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Archives of Biochemistry and Biophysics 602 (2016) 116e126

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Crystallographic studies on protein misfolding: Domain swapping and

amyloid formation in the SH3 domain
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
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.
SH3 domain

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

valuable structural information include high-resolution electron
Since Annsen's pioneering work on protein folding a number of microscopy, solid state NMR and other NMR techniques, Fourier
research works have studied how native structure acquisition is transform infrared spectroscopy, circular dichroism and electron
inuenced by protein sequence and the milieu conditions [1]. paramagnetic resonance [10]. Even though the information pro-
Failure in the folding process of a protein may result in the devel- vided by these techniques has allowed researchers to obtain a
opment of protein deposition diseases. Creutzfeldt-Jakob disease detailed view of several amyloid structures, the mechanism by
[2] was the rst one to illustrate that a misfolded protein can be the which amyloidogenic proteins become unfolded and initiate their
origin of some diseases. To date over 30 major human diseases have aggregation process is still unknown. The development of models
been connected to amyloid formation; these include both geneti- to explain this process is particularly challenging due to the dif-
cally and non-genetically related diseases, some of which are culty in obtaining high-resolution structural information, and this
transmissible [3,4]. In addition, some amyloids have been classied is where structural studies on three-dimensional (3D) domain-
as functional amyloids and reported to play a role in diverse swapped proteins can help.
physiological processes [5e8]. 3D domain swapping is a phenomenon that occurs when two or
X-ray diffraction experiments conducted by Astbury and Dick- more molecules of a partially unfolded protomer form homo-
inson in the 1930s rst showed the typical cross-b ber diffraction dimers, or higher-order oligomers, by exchanging protein domains
pattern displayed by amyloid bers [9]. Nevertheless, structural or secondary structure elements. The formation of these oligomers
information on amyloids is still problematic because the two has been proposed as a possible mechanism underlying the
structural techniques that provide high-resolution data, nuclear appearance of early aggregates of amyloid bril [11]. Some well-
magnetic resonance (NMR) and X-ray crystallography, are not characterized amyloid-forming globular proteins also form inter-
applicable to these aggregates owing to their large size and the 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
E-mail address:
0003-9861/ 2016 Elsevier Inc. All rights reserved.
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almost any protein may oligomerize via this mechanism [17]. The structures available for a complete amyloid bril, for example
swapped-oligomer appears as an alternative mechanism for pro- those of the functional prion HET-s from the fungi Podospora
tomer folding, possibly driven by a lower energy of self-associated anserina [25] and the b-amyloid (1e42) bril [23,24,26]. Never-
species compared with the single folded chain, or alternatively by theless, most of the crystallographic information about amyloid
the faster formation of the oligomer and its later stabilization. Most aggregates is limited to short peptide sequences that are able to
of the hydrophobic-core interactions of the swapped oligomer are form amyloid and crystallize at the same time. Eisenberg's group
already present in the protomer, and the secondary structure ele- solved the crystallographic structure of about one hundred amyloid
ments are often conserved [18]. On the other hand, major changes forming peptides derived from Sup35 [27,28], transthyretin [29], b-
are present in the so-called hinge loops, which facilitate the pro- amyloid [30] and insulin [31], among others. These structures
tomer's opening. revealed shared characteristics, as is the case of the cross b-sheet
Nowadays, the structures of several domain-swapping/amyloid- structures [32], the dry interface between sheets [27] and the steric
forming proteins are available, and although the role of 3D domain zipper [28]. They also show that the remarkable stability of these
swapping is not clear, the structural information obtained from bers is reached through near optimal values for main-chain
these intertwined proteins may help to elucidate their plausible dihedral angles, along with an extensive packing of hydrophobic
functional role and, in addition, to clarify the complex mechanism side chains and salt bridge/hydrogen bond formation between the
of amyloidogenesis. Furthermore, these oligomeric structures peptides (Fig. 1).
