Article
A Bacteriophage Capsid Protein Provides a
General Amyloid Interaction Motif (GAIM)
That Binds and Remodels Misfolded
Protein Assemblies
Rajaraman Krishnan 1 , Haim Tsubery 1 , Ming Y. Proschitsky 1 , Eva Asp 1 ,
Michal Lulu 1 , Sharon Gilead 1 , Myra Gartner 1 , Jonathan P. Waltho 2, 3 ,
Peter J. Davis 3 , Andrea M. Hounslow 3 , Daniel A. Kirschner 4 , Hideyo Inouye 4 ,
David G. Myszka 5 , Jason Wright 1 , Beka Solomon 6 and Richard A. Fisher 1
1 - Neurophage Pharmaceuticals, 222 Third Street, Suite 3120, Cambridge, MA 02142, USA
2 - Faculty of Life Sciences, Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street,
Manchester M1 7DN, UK
3 - Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK
4 - Department of Biology, Boston College, Chestnut Hill, MA 02467, USA
5 - Biosensor Tools LLC, 1588 East Connecticut Drive, Salt Lake City, UT 84103, USA
6 - Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Tel Aviv 69978, Israel
Correspondence to Rajaraman Krishnan and Richard A. Fisher: [email protected];
[email protected]
http://dx.doi.org/10.1016/j.jmb.2014.04.015
Edited by R. Wetzel
Abstract
Misfolded protein aggregates, characterized by a canonical amyloid fold, play a central role in the
pathobiology of neurodegenerative diseases. Agents that bind and sequester neurotoxic intermediates of
amyloid assembly, inhibit the assembly or promote the destabilization of such protein aggregates are in
clinical testing. Here, we show that the gene 3 protein (g3p) of filamentous bacteriophage mediates potent
generic binding to the amyloid fold. We have characterized the amyloid binding and conformational
remodeling activities using an array of techniques, including X-ray fiber diffraction and NMR. The mechanism
for g3p binding with amyloid appears to reflect its physiological role during infection of Escherichia coli, which
is dependent on temperature-sensitive interdomain unfolding and cis–trans prolyl isomerization of g3p. In
addition, a natural receptor for g3p, TolA-C, competitively interferes with Aβ binding to g3p. NMR studies
show that g3p binding to Aβ fibers is predominantly through middle and C-terminal residues of the Aβ subunit,
indicating β strand–g3p interactions. A recombinant bivalent g3p molecule, an immunoglobulin Fc (Ig)
fusion of the two N-terminal g3p domains, (1) potently binds Aβ fibers (fAβ) (KD = 9.4 nM); (2); blocks
fAβ assembly (IC50 ~ 50 nM) and (3) dissociates fAβ (EC50 = 40–100 nM). The binding of g3p to
misfolded protein assemblies is generic, and amyloid-targeted activities can be demonstrated using other
misfolded protein systems. Taken together, our studies show that g3p(N1N2) acts as a general amyloid
interaction motif.
© 2014 Elsevier Ltd. All rights reserved.
Introduction
In neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease and numerous non-AD dementias, there is progressive
accumulation of extracellