded conformations in the
acid-unfolded state, but not 3D domain-swapped forms [105e110].
Besides, NMR and electrospray ionization mass spectrometry
analysis of H/D exchange allowed identication of those residues
involved in oligomeric and bril core structures: residues
belonging to the RT loop and adjacent to the diverging b-turn in the
native state play a key role in the conversion of the soluble PI3K-
SH3 into amyloid brils [111].

Fig. 4. Intertwined dimers of the SH3 domains. (A) Eps8-SH3 domain (PDB code 1I07). Relevant residues are labelled in red (chain A, blue) and in black (chain B, pale cyan). The
intertwined dimer formation is stabilized through aromatic stacking interactions from the same region of the partner molecule (F52/W40) and salt bridges (D35/R18/E22). (B)
p47phox -SH3 domain. This intertwined dimer structure was solved in the context of the full protein, which is also shown in transparent mode (PDB code 1UEC). (C) CRKL C-terminal
SH3. The opening of the hinge loop generates a new extended b-strand (b*) with hydrogen bonds between residues of both chains. A careful examination of the packing of the SH3
molecules in the unit cell shows additional contacts between C13 of symmetry related molecules. The symmetry related dimer is also shown in transparent mode to indicate the
disulde bond between cysteine residues of the symmetry related chains (shown in the inset) (PDB code 2BZY). (D) Intertwined dimer of the c-Src-SH3 domain (PDB code 4JZ3). The
PEG molecule that favors the formation of the intertwined dimer is also shown.
122
mara-Artigas / Archives of Biochemistry and Biophysics 602 (2016) 116e126
A. Ca
A 3D model structure of the PI3K-SH3 domain amyloid bers has
been determined by cryoelectron microscopy [108]. As in the NMR
studies, the structural features of these bers indicated that this
SH3 domain needs to be partially unfolded to adopt the amyloid
form. On the other hand, NMR studies performed by Bayro et al.
[105,106] showed that the secondary structure of the bers was
different from the native fold: b1, b2 and b3 were preserved in the
bers as part of a longer b-strand, but not b4 and b5, which adopted
a random coil conformation. These modications also affected the
n-Src, RT loops and the diverging b-turn, which also adopted a rigid
b-strand conformation.
The most important difference in the PI3K-SH3 domain as
compared with other SH3 domains is the presence of a longer n-Src
loop. To address the possible role of this loop in the amyloidogenic
behavior of the PI3K-SH3 domain, Ventura et al. [112] constructed a
chimeric protein by the insertion of the n-Src loop of the PI3K-SH3
domain into the a-spectrin SH3 domain (a-Spc-SH3). When these
experiments were conducted, the a-Spc-SH3 was considered a
non-amyloidogenic protein, but nowadays we know that this SH3
also forms amyloids. Morel et al. [98] described the formation of
amyloid bers in the N47A mutant of the a-Spc-SH3 after incuba-
tion for several days at pH 3.0 and 37  C at high protein concen-
tration. The mutated residue belongs to the distal loop, which is
involved in the early folding events in this SH3 and destabilizes the
folding nucleus [98,113]. In most of the structures of the a-Spc-SH3,
residue N47 is placed in a disallowed region of the Ramachandran
plot. Also, it is worth mentioning that in most of the crystallo-
graphic structures some residues at the distal loop of the a-Spc-SH3
show weak electron density in the difference maps. This behavior
might be attributed to exibility or to heterogeneous conforma-
tions in this loop [114e117].
Although dimers and other higher order oligomers have been
described in the a-Spc-SH3 upon aggregation conditions, no
domain-swapped forms have been isolated [118,119]. As well as
N47A mutant, WT and other a-Spc-SH3 mutants can form amyloid
at acidic pH, but their formation is slower [120,121]. A detailed
study of the environmental conditions affecting the nucleation of
amyloid brils in this SH3 domain revealed that partial unfolding of
the native state and oligomerization trigger the formation of am-
yloid bers [122].
As the folding/unfolding process of the a-Spc-SH3 is one of the
best characterized [114,123e127], this SH3 domain has been used
as a model to study amyloid formation. Ventura et al. also per-
formed studies on the correlation between in vivo and in vitro ag-
gregation of this SH3 domain and suggested that the rules that
govern in vitro aggregation might also be valid in vivo [128]. In
addition, the role of amyloidogenic sequences has been studied by
inserting them into the a-Spc-SH3. As an example, to validate the
amyloid stretch hypothesis, Esteras-Chopo et al. [129] performed
the insertion of the designed high amyloidogenic sequence (STVIIE)
and an amyloidogenic six-residue fragment of the Ab-amyloid
peptide (16KLVFFA21) in the amino- and carboxyl terminal, as well
as in the core, of the a-Spc-SH3. The results of this work showed
that short amyloid stretches accessible for intermolecular in-
teractions trigger the self-assembly reaction.
Another SH3 domain that has been found to form amyloids is
the c-Yes-SH3 domain. These amyloids were obtained in the same
conditions as described for the previous SH3 domains (pH 3.0) [95],
but in this case the amyloid appears after a few days of incubation
at room temperature in the absence of salts and at low protein
concentration. Although most of the SH3 forming amyloids show a
pH-dependence and a preference for acidic or mild-acidic pH, a
destabilizing C-terminal truncated mutant of the Fyn-SH3 domain
(A39V/N53P/V55L/D(57e60) mutant) forms amyloid bers at
neutral pH [96]. This mutant was designed from data obtained from
relaxation dispersion NMR spectroscopy, which allowed re-
searchers to determine the structure of a low-populated on-
pathway folding intermediate of the Fyn-SH3 A39V/N53P/V55L
mutant (PDB entry 2L2P) and to study the aggregation prone re-
gions of this SH3 domain [96]. Kay et al. proposed that aggregation
in these domains does not proceed via global unfolding, but rather
by locally unfolded conformations accessed through thermal
uctuations.
1.7. The c-Src-SH3: a model for 3D domain swapping and amyloid
formation
To date, c-Src-SH3 is the only SH3 domain that has been re-
ported to form intertwined dimers and amyloid brils [55]. Though
the structure of this domain was rst solved by NMR in 1992 [130],
the rst crystallographic structure was not described until 2009
[57]. The crystallographic structure of the Q128R mutant was a
dimer where, as in the Eps8-SH3 intertwined dimer, the C-terminal
b-strands b3-b5 of two different protomers were switched. Several
years later, the structure of the WT and other mutants showed the
same intertwined dimers (Fig. 4D) [55]. A detailed analysis of the
potential salt bridges/hydrogen bonds between acidic residues
demonstrated a role of these interactions in the pH-dependence of
the intertwined dimer formation. In this case, the intertwined
structure was solved from crystals grown at mild acidic pH, while
the monomeric structure was obtained at neutral pH (Table 1).
The addition of different substances to the solution of the c-Src-
SH3 domain favors the equilibrium in the direction of the oligomer
formation or its dissociation. As an example, low molecular weight
PEGs favor dimerization, but the binding of proline rich motifs
peptides reverts the process: DLS experiments showed that the
addition of proline rich peptides in a 1:1 ratio to a solution con-
taining the intertwined dimer in presence of PEG300 results in the
formation of the monomer in a few minutes [55,104]. Furthermore,
the crystallographic structures of this SH3 domain in complex with
the high afnity synthetic peptides VSL12 (class I) and APP12 (class
II) showed the characteristic globular fold of the SH3 domain bound
to the peptides, even though the crystals were obtained at mild
acidic pH (5.0) and in the presence of PEG300 (10%) [103].
Interestingly, several years before the crystal structure of the
intertwined dimer was solved, Ding and co-workers predicted its
formation by molecular dynamics calculations [131]. In this study
the authors proposed that, in some cases, the formation of an open
monomer could yield amyloid brils if the swapping between
different protomers is not reciprocal but propagational [132,133].
This kind of assembly into amyloid brils has been proposed to be
behind the mechanism suggested for some domain-swapping/
amyloid-forming proteins, such as GB1 [90]. Nowadays, most of
the experimental evidence suggests that this model is not correct
[90,134]. As we mention above, the structural information on the
PI3K-SH3 amyloid brils indicates that the secondary structure of
the bers is different from the native fold [105,106].
Mutagenesis can help to understand the role of specic residues,
or groups of residues, that are important for domain swapping and
amyloid formation. We have studied the aggregation process of
several mutants of the residue Q128, at the distal loop, by different
techniques. Our results showed that amyloid formation was
dependent on the residue at this position, while 3D domain
swapping was not affected [55]. The single mutation of Q128 to
lysine, arginine or glutamate affects the protein stability in a pH-
dependent manner that is related to the charges of the residues
present in the pocket formed by the distal loop and the diverging b-
turn. The three mutants and the WT protein form intertwined di-
mers at mild acidic pH, but while the WT protein forms amyloids at
the same pH, the glutamate mutant only forms amyloids at neutral

mara-Artigas / Archives of Biochemistry and Biophysics 602 (2016) 116e126
A. Ca
pH (Table 1) (Fig. 5). This further indicates a key role of the residue
at position 128 in the folding and formation of amyloid aggregates
[55].
Residue Q128, like N47 in the a-spectrin SH3 domain, is located
at the distal loop and it is also involved in early folding events. In
addition, the high-resolution structures of the c-Src-SH3 (WT and
mutants) revealed the presence of several well-conserved water
molecules that might have a role in the protein folding process
[135]. This water network is present in the monomeric and the
intertwined dimer and facilitates the interactions among amino
acid residues at the distal loop and the b-diverging turn. X-ray
crystallography is the only technique that allows an accurate
modelling of the solvent and, together with the information pro-
vided by NMR techniques, it may prove essential in analyzing the
structural determinants of domain swapping and amyloid forma-
tion. Interestingly, the structure of the monomeric form of the c-
Src-SH3 showed two molecules in the asymmetric unit (chain A
and B) and each molecule displayed a different arrangement of the
hydrogen-bond network around the distal loop and the diverging
b-turn: in chain A, E106 is hydrogen bound to S123; while in chain
B this residue is hydrogen bound to T125. The hydrogen-bond be-
tween E106 and S123 has previously been reported to play a key
role in the folding reaction of the c-Src-SH3, bringing together the
distal loop and the diverging b-turn [136]. Changes in this hydrogen
bond seem to be correlated with the conformational changes and
the water network arrangement around the diverging b-turn and
the distal loop. Interestingly, the structure of the intermediate state
of folding of the Fyn-SH3 A39V/N53P/V55L mutant (PDB entry
2L2P) shows equivalent hydrogen bond interactions [96]. Differ-
ences between the chains of the WT c-Src-SH3 are also found in the
n-Src loop: chain A shows an ordered loop, while some residues in
chain B have not been modelled. The same features have been
found in the monomeric structure of the c-Src-SH3 mutant Q128E
(PDB entry 4OMO). Similar behavior has also been found in the
crystal structure of the WT Fyn-SH3 domain (PDB entry 3UA6),
which also has two molecules in the asymmetric unit [137]. The
crystal structure of the c-Yes-SH3 has only one molecule in the
asymmetric unit, but residues at the n-Src loop also show high B-
factors. All these ndings seem to indicate high exibility in the n-
Fig. 5. Amyloid brils of the c-Src-SH3 domain at neutral pH. Negatively stained
TEM image illustrating the c-Src-SH3 Q128E mutant incubated for two weeks in 0.1 M
Hepes 0.1 M sodium chloride (pH 7.0) at 37  C is shown, indicating the presence of long
amyloid brils.
123
Src loop in all the structures of these amyloid-forming members of
the Src tyrosine kinase family.
As the hinge loop plays an important role in the opening of the
monomer and subsequent formation of the intertwined structure,
our group designed several chimeric proteins to examine if the
determinant of the formation of these structures is inherent to the
sequence of the hinge loop, the n-Src loop, or neither. In the c-Src-
[RT/n-Src-Abl]-SH3 domain (SRC-2X) the amino acid residues of
the RT and n-Src loop of the c-Src-SH3 domain were replaced by
those present in the SH3 domain of the Abl tyrosine kinase. To date,
although peptides related to the diverging b-turn of this SH3
domain have been proven to form amyloids [138], the whole SH3
domain has not been reported to form either intertwined dimers or
amyloids. Unexpectedly, the resulting chimera SRC-2X crystallized
in a domain-swapped dimer where both RT and n-Src loops act as
hinge loops [56]. Domain swapping through two different hinge
loops has been described for ribonuclease A and cyanovirin-N, but
in these proteins the double-swapped form results in the formation
of trimers and tetramers [60,61]. To the best of our knowledge, this
is the rst time that the simultaneous opening of two loops of a
protein has resulted in the formation of a domain-swapped dimer.
1.8. SH3 domains as a model to study 3D domain swapping and
amyloid formation
To date, there is no evidence of any biological relevance of 3D
domain swapping and amyloid formation in the SH3 domain, but
certain characteristics of this small protein make it an ideal model
for studying both processes. The SH3 domain is characterized by
the lack of prosthetic groups and disulde bridges. It is easy to
purify and is also soluble over a broad range of pH and conditions.
For all these reasons, SH3 domains from different sources have
been studied extensively in the eld of protein folding and protein
interactions, and there is a vast amount of thermodynamic, kinetic
and structural information from both experimental and computa-
tional approaches [114,123e126,131,139e144]. These studies indi-
cate that the transition state of folding in these domains is mainly
organized around the hairpin formed by b3 and b4 strands, which
contains the distal loop (Fig. 3). Traditionally the unfolding process
of most of the SH3 domains has been analyzed using the two-state
model, but recently a more complex scenario has been drawn
[96,136,145,146]. Molecular-dynamics calculations also predicted
this complexity and some ndings suggest that SH3 domains may
exhibit stable intermediates under conditions that stabilize the
native state [66,147,148]. These folding intermediates have also
been analyzed by NMR techniques [136,145,146,149].
Most of the SH3 domains have a conserved acidic residue,
aspartate/glutamate, in the diverging b-turn and a serine/threo-
nine residue in the distal loop that form a hydrogen bond in the
folded state of the protein. The formation of this hydrogen bond
is a key step in the folding process of the SH3 domain. The n-Src
loop, in its role as hinge loop, mediates the approach of the distal
loop to the diverging turn in the folding process. The exibility of
the loop facilitates the folding-unfolding process and, in most of
the crystallographic structures of SH3 domains, is reected in
high B-factors for residues at the n-Src loop, which in some cases
has not been modelled [55,95,137]. Additionally, together with
the RT-loop, the n-Src loop plays a central role in the specicity
of the binding of proline rich motifs, and upon binding it shows
signicant conformational changes [103]. However, in the amy-
loid brils of the PI3K-SH3 domain, these exible regions have
been shown to adopt rigid b-strand conformations [106]. More-
over, the crystallographic structures of the domain-swapped di-
mers of SH3 domains show reduced mobility of the residues at
the hinge loops, which, analogously to the amyloid ber of the
124
mara-Artigas / Archives of Biochemistry and Biophysics 602 (2016) 116e126
A. Ca
PI3K-SH3, adopt an extended conformation. By way of example,
the monomeric structure of the CRKL C-terminal SH3 domain
shows high B-factor values for the RT loop residues (PDB code
2BZX), but upon opening of the protomer these residues adopt a
b-strand conformation (Fig. 4C) [93].
The molecular mechanism that initiates the amyloid formation
process is not well established. Some authors claim that the
structural environment of the amyloidogenic sequences plays a key
role in whether or not an amyloid-prone sequence can productively
trigger the amyloid self-assembly process. In most of the SH3 do-
mains the rst strand is hydrophobic and aggregation prone upon
exposition to the solvent [150]. Thus, changes in the solution con-
ditions that expose the b1 strand might trigger the amyloid for-
mation. The structure of the Fyn-SH3 domain on-pathway folding
intermediate showed that strand b5 is disordered and b1 becomes
exposed and involved in non-native contacts, and the destabilizing
C-terminal truncated mutant of the Fyn-SH3 domain (A39V/N53P/
V55L/D(57e60) mutant) was found to form amyloids [96].
The pH value of the solution plays a key role in the formation
of domain-swapped dimers and amyloids. The a-Spc-SH3 N47A
mutant forms amyloid in a narrow range of pH (pH 2.8e3.5) and
this behavior has been attributed to the protonation of E7, which
is expected to be unprotonated at pH > 4.0 [151]. This residue is
close to the most aggregation prone region of the a-Spc-SH3,
which comprises b-strand b1 (predicted using TANGO algorithm
[152]). Charged residues have also been reported to play an
important role in bril formation as they can act as gatekeepers:
residues with the same charge show repulsive interactions upon
self-assembly that prevent protein aggregation. Aggregation an-
alyses performed using TANGO showed that arginine and lysine
residues act as better gatekeepers than the acidic residues
glutamate and aspartate [153]. In addition to their charge, their
side chains have much larger conformational entropy. To date no
amyloid formation has been found in the arginine and lysine
mutants at position 128 of the c-Src-SH3 domain, while the WT
and the glutamate mutant form amyloid at different pH (5.0 and
7.0, respectively) [55].
In conclusion, partially unfolded states, such as those present
in domain-swapped proteins, may play a role in the initiation of
amyloid aggregates: these intermediates are available to interact
with a neighboring molecule in the same or fully unfolded state.
As pointed out above, the c-Src-SH3 forms amyloid at different
pH, 7.0 and 5.0, depending on whether the protein is a monomer
or an intertwined dimer in solution, respectively. The formation
of amyloid of the WT variant is characterized by rapid formation
of the aggregates at high protein concentration (>20 mg/mL),
moderate temperatures (25  C) and low ionic strength. Under
these conditions, the domain-swapped oligomer appears at
concentration higher than 10 mg/mL. A similar behavior has been
described for the formation of amyloid brils from domain-
swapped dimers of the GB1 [154], where bril formation takes
place easily for those mutants that exhibit domain-swapped di-
mers. However, all evidence indicates that amyloid formation in
the c-Src-SH3 might proceed via two distinct pathways at mildly
acidic pH as compared to neutral pH [55]. It is well established
that the mechanism underlying amyloid conversion has the same
kinetic mechanism as crystal formation: nucleation and growth
[155,156]. At acidic pH, the domain-swapped form might play a
role in the formation of amyloid aggregates by acting as a
nucleating particle [157]. On the contrary, the formation of am-
yloid bers by the Q128E mutant at neutral pH behaves like a
nucleated polymerization, with a long lag phase [158,159].
Further studies with these modular domains might help to clarify
the mechanism of 3D domain swapping and to unravel its
involvement in the early steps of the formation of amyloid brils.
Acknowledgements
This research was funded by the Spanish Ministry of Economy
and Competitiveness (Spain) and FEDER (EU) [BIO2012-39922-
C02-01/02].
References
[1] C.B. Annsen, E. Haber, M. Sela, F.H. White Jr., Proc. Natl. Acad. Sci. U. S. A. 47
(1961) 1309e1314.
[2] S.B. Prusiner, R.A. Barry, M.P. McKinley, C.G. Bellinger, R.K. Meyer,
S.J. DeArmond, D.T. Kingsbury, Microbiol. Sci. 2 (1985) 33e39.
[3] J. Greenwald, R. Riek, J. Mol. Biol. 421 (2012) 417e426.
[4] F. Chiti, C.M. Dobson, Annu. Rev. Biochem. 75 (2006) 333e366.
[5] J. Greenwald, R. Riek, Structure 18 (2010) 1244e1260.
[6] L. Breydo, V.N. Uversky, FEBS Lett. 589 (2015) 2640e2648.
[7] K.W. Tipping, P. van Oosten-Hawle, E.W. Hewitt, S.E. Radford, Trends Bio-
chem. Sci. 40 (2015) 719e727.
[8] D.M. Fowler, A.V. Koulov, W.E. Balch, J.W. Kelly, Trends Biochem. Sci. 32
(2007) 217e224.
[9] W.T. Astbury, S. Dickinson, K. Bailey, Biochem. J. 29 (1935) 2351e2360.
[10] B.H. Toyama, J.S. Weissman, Annu. Rev. Biochem. 80 (2011) 557e585.
[11] M.J. Bennett, M.R. Sawaya, D. Eisenberg, Structure 14 (2006) 811e824.
[12] I. Hafner-Bratkovic, R. Bester, P. Pristovsek, L. Gaedtke, P. Veranic,
J. Gaspersic, M. Mancek-Keber, M. Avbelj, M. Polymenidou, C. Julius,
A. Aguzzi, I. Vorberg, R. Jerala, J. Biol. Chem. 286 (2011) 12149e12156.
[13] K.J. Knaus, M. Morillas, W. Swietnicki, M. Malone, W.K. Surewicz, V.C. Yee,
Nat. Struct. Biol. 8 (2001) 770e774.
[14] R. Janowski, M. Kozak, M. Abrahamson, A. Grubb, M. Jaskolski, Proteins 61
(2005) 570e578.
[15] M. Nilsson, X. Wang, S. Rodziewicz-Motowidlo, R. Janowski, V. Lindstrom,
P. Onnerfjord, G. Westermark, Z. Grzonka, M. Jaskolski, A. Grubb, J. Biol.
Chem. 279 (2004) 24236e24245.
[16] M. Wahlbom, X. Wang, V. Lindstrom, E. Carlemalm, M. Jaskolski, A. Grubb,
J. Biol. Chem. 282 (2007) 18318e18326.
[17] Y. Huang, H. Cao, Z. Liu, Proteins 80 (2012) 1610e1619.
[18] S. Yang, H. Levine, J.N. Onuchic, J. Mol. Biol. 352 (2005) 202e211.
[19] B. Caughey, P.T. Lansbury, Annu. Rev. Neurosci. 26 (2003) 267e298.
[20] F. Rousseau, H. Wilkinson, J. Villanueva, L. Serrano, J.W. Schymkowitz,
L.S. Itzhaki, J. Mol. Biol. 363 (2006) 496e505.
[21] M. Fandrich, J. Mol. Biol. 421 (2012) 427e440.
[22] D. Eisenberg, M. Jucker, Cell 148 (2012) 1188e1203.
[23] T. Luhrs, C. Ritter, M. Adrian, D. Riek-Loher, B. Bohrmann, H. Dobeli,
D. Schubert, R. Riek, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 17342e17347.
[24] W. Qiang, W.M. Yau, Y. Luo, M.P. Mattson, R. Tycko, Proc. Natl. Acad. Sci. U. S.
A. 109 (2012) 4443e4448.
[25] H. Van Melckebeke, C. Wasmer, A. Lange, E. Ab, A. Loquet, A. Bockmann,
B.H. Meier, J. Am. Chem. Soc. 132 (2010) 13765e13775.
[26] Y. Xiao, B. Ma, D. McElheny, S. Parthasarathy, F. Long, M. Hoshi, R. Nussinov,
Y. Ishii, Nat. Struct. Mol. Biol. 22 (2015) 499e505.
[27] M. Balbirnie, R. Grothe, D.S. Eisenberg, Proc. Natl. Acad. Sci. U. S. A. 98 (2001)
2375e2380.