deserve attention since some studies on deposition diseases have The structural studies also revealed conformational diversity in
attributed the toxicity to oligomeric assemblies of the protein, prion proteins [33] and other amyloid systems [28,34,35]. This
rather than to the docking of mature brils [19e21]. 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-
1.1. Structural information on amyloids mation can occur when the native globular fold of a protein is desta-
bilized under conditions in which noncovalent interactions still remain
High-resolution structural information on amyloids is limited, favorable [36]. In this way, manipulating the pH, temperature and
but recent technological advances and the use of structural ap- salt concentration gives the possibility of producing amyloids un-
proaches in concert resulted in a more detailed picture of these der controlled conditions that might result in morphologically
aggregates. Most of this information comes from the study of some different structures. In addition, it provides the opportunity to
well-characterized amyloid-forming proteins, for example Sup35 investigate the molecular basis of amyloid formation in different
prion proteins, Alzheimer's disease b-amyloid, Parkinson's disease proteins.
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 1.2. Structural information on 3D domain-swapped proteins
be in-register or out of register. In addition, there are several ter-
tiary structures for these b-sheets: b-sandwich; b-solenoid, which Although domain swapping was rst proposed in the 1960s to
can form a b-helix or a b-roll [22]. Fig. 1 illustrates the structure of explain the behavior of a ribonuclease A dimer [37], no structural
Alzheimer's b-amyloid brils. evidence for these oligomers was obtained until the 1990s [38]. The
To date, solid-state NMR provides the few high-resolution 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.
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from structural data [39]. In fact, it was the rst protein structure into account the interchanged segment of the protein. Interestingly,
determined in both the monomeric and domain-swapped dimer these authors found that of all the structures included in this study
forms (Fig. 2A) [40]. In this protein the segment swapped between only three interchanged a protein segment that it is a structural
protomers was a structural domain, but 3D domain swapping ter- domain: diphtheria toxin [39], bB2-crystallin [51] and CIB1 [52].
minology has been applied to the subsequently discovered inter- The web database ProSwap ( com-
twined proteins, even in those cases where only a secondary piles 3D domain-swapped structures up to 2011. Shameer et al. [53]
structure element (not regarded as a structural domain) is the also developed a web page to compile all the 3D domain-swapped
interchanged segment. One example is ribonuclease A, which ex- proteins described up to 2010 (
changes the amino terminal a-helix and/or the carboxyl terminal b- Unfortunately, due to the complexity of the search, these web pages
strand [41,42] (Fig. 2B). have not been updated.
Eisenberg and co-workers developed a terminology to describe Another attempt to classify domain-swapped proteins was
the 3D domain-swapping phenomenon [46,47]. They dened three performed by Huang et al. [17], who also performed a detailed
different classes of domain swapping taking into account the analysis of 500 structures at the PDB to identify their special fea-
availability of the structures of the swapped and un-swapped tures. This structural analysis brought to light the diversity of the
forms: i) bona de domain swapping: the dimeric and monomeric domain-swapped structures. Among the bona de domain-
structures of the protein are known; ii) quasi domain swapping: the swapped proteins, sizes vary from the Eps8-SH3 domain (60
structure has a homologous monomer; and iii) candidates for amino acid residues) to the diphtheria toxin (535 amino acid resi-
domain swapping: no monomer structure is available. The poly- dues) for the smallest and largest proteins, respectively. There is no
peptide chain conformation adopted by the domain-swapped preferred fold among the known 3D domain-swapped proteins:
oligomer is called open monomer, while the monomeric confor- some are all a, others all b, and most of them show both elements of
mation is called closed monomer. Each form shows different in- secondary structure in their fold. Sometimes the extent of the
terfaces: the closed interface or primary interface is the secondary structure element interchanged is just a short segment
intramolecular interface, which is the same in the oligomer as in of the polypeptide chain or a single element of secondary structure
the monomer but is formed by different chains; the open interface [41,42]. In other proteins the exchange of secondary structure is
or secondary interface is unique to the oligomeric form [46,48,49]. complete, as for example the SH3 domain [54e57]. Most frequently,
An analysis of the bona de and quasi domain-swapped struc- domain swapping results in an intertwined dimer, but sometimes it
tures deposited at the Protein Data Bank (PDB) shows that more appears as an intertwined trimer or tetramer [58,59] and some
than one hundred proteins oligomerize by this mechanism. Some proteins exhibit multiple swapping modes [60,61] (Fig. 2B).
structures have been solved by means of NMR techniques (<10%), In order to be able to oligomerize by 3D domain swapping, a
but most of them have been solved by X-ray crystallography. protein must have a region of the polypeptide chain that can be
Recently, MacKinnon and Wodak [50] have performed an extensive exchanged between monomers once the conformational change of
study on the interfaces of multi-domain homo-oligomeric proteins. the hinge loop takes place. This loop must have suitable length and
They found that intertwined oligomers are very common in exibility to allow the placement of the swapped region in the open
homomeric association, and they performed a classication taking 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.
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swapping have focused on the hinge loop by performing muta- amino acid deletion and suggested that domain swapping of the
genesis of some critical residues [60] or shortening/lengthening it major homology region (MHR) segment of adjacent Gag molecules
[49]. The manipulation of the hinge loop might result in changes in might play a role in viral assembly. In contrast, a biophysical
the behavior of these domain-swapped oligomers as it affects its analysis of the MHR motif indicates that domain-swapped dimers
exibility [62] or the propensity of the residues to stabilize the b- are not present in the nal assembly of the HIV capsid and this
turn [63,64]. Sometimes the manipulation of a loop in a monomeric reaction might be an evolutionary remnant of the biologically
protein results in the formation of a de novo domain-swapped relevant dimerization of some oligomeric proteins, such as the
oligomer. As an example, the articial insertion of a long gluta- homologous mammalian SCAN domain [85,86]. Nevertheless, the
mine repeats in the inhibitory loop of the chymotrypsin inhibitor 2, role of domain swapping in capsid assembly is reinforced by the
a monomeric protein, resulting in an intertwined dimer [65]. crystal structure of the icosahedral rice yellow mottle virus and
Other experimental and computational evidence demonstrated biochemical analysis, which justies an increase in the stability of
that the hinge loop is not the only factor responsible for oligo- this capsid as compared to other homologous protein capsids that
merization [66]. Point mutations in the hydrophobic core of the do not show this phenomenon [87].
protein and in amino acid residues stabilizing the secondary Without doubt, the most widely claimed function of domain-
interface also affect domain swapping [55,67,68]. swapped oligomers is their plausible participation in the rst
steps of the development of higher-order oligomers that lead to the
1.3. Functional role of 3D domain swapping formation of amyloids. The rst experimental evidence was
brought to light by the intertwined dimer of cystatin C, which was a
For many years, domain swapping has been considered an known amyloidogenic protein [88,89]. At the same time, a model
in vitro artefact [69]. The high protein concentrations used in the for amyloid formation by a polar zipper with 3D domain swapping
crystallization setups and the presence of additives can promote 3D was proposed for ribonuclease A and other proteins [42].
domain swapping by introducing unfavorable protein-solvent in- At present, many of the known domain-swapped proteins have
teractions, but similar conditions might also take place in vivo. As been described to form amyloid aggregates. Although there is some
the number of 3D domain-swapped structures available increases, controversy regarding the relationship between 3D domain
there is growing evidence of the biological relevance of some of swapping and amyloid formation [90], the structural information
these oligomers. Moreover, there are cases where only the inter- available on different swapped proteins is of great value to analyze
twined oligomer of the protein is known, and it is the functional the rst steps of oligomer formation. Unveiling the mechanism of
form [70e73]. the rst steps of this misfolding phenomenon could help to
It has been suggested that domain swapping plays a role in the determine why a specic protein changes its folding preferences
evolution of oligomeric proteins [74,75]. These proteins are present and how oligomerization can account for the additional stabiliza-
in all organisms and show structural and functional advantages tion factors. In this way, structural information on 3D domain-
compared to monomeric ones: oligomeric proteins are commonly swapped proteins may provide a key to build models of protein
more stable and, from a functional point of view, oligomerization misfolding. Certainly, additional thermodynamic and kinetic in-
can bear some control mechanism that improves the accessibility formation on the folding process can help to develop these models.