[28] M.R. Sawaya, S. Sambashivan, R. Nelson, M.I. Ivanova, S.A. Sievers,
M.I. Apostol, M.J. Thompson, M. Balbirnie, J.J. Wiltzius, H.T. McFarlane,
A.O. Madsen, C. Riekel, D. Eisenberg, Nature 447 (2007) 453e457.
[29] L. Saelices, L.M. Johnson, W.Y. Liang, M.R. Sawaya, D. Cascio, P. Ruchala,
J. Whitelegge, L. Jiang, R. Riek, D.S. Eisenberg, J. Biol. Chem. 290 (2015)
28932e28943.
[30] J.P. Colletier, A. Laganowsky, M. Landau, M. Zhao, A.B. Soriaga,
L. Goldschmidt, D. Flot, D. Cascio, M.R. Sawaya, D. Eisenberg, Proc. Natl. Acad.
Sci. U. S. A. 108 (2011) 16938e16943.
[31] M.I. Ivanova, S.A. Sievers, M.R. Sawaya, J.S. Wall, D. Eisenberg, Proc. Natl.
Acad. Sci. U. S. A. 106 (2009) 18990e18995.
[32] R. Nelson, M.R. Sawaya, M. Balbirnie, A.O. Madsen, C. Riekel, R. Grothe,
D. Eisenberg, Nature 435 (2005) 773e778.
[33] K.S. MacLea, K.R. Paul, Z. Ben-Musa, A. Waechter, J.E. Shattuck, M. Gruca,
E.D. Ross, Mol. Cell. Biol. 35 (2015) 899e911.
[34] J.J. Wiltzius, M. Landau, R. Nelson, M.R. Sawaya, M.I. Apostol, L. Goldschmidt,
A.B. Soriaga, D. Cascio, K. Rajashankar, D. Eisenberg, Nat. Struct. Mol. Biol. 16
(2009) 973e978.
[35] T. Hard, J. Phys. Chem. Lett. 5 (2014) 607e614.
[36] F. Chiti, P. Webster, N. Taddei, A. Clark, M. Stefani, G. Ramponi, C.M. Dobson,
Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 3590e3594.
[37] A.M. Cresteld, W.H. Stein, S. Moore, Archives Biochem. Biophys. Suppl. 1
(1962) 217e222.
[38] L. Mazzarella, S. Capasso, D. Demasi, G. Di Lorenzo, C.A. Mattia, A. Zagari, Acta
Crystallogr. Sect. D. Biol. Crystallogr. 49 (1993) 389e402.
[39] M.J. Bennett, S. Choe, D. Eisenberg, Protein Sci. A Publ. Protein Soc. 3 (1994)
1444e1463.
[40] M.J. Bennett, S. Choe, D. Eisenberg, Proc. Natl. Acad. Sci. U. S. A. 91 (1994)
3127e3131.
[41] S. Adinol, R. Piccoli, F. Sica, L. Mazzarella, FEBS Lett. 398 (1996) 326e332.

mara-Artigas / Archives of Biochemistry and Biophysics 602 (2016) 116e126
A. Ca
[42] Y. Liu, G. Gotte, M. Libonati, D. Eisenberg, Nat. Struct. Biol. 8 (2001) 211e214.
[43] M.J. Bennett, D. Eisenberg, Protein Sci. A Publ. Protein Soc. 3 (1994)
1464e1475.
[44] D.J. Boerema, V.A. Tereshko, S.B. Kent, Biopolymers 90 (2008) 278e286.
[45] Y. Liu, P.J. Hart, M.P. Schlunegger, D. Eisenberg, Proc. Natl. Acad. Sci. U. S. A.
95 (1998) 3437e3442.
[46] M.J. Bennett, M.P. Schlunegger, D. Eisenberg, Protein Sci. A Publ. Protein Soc.
4 (1995) 2455e2468.
[47] Y. Liu, D. Eisenberg, Protein Sci. A Publ. Protein Soc. 11 (2002) 1285e1299.
[48] M.P. Schlunegger, M.J. Bennett, D. Eisenberg, Adv. Protein Chem. 50 (1997)
61e122.
[49] F. Rousseau, J. Schymkowitz, L.S. Itzhaki, Adv. Exp. Med. Biol. 747 (2012)
137e152.
[50] S.S. MacKinnon, S.J. Wodak, J. Mol. Biol. 427 (2015) 350e370.
[51] M.A. Smith, O.A. Bateman, R. Jaenicke, C. Slingsby, Protein Sci. A Publ. Protein
Soc. 16 (2007) 615e625.
[52] C.J. Blamey, C. Ceccarelli, U.P. Naik, B.J. Bahnson, Protein Sci. A Publ. Protein
Soc. 14 (2005) 1214e1221.
[53] K. Shameer, P.N. Shingate, S.C. Manjunath, M. Karthika, G. Pugalenthi,
R. Sowdhamini, Database J. Biol. Databases Curation 2011 (2011) bar042.
[54] K.V. Kishan, G. Scita, W.T. Wong, P.P. Di Fiore, M.E. Newcomer, Nat. Struct.
Biol. 4 (1997) 739e743.
[55] J. Bacarizo, S. Martinez-Rodriguez, J.M. Martin-Garcia, M. Andujar-Sanchez,
E. Ortiz-Salmeron, J.L. Neira, A. Camara-Artigas, PLoS One 9 (2014) e113224.
[56] A. Camara-Artigas, S. Martinez-Rodriguez, E. Ortiz-Salmeron, J.M. Martin-
Garcia, J. Struct. Biol. 186 (2014) 195e203.
[57] A. Camara-Artigas, J.M. Martin-Garcia, B. Morel, J. Ruiz-Sanz, I. Luque, FEBS
Lett. 583 (2009) 749e753.