and specicity of active sites. Thus, domain swapping has been There are a few 3D domain-swapped proteins forming intertwined
described to participate in the appearance of new binding sites, oligomers and amyloids that have been extensively studied from
such as in the coagulation factor IX binding protein, where a Mg2 the thermodynamic and kinetic points of view. One of these pro-
ion is bound to the domain-swapped dimer [76]. These ions have teins is the SH3 domain of the c-Src tyrosine kinase. Recently, our
been shown to maintain the native conformation and in vivo group has demonstrated that this protein forms amyloid brils in a
function of factor IX intertwined Gla domain during blood coagu- pH-dependent manner that it is related to the nature of the residue
lation [77]. Another example is the human zinc metalloenzyme at position 128 [55].
glyoxalase I, where the active site zinc ion interacts with both
chains in the intertwined dimer [78]. This protein is homologous 1.4. 3D domain swapping and amyloid formation in SH3 domains
with the bleomycin resistance protein, which has been proposed to
arise from gene duplication and is only known in its intertwined The SH3 domain is a small modular domain (~60-residue long)
form [79,80]. present in a large number of proteins. It is an all-b protein where
Another putative function of intertwined proteins is the for- the b-strands form an open b-barrel structure. Comparison of
mation of allosteric sites in enzymes, for example the type II re- different SH3 domains reveals that the hydrophobic core of these
striction endonuclease SgrAI, which forms a tetramer by swapping domains is mainly conserved, but the sequence and conformation
the amino-terminal 24 amino acid residues [81]. The formation of of their loops show considerable variability (Fig. 3). This small
an allosteric site was also proposed for the domain-swapped bovine modular domain is also one of the most widely present in all ge-
seminal ribonuclease [82]. In this case, the two chains forming the nomes, and it is found in different kinds of proteins that develop
intertwined dimer exchange their amino-terminal a-helix and a key functions in the cell [91].
new nucleotide-binding site is generated in a cavity at the interface To date, only four SH3 domains have been described to form
between the two subunits. Another example is the survival protein domain-swapped dimers: Eps8 [54], p47phox [92], the C-terminal
SurE from Salmonella typhimurium, whose dimers are formed by SH3 domain of CRKL [93] and c-Src [57,94]. However, the diverse
the swapping of the carboxyl-terminal a-helix and a loop with two nature of these SH3 domains suggests that other SH3 domains
b-strands [83]. These examples indicate that oligomerization might also show 3D domain swapping. On the other hand, amyloid
through 3D domain swapping may play a role in the modulation of formation has been experimentally described in ve SH3 domains:
the catalytic activity of some enzymes. c-Yes [95], Fyn [96], c-Src [55], PI3K [97] and a-spectrin [98]. c-Yes,
Structural evidence also put forward the involvement of 3D Fyn and c-Src SH3 domains are part of tyrosine kinases that belong
domain swapping in the formation of supra-macromolecular ag- to the Src family and show a high degree of sequence homology
gregates such as those appearing in viral capsid structures. Ivanov (Fig. 3). Among all the SH3 domains, the c-Src-SH3 is the only one
et al. [84] reported the crystal structure of a distinct domain- that has been described to form both amyloid and 3D domain-
swapped variant of the HIV-1 CA-CTD dimer stabilized by a single swapped dimers. Table 1 compiles the above information and
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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

relevant references. loop acts as hinge loop and the canonical SH3 fold is formed by
interchanging the C-terminal b-strands b3-b5 between the two
1.5. 3D domain swapping in SH3 domain 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
The rst 3D domain-swapped SH3 domain described was Eps8 availability of the structures of both forms allowed them to study
(Eps8-SH3), an oncoprotein that participates in v-Src-induced the factors that favor the formation of the domain-swapped dimer.
cellular transformation. The crystallographic structure of this SH3 The main difference was that each crystal form was obtained at a
domain was solved in the triclinic space group P1, and the asym- different pH: the monomeric and dimeric forms were obtained at
metric unit was made up of two chains of the domain which formed pH 4.0 and 7.0, respectively. Analysis of the potential salt bridges/
an intertwined dimer. In this domain-swapped structure, the n-Src 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
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of these interactions in the formation of the intertwined dimer. each form seems to be favored at different pH values. In this way,
Additionally, the formation of the dimer resulted in the burial of a the monomeric and dimeric form crystal structures of the CRKL C-
surface area of 4000 2, and Kishan et al. also suggested that the terminal SH3 domain were obtained at slightly different pH (6.5
stacking of aromatic residues W40 and F52 plays a role in the and 5.6, respectively) and ionic strength [93]. The intertwined
stabilization of the intertwined dimer. dimer of p47phox seems to arise from the quite slow conversion
Owing to its small size, the SH3 domain is an ideal model to from monomeric to dimeric form under the crystallization condi-
study protein folding by molecular dynamics. Yang et al. [66] tions [92,100,101]. The structure of the monomeric form was solved
applied the symmetrized Go -type potential model to the Eps8- by NMR at pH 6.5 in complex with a proline rich motif peptide
SH3 domain to determine how domain-swapped proteins un- [102]. The pH and the presence of the proline rich motif peptide
dergo a transition from the monomeric to the swapped confor- bound to the N-terminal SH3 domain of the p47phox might stabilize
mation. To establish whether the local properties of the hinge loop the monomeric form, as occurs in the c-Src-SH3 domain
were responsible for the domain swapping, these authors increased [55,103,104].
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, 1.6. Amyloid formation in SH3 domains
with the experimentally observed domain-swapped dimer at the
highest representation. They concluded that under conditions fa- The phosphatidylinositol 3-kinase (PI3K) SH3 domain was the
voring intermolecular interactions, swapping is a means of rst one described to form amyloid [97]. This amyloid is obtained at
reducing energy frustration and is determined by the native-state acidic pH (2.0) and moderate temperatures (25e37  C) and it is one
topology rather than local signals at the hinge region. of the best characterized from the structural point of view. Studies
Indeed, other domain-swapped SH3 domains have different performed at different pH showed that amyloid bril formation
hinge loops. The structure of the tandem SH3 domains of the proceeds through different types of intermediates. NMR experi-
p47phox, a cytosolic regulatory component of NADPH oxidase, was ments showed the presence of partly folded conformations in the
solved by Groemping et al. [100] as an intertwined dimer of the N- acid-unfolded state, but not 3D domain-swapped forms [105e110].
terminal SH3 domain (SH3-A, residues 159e213), where the distal Besides, NMR and electrospray ionization mass spectrometry
loop acts as hinge loop (Fig. 4B). However, in the Crk-like (CRKL) C- analysis of H/D exchange allowed identication of those residues
terminal SH3 domain it is the RT loop that acts as hinge loop and involved in oligomeric and bril core structures: residues
strand b1 is interchanged between neighbouring monomers belonging to the RT loop and adjacent to the diverging b-turn in the
(Fig. 4C) [93]. In these SH3 domains, as in the Eps8-SH3, the native state play a key role in the conversion of the soluble PI3K-
monomeric and dimeric species are in equilibrium in solution and 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.