[58] S. Hirota, Y. Hattori, S. Nagao, M. Taketa, H. Komori, H. Kamikubo, Z. Wang,
I. Takahashi, S. Negi, Y. Sugiura, M. Kataoka, Y. Higuchi, Proc. Natl. Acad. Sci.
U. S. A. 107 (2010) 12854e12859.
[59] I. Zegers, J. Deswarte, L. Wyns, Proc. Natl. Acad. Sci. U. S. A. 96 (1999)
818e822.
[60] L.M. Koharudin, L. Liu, A.M. Gronenborn, Proc. Natl. Acad. Sci. U. S. A. 110
(2013) 7702e7707.
[61] G. Gotte, M. Libonati, J. Biol. Chem. 279 (2004) 36670e36679.
[62] F. Rousseau, J.W. Schymkowitz, H.R. Wilkinson, L.S. Itzhaki, Proc. Natl. Acad.
Sci. U. S. A. 98 (2001) 5596e5601.
[63] M. Orlikowska, E. Jankowska, R. Kolodziejczyk, M. Jaskolski, A. Szymanska,
J. Struct. Biol. 173 (2011) 406e413.
[64] A. Szymanska, E. Jankowska, M. Orlikowska, I. Behrendt, P. Czaplewska,
S. Rodziewicz-Motowidlo, Front. Mol. Neurosci. 5 (2012) 82.
[65] Y.W. Chen, K. Stott, M.F. Perutz, Proc. Natl. Acad. Sci. U. S. A. 96 (1999)
1257e1261.
[66] S. Yang, S.S. Cho, Y. Levy, M.S. Cheung, H. Levine, P.G. Wolynes, J.N. Onuchic,
Proc. Natl. Acad. Sci. U. S. A. 101 (2004) 13786e13791.
[67] F.L. Sirota, S. Hery-Huynh, S. Maurer-Stroh, S.J. Wodak, Proteins 72 (2008)
88e104.
[68] J.W. O'Neill, D.E. Kim, K. Johnsen, D. Baker, K.Y. Zhang, Structure 9 (2001)
1017e1027.
[69] M.E. Newcomer, Curr. Opin. Struct. Biol. 12 (2002) 48e53.
[70] H. Raaijmakers, O. Vix, I. Toro, S. Golz, B. Kemper, D. Suck, EMBO J. 18 (1999)
1447e1458.
[71] B. Bagautdinov, Acta Crystallogr. Sect. F. Struct. Biol. Commun. 70 (2014)
404e413.
[72] A. Sato, S. Yokotani, T. Tadokoro, S. Tanaka, C. Angkawidjaja, Y. Koga,
K. Takano, S. Kanaya, J. Synchrotron Radiat. 18 (2011) 6e10.
[73] J.L. Llacer, A. Contreras, K. Forchhammer, C. Marco-Marin, F. Gil-Ortiz,
R. Maldonado, I. Fita, V. Rubio, Proc. Natl. Acad. Sci. U. S. A. 104 (2007)
17644e17649.
[74] M.H. Ali, B. Imperiali, Bioorg. Med. Chem. 13 (2005) 5013e5020.
[75] A.M. Gronenborn, Curr. Opin. Struct. Biol. 19 (2009) 39e49.
[76] H. Mizuno, Z. Fujimoto, M. Koizumi, H. Kano, H. Atoda, T. Morita, J. Mol. Biol.
289 (1999) 103e112.
[77] Y. Shikamoto, T. Morita, Z. Fujimoto, H. Mizuno, J. Biol. Chem. 278 (2003)
24090e24094.
[78] A.D. Cameron, B. Olin, M. Ridderstrom, B. Mannervik, T.A. Jones, EMBO J. 16
(1997) 3386e3395.
[79] P. Dumas, M. Bergdoll, C. Cagnon, J.M. Masson, EMBO J. 13 (1994)
2483e2492.
[80] M. Ridderstrom, B. Mannervik, Biochem. J. 316 (3) (1996) 1005e1006.
[81] C.K. Park, H.K. Joshi, A. Agrawal, M.I. Ghare, E.J. Little, P.W. Dunten,
J. Bitinaite, N.C. Horton, PLoS Biol. 8 (2010) e1000554.
[82] L. Vitagliano, S. Adinol, F. Sica, A. Merlino, A. Zagari, L. Mazzarella, J. Mol.
Biol. 293 (1999) 569e577.
[83] Y.K. Mathiharan, H.S. Savithri, M.R. Murthy, Acta Crystallogr. Sect. D. Biol.
Crystallogr. 71 (2015) 1812e1823.
[84] D. Ivanov, O.V. Tsodikov, J. Kasanov, T. Ellenberger, G. Wagner, T. Collins,
Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 4353e4358.
[85] D. Ivanov, J.R. Stone, J.L. Maki, T. Collins, G. Wagner, Mol. Cell 17 (2005)
137e143.
[86] R. Bocanegra, M.A. Fuertes, A. Rodriguez-Huete, J.L. Neira, M.G. Mateu, Bio-
phys. J. 108 (2015) 338e349.
[87] C. Qu, L. Liljas, N. Opalka, C. Brugidou, M. Yeager, R.N. Beachy, C.M. Fauquet,
J.E. Johnson, T. Lin, Structure 8 (2000) 1095e1103.
125
[88] R. Janowski, M. Kozak, E. Jankowska, Z. Grzonka, A. Grubb, M. Abrahamson,
M. Jaskolski, Nat. Struct. Biol. 8 (2001) 316e320.
[89] M. Jaskolski, Acta Biochim. Pol. 48 (2001) 807e827.
[90] P.C. van der Wel, Prion 6 (2012) 211e216.
[91] B.K. Kay, FEBS Lett. 586 (2012) 2606e2608.
[92] S. Yuzawa, N.N. Suzuki, Y. Fujioka, K. Ogura, H. Sumimoto, F. Inagaki, Genes
cells Devoted Mol. Cell. Mech. 9 (2004) 443e456.
[93] M. Harkiolaki, R.J. Gilbert, E.Y. Jones, S.M. Feller, Structure 14 (2006)
1741e1753.