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A 3D model structure of the PI3K-SH3 domain amyloid bers has relaxation dispersion NMR spectroscopy, which allowed re-
been determined by cryoelectron microscopy [108]. As in the NMR searchers to determine the structure of a low-populated on-
studies, the structural features of these bers indicated that this pathway folding intermediate of the Fyn-SH3 A39V/N53P/V55L
SH3 domain needs to be partially unfolded to adopt the amyloid mutant (PDB entry 2L2P) and to study the aggregation prone re-
form. On the other hand, NMR studies performed by Bayro et al. gions of this SH3 domain [96]. Kay et al. proposed that aggregation
[105,106] showed that the secondary structure of the bers was in these domains does not proceed via global unfolding, but rather
different from the native fold: b1, b2 and b3 were preserved in the by locally unfolded conformations accessed through thermal
bers as part of a longer b-strand, but not b4 and b5, which adopted uctuations.
a random coil conformation. These modications also affected the
n-Src, RT loops and the diverging b-turn, which also adopted a rigid 1.7. The c-Src-SH3: a model for 3D domain swapping and amyloid
b-strand conformation. formation
The most important difference in the PI3K-SH3 domain as
compared with other SH3 domains is the presence of a longer n-Src To date, c-Src-SH3 is the only SH3 domain that has been re-
loop. To address the possible role of this loop in the amyloidogenic ported to form intertwined dimers and amyloid brils [55]. Though
behavior of the PI3K-SH3 domain, Ventura et al. [112] constructed a the structure of this domain was rst solved by NMR in 1992 [130],
chimeric protein by the insertion of the n-Src loop of the PI3K-SH3 the rst crystallographic structure was not described until 2009
domain into the a-spectrin SH3 domain (a-Spc-SH3). When these [57]. The crystallographic structure of the Q128R mutant was a
experiments were conducted, the a-Spc-SH3 was considered a dimer where, as in the Eps8-SH3 intertwined dimer, the C-terminal
non-amyloidogenic protein, but nowadays we know that this SH3 b-strands b3-b5 of two different protomers were switched. Several
also forms amyloids. Morel et al. [98] described the formation of years later, the structure of the WT and other mutants showed the
amyloid bers in the N47A mutant of the a-Spc-SH3 after incuba- same intertwined dimers (Fig. 4D) [55]. A detailed analysis of the
tion for several days at pH 3.0 and 37  C at high protein concen- potential salt bridges/hydrogen bonds between acidic residues
tration. The mutated residue belongs to the distal loop, which is demonstrated a role of these interactions in the pH-dependence of
involved in the early folding events in this SH3 and destabilizes the the intertwined dimer formation. In this case, the intertwined
folding nucleus [98,113]. In most of the structures of the a-Spc-SH3, structure was solved from crystals grown at mild acidic pH, while
residue N47 is placed in a disallowed region of the Ramachandran the monomeric structure was obtained at neutral pH (Table 1).
plot. Also, it is worth mentioning that in most of the crystallo- The addition of different substances to the solution of the c-Src-
graphic structures some residues at the distal loop of the a-Spc-SH3 SH3 domain favors the equilibrium in the direction of the oligomer
show weak electron density in the difference maps. This behavior formation or its dissociation. As an example, low molecular weight
might be attributed to exibility or to heterogeneous conforma- PEGs favor dimerization, but the binding of proline rich motifs
tions in this loop [114e117]. peptides reverts the process: DLS experiments showed that the
Although dimers and other higher order oligomers have been addition of proline rich peptides in a 1:1 ratio to a solution con-
described in the a-Spc-SH3 upon aggregation conditions, no taining the intertwined dimer in presence of PEG300 results in the
domain-swapped forms have been isolated [118,119]. As well as formation of the monomer in a few minutes [55,104]. Furthermore,
N47A mutant, WT and other a-Spc-SH3 mutants can form amyloid the crystallographic structures of this SH3 domain in complex with
at acidic pH, but their formation is slower [120,121]. A detailed the high afnity synthetic peptides VSL12 (class I) and APP12 (class
study of the environmental conditions affecting the nucleation of II) showed the characteristic globular fold of the SH3 domain bound
amyloid brils in this SH3 domain revealed that partial unfolding of to the peptides, even though the crystals were obtained at mild
the native state and oligomerization trigger the formation of am- acidic pH (5.0) and in the presence of PEG300 (10%) [103].
yloid bers [122]. Interestingly, several years before the crystal structure of the
As the folding/unfolding process of the a-Spc-SH3 is one of the intertwined dimer was solved, Ding and co-workers predicted its
best characterized [114,123e127], this SH3 domain has been used formation by molecular dynamics calculations [131]. In this study
as a model to study amyloid formation. Ventura et al. also per- the authors proposed that, in some cases, the formation of an open
formed studies on the correlation between in vivo and in vitro ag- monomer could yield amyloid brils if the swapping between
gregation of this SH3 domain and suggested that the rules that different protomers is not reciprocal but propagational [132,133].
govern in vitro aggregation might also be valid in vivo [128]. In This kind of assembly into amyloid brils has been proposed to be
addition, the role of amyloidogenic sequences has been studied by behind the mechanism suggested for some domain-swapping/
inserting them into the a-Spc-SH3. As an example, to validate the amyloid-forming proteins, such as GB1 [90]. Nowadays, most of
amyloid stretch hypothesis, Esteras-Chopo et al. [129] performed the experimental evidence suggests that this model is not correct
the insertion of the designed high amyloidogenic sequence (STVIIE) [90,134]. As we mention above, the structural information on the
and an amyloidogenic six-residue fragment of the Ab-amyloid PI3K-SH3 amyloid brils indicates that the secondary structure of
peptide (16KLVFFA21) in the amino- and carboxyl terminal, as well the bers is different from the native fold [105,106].
as in the core, of the a-Spc-SH3. The results of this work showed Mutagenesis can help to understand the role of specic residues,
that short amyloid stretches accessible for intermolecular in- or groups of residues, that are important for domain swapping and
teractions trigger the self-assembly reaction. amyloid formation. We have studied the aggregation process of
Another SH3 domain that has been found to form amyloids is several mutants of the residue Q128, at the distal loop, by different
the c-Yes-SH3 domain. These amyloids were obtained in the same techniques. Our results showed that amyloid formation was
conditions as described for the previous SH3 domains (pH 3.0) [95], dependent on the residue at this position, while 3D domain
but in this case the amyloid appears after a few days of incubation swapping was not affected [55]. The single mutation of Q128 to
at room temperature in the absence of salts and at low protein lysine, arginine or glutamate affects the protein stability in a pH-
concentration. Although most of the SH3 forming amyloids show a dependent manner that is related to the charges of the residues
pH-dependence and a preference for acidic or mild-acidic pH, a present in the pocket formed by the distal loop and the diverging b-
destabilizing C-terminal truncated mutant of the Fyn-SH3 domain turn. The three mutants and the WT protein form intertwined di-
(A39V/N53P/V55L/D(57e60) mutant) forms amyloid bers at mers at mild acidic pH, but while the WT protein forms amyloids at
neutral pH [96]. This mutant was designed from data obtained from the same pH, the glutamate mutant only forms amyloids at neutral
mara-Artigas / Archives of Biochemistry and Biophysics 602 (2016) 116e126
A. Ca 123

pH (Table 1) (Fig. 5). This further indicates a key role of the residue Src loop in all the structures of these amyloid-forming members of
at position 128 in the folding and formation of amyloid aggregates the Src tyrosine kinase family.