[94] J. Bacarizo, J.L. Neira, M. Andujar-Sa
nchez, E. Ortiz-Salmero
n, A. Ca
mara-
Artigas, Submitted to Biochemistry (2013).
[95] J.M. Martin-Garcia, I. Luque, P.L. Mateo, J. Ruiz-Sanz, A. Camara-Artigas, FEBS
Lett. 581 (2007) 1701e1706.
[96] P. Neudecker, P. Robustelli, A. Cavalli, P. Walsh, P. Lundstrom, A. Zarrine-
Afsar, S. Sharpe, M. Vendruscolo, L.E. Kay, Science 336 (2012) 362e366.
[97] J.I. Guijarro, M. Sunde, J.A. Jones, I.D. Campbell, C.M. Dobson, Proc. Natl. Acad.
Sci. U. S. A. 95 (1998) 4224e4228.
[98] B. Morel, S. Casares, F. Conejero-Lara, J. Mol. Biol. 356 (2006) 453e468.
[99] K.V. Kishan, M.E. Newcomer, T.H. Rhodes, S.D. Guilliot, Protein Sci. A Publ.
Protein Soc. 10 (2001) 1046e1055.
[100] Y. Groemping, K. Lapouge, S.J. Smerdon, K. Rittinger, Cell 113 (2003)
343e355.
[101] S. Yuzawa, K. Ogura, M. Horiuchi, N.N. Suzuki, Y. Fujioka, M. Kataoka,
H. Sumimoto, F. Inagaki, J. Biol. Chem. 279 (2004) 29752e29760.
[102] K. Ogura, I. Nobuhisa, S. Yuzawa, R. Takeya, S. Torikai, K. Saikawa,
H. Sumimoto, F. Inagaki, J. Biol. Chem. 281 (2006) 3660e3668.
[103] J. Bacarizo, A. Camara-Artigas, Acta Crystallogr. Sect. D. Biol. Crystallogr. 69
(2013) 756e766.
[104] J. Bacarizo, S. Martinez-Rodriguez, A. Camara-Artigas, J. Struct. Biol. 189
(2015) 67e72.
[105] M.J. Bayro, G.T. Debelouchina, M.T. Eddy, N.R. Birkett, C.E. MacPhee,
M. Rosay, W.E. Maas, C.M. Dobson, R.G. Grifn, J. Am. Chem. Soc. 133 (2011)
13967e13974.
[106] M.J. Bayro, T. Maly, N.R. Birkett, C.E. Macphee, C.M. Dobson, R.G. Grifn,
Biochemistry 49 (2010) 7474e7484.
[107] J. Zurdo, J.I. Guijarro, J.L. Jimenez, H.R. Saibil, C.M. Dobson, J. Mol. Biol. 311
(2001) 325e340.
[108] J.L. Jimenez, J.I. Guijarro, E. Orlova, J. Zurdo, C.M. Dobson, M. Sunde,
H.R. Saibil, EMBO J. 18 (1999) 815e821.
[109] A. Orte, N.R. Birkett, R.W. Clarke, G.L. Devlin, C.M. Dobson, D. Klenerman,
Proc. Natl. Acad. Sci. U. S. A. 105 (2008) 14424e14429.
[110] Y. Yu, Y. Wang, J. He, Y. Liu, H. Li, H. Zhang, Y. Song, J. Biomol. Struct. Dyn. 27
(2010) 641e649.
[111] N. Carulla, M. Zhou, M. Arimon, M. Gairi, E. Giralt, C.V. Robinson,
C.M. Dobson, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) 7828e7833.
[112] S. Ventura, E. Lacroix, L. Serrano, J. Mol. Biol. 322 (2002) 1147e1158.
[113] D.K. Klimov, D. Thirumalai, J. Mol. Biol. 317 (2002) 721e737.
[114] M.C. Vega, J.C. Martinez, L. Serrano, Protein Sci. A Publ. Protein Soc. 9 (2000)
2322e2328.
[115] A. Camara-Artigas, M. Andujar-Sanchez, E. Ortiz-Salmeron, C. Cuadri,
E.S. Cobos, J.M. Martin-Garcia, Acta Crystallogr. Sect. F Struct. Biol. Cryst.
Commun. 66 (2010) 1023e1027.
[116] A. Camara-Artigas, J.A. Gavira, S. Casares, J.M. Garcia-Ruiz, F. Conejero-Lara,
J.P. Allen, J.C. Martinez, Acta Crystallogr. Sect. D. Biol. Crystallogr. 67 (2011)
189e196.
[117] A. Camara-Artigas, M. Andujar-Sanchez, E. Ortiz-Salmeron, C. Cuadri,
S. Casares, Acta Crystallogr. Sect. D. Biol. Crystallogr. 65 (2009) 1247e1252.
[118] D. Ruzafa, B. Morel, L. Varela, A.I. Azuaga, F. Conejero-Lara, PLoS One 7 (2012)
e49690.
[119] F. Castello, S. Casares, M.J. Ruedas-Rama, A. Orte, J. Phys. Chem. B 119 (2015)
8260e8267.
[120] D. Ruzafa, L. Varela, A.I. Azuaga, F. Conejero-Lara, B. Morel, Phys. Chem.
Chem. Phys. PCCP 16 (2014) 2989e3000.
[121] L. Varela, B. Morel, A.I. Azuaga, F. Conejero-Lara, FEBS Lett. 583 (2009)
801e806.
[122] B. Morel, L. Varela, A.I. Azuaga, F. Conejero-Lara, Biophysi. J. 99 (2010)
3801e3810.
[123] S. Casares, O. Lopez-Mayorga, M.C. Vega, A. Camara-Artigas, F. Conejero-Lara,
Proteins 67 (2007) 531e547.
[124] J.C. Martinez, L. Serrano, Nat. Struct. Biol. 6 (1999) 1010e1016.
[125] J.C. Martinez, M.T. Pisabarro, L. Serrano, Nat. Struct. Biol. 5 (1998) 721e729.