[55]. As the hinge loop plays an important role in the opening of the
Residue Q128, like N47 in the a-spectrin SH3 domain, is located monomer and subsequent formation of the intertwined structure,
at the distal loop and it is also involved in early folding events. In our group designed several chimeric proteins to examine if the
addition, the high-resolution structures of the c-Src-SH3 (WT and determinant of the formation of these structures is inherent to the
mutants) revealed the presence of several well-conserved water sequence of the hinge loop, the n-Src loop, or neither. In the c-Src-
molecules that might have a role in the protein folding process [RT/n-Src-Abl]-SH3 domain (SRC-2X) the amino acid residues of
[135]. This water network is present in the monomeric and the the RT and n-Src loop of the c-Src-SH3 domain were replaced by
intertwined dimer and facilitates the interactions among amino those present in the SH3 domain of the Abl tyrosine kinase. To date,
acid residues at the distal loop and the b-diverging turn. X-ray although peptides related to the diverging b-turn of this SH3
crystallography is the only technique that allows an accurate domain have been proven to form amyloids [138], the whole SH3
modelling of the solvent and, together with the information pro- domain has not been reported to form either intertwined dimers or
vided by NMR techniques, it may prove essential in analyzing the amyloids. Unexpectedly, the resulting chimera SRC-2X crystallized
structural determinants of domain swapping and amyloid forma- in a domain-swapped dimer where both RT and n-Src loops act as
tion. Interestingly, the structure of the monomeric form of the c- hinge loops [56]. Domain swapping through two different hinge
Src-SH3 showed two molecules in the asymmetric unit (chain A loops has been described for ribonuclease A and cyanovirin-N, but
and B) and each molecule displayed a different arrangement of the in these proteins the double-swapped form results in the formation
hydrogen-bond network around the distal loop and the diverging of trimers and tetramers [60,61]. To the best of our knowledge, this
b-turn: in chain A, E106 is hydrogen bound to S123; while in chain is the rst time that the simultaneous opening of two loops of a
B this residue is hydrogen bound to T125. The hydrogen-bond be- protein has resulted in the formation of a domain-swapped dimer.
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 1.8. SH3 domains as a model to study 3D domain swapping and
distal loop and the diverging b-turn [136]. Changes in this hydrogen amyloid formation
bond seem to be correlated with the conformational changes and
the water network arrangement around the diverging b-turn and To date, there is no evidence of any biological relevance of 3D
the distal loop. Interestingly, the structure of the intermediate state domain swapping and amyloid formation in the SH3 domain, but
of folding of the Fyn-SH3 A39V/N53P/V55L mutant (PDB entry certain characteristics of this small protein make it an ideal model
2L2P) shows equivalent hydrogen bond interactions [96]. Differ- for studying both processes. The SH3 domain is characterized by
ences between the chains of the WT c-Src-SH3 are also found in the the lack of prosthetic groups and disulde bridges. It is easy to
n-Src loop: chain A shows an ordered loop, while some residues in purify and is also soluble over a broad range of pH and conditions.
chain B have not been modelled. The same features have been For all these reasons, SH3 domains from different sources have
found in the monomeric structure of the c-Src-SH3 mutant Q128E been studied extensively in the eld of protein folding and protein
(PDB entry 4OMO). Similar behavior has also been found in the interactions, and there is a vast amount of thermodynamic, kinetic
crystal structure of the WT Fyn-SH3 domain (PDB entry 3UA6), and structural information from both experimental and computa-
which also has two molecules in the asymmetric unit [137]. The tional approaches [114,123e126,131,139e144]. These studies indi-
crystal structure of the c-Yes-SH3 has only one molecule in the cate that the transition state of folding in these domains is mainly
asymmetric unit, but residues at the n-Src loop also show high B- organized around the hairpin formed by b3 and b4 strands, which
factors. All these ndings seem to indicate high exibility in the n- 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
Fig. 5. Amyloid brils of the c-Src-SH3 domain at neutral pH. Negatively stained been shown to adopt rigid b-strand conformations [106]. More-
TEM image illustrating the c-Src-SH3 Q128E mutant incubated for two weeks in 0.1 M over, the crystallographic structures of the domain-swapped di-
Hepes 0.1 M sodium chloride (pH 7.0) at 37  C is shown, indicating the presence of long mers of SH3 domains show reduced mobility of the residues at
amyloid brils.
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, Acknowledgements

the monomeric structure of the CRKL C-terminal SH3 domain
shows high B-factor values for the RT loop residues (PDB code This research was funded by the Spanish Ministry of Economy
2BZX), but upon opening of the protomer these residues adopt a and Competitiveness (Spain) and FEDER (EU) [BIO2012-39922-
b-strand conformation (Fig. 4C) [93]. C02-01/02].
The molecular mechanism that initiates the amyloid formation
process is not well established. Some authors claim that the
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