[126] A.R. Viguera, J.C. Martinez, V.V. Filimonov, P.L. Mateo, L. Serrano, Biochem-
istry 33 (1994) 2142e2150.
[127] A.M. Fernandez-Escamilla, M.S. Cheung, M.C. Vega, M. Wilmanns,
J.N. Onuchic, L. Serrano, Proc. Natl. Acad. Sci. U. S. A. 101 (2004) 2834e2839.
[128] A. Espargaro, V. Castillo, N.S. de Groot, S. Ventura, J. Mol. Biol. 378 (2008)
1116e1131.
[129] A. Esteras-Chopo, L. Serrano, M. Lopez de la Paz, Proc. Natl. Acad. Sci. U. S. A.
102 (2005) 16672e16677.
[130] H. Yu, M.K. Rosen, T.B. Shin, C. Seidel-Dugan, J.S. Brugge, S.L. Schreiber, Sci-
ence 258 (1992) 1665e1668.
[131] F. Ding, N.V. Dokholyan, S.V. Buldyrev, H.E. Stanley, E.I. Shakhnovich, J. Mol.
Biol. 324 (2002) 851e857.
[132] F. Ding, N.V. Dokholyan, S.V. Buldyrev, H.E. Stanley, E.I. Shakhnovich, J. Mol.
Biol. 324 (2002) 851e857.
126
mara-Artigas / Archives of Biochemistry and Biophysics 602 (2016) 116e126
A. Ca
[133] F. Ding, K.C. Prutzman, S.L. Campbell, N.V. Dokholyan, Structure 14 (2006)
5e14.
[134] J. Li, C.L. Hoop, R. Kodali, V.N. Sivanandam, P.C. van der Wel, J. Biol. Chem.
286 (2011) 28988e28995.
[135] W. Guo, S. Lampoudi, J.E. Shea, Biophys. J. 85 (2003) 61e69.
[136] V.P. Grantcharova, D.S. Riddle, D. Baker, Proc. Natl. Acad. Sci. U. S. A. 97
(2000) 7084e7089.
[137] J.M. Martin-Garcia, I. Luque, J. Ruiz-Sanz, A. Camara-Artigas, Acta Crystallogr.
Sect. D. Biol. Crystallogr. 68 (2012) 1030e1040.
[138] D. Lapidus, V. Duka, V. Stonkus, C. Czaplewski, A. Liwo, S. Ventura, I. Liepina,
Biopolymers 98 (2012) 557e566.
[139] V.P. Grantcharova, D. Baker, Biochemistry 36 (1997) 15685e15692.
[140] E.S. Cobos, V.V. Filimonov, M.C. Vega, P.L. Mateo, L. Serrano, J.C. Martinez,
J. Mol. Biol. 328 (2003) 221e233.
[141] V.V. Filimonov, A.I. Azuaga, A.R. Viguera, L. Serrano, P.L. Mateo, Biophys.
Chem. 77 (1999) 195e208.
[142] W.T. Chu, J.L. Zhang, Q.C. Zheng, L. Chen, H.X. Zhang, e64886, PLoS One 8
(2013).
[143] D. Long, G. Bouvignies, L.E. Kay, Proc. Natl. Acad. Sci. U. S. A. 111 (2014)
8820e8825.
[144] I.V. Kalgin, M. Karplus, S.F. Chekmarev, J. Phys. Chem. B 113 (2009)
12759e12772.
[145] T. Kortemme, M.J. Kelly, L.E. Kay, J. Forman-Kay, L. Serrano, J. Mol. Biol. 297
(2000) 1217e1229.
[146] Y.K. Mok, C.M. Kay, L.E. Kay, J. Forman-Kay, J. Mol. Biol. 289 (1999) 619e638.
[147] F. Ding, W. Guo, N.V. Dokholyan, E.I. Shakhnovich, J.E. Shea, J. Mol. Biol. 350
(2005) 1035e1050.
[148] J.M. Borreguero, F. Ding, S.V. Buldyrev, H.E. Stanley, N.V. Dokholyan, Biophys.
J. 87 (2004) 521e533.
[149] D.M. Korzhnev, L.E. Kay, Accounts Chem. Res. 41 (2008) 442e451.
[150] O. Conchillo-Sole, N.S. de Groot, F.X. Aviles, J. Vendrell, X. Daura, S. Ventura,
BMC Bioinforma. 8 (2007) 65.
[151] H. Krobath, S.G. Estacio, P.F. Faisca, E.I. Shakhnovich, J. Mol. Biol. 422 (2012)
705e722.
[152] A.M. Fernandez-Escamilla, F. Rousseau, J. Schymkowitz, L. Serrano, Nat.
Biotechnol. 22 (2004) 1302e1306.
[153] F. Rousseau, J. Schymkowitz, L. Serrano, Curr. Opin. Struct. Biol. 16 (2006)
118e126.
[154] J.M. Louis, I.J. Byeon, U. Baxa, A.M. Gronenborn, J. Mol. Biol. 348 (2005)
687e698.
[155] J.D. Harper, P.T. Lansbury Jr., Annu. Rev. Biochem. 66 (1997) 385e407.
[156] M. Ivanova, R. Jasuja, L. Krasnosselskaia, R. Josephs, Z. Wang, M. Ding,
K. Horiuchi, K. Adachi, F.A. Ferrone, J. Mol. Biol. 314 (2001) 851e861.
[157] A.R. Hurshman, J.T. White, E.T. Powers, J.W. Kelly, Biochemistry 43 (2004)
7365e7381.
[158] S.I. Cohen, M. Vendruscolo, C.M. Dobson, T.P. Knowles, J. Chem. Phys. 135
(2011) 065106.
[159] S.I. Cohen, M. Vendruscolo, M.E. Welland, C.M. Dobson, E.M. Terentjev,
T.P. Knowles, J. Chem. Phys. 135 (2011) 065105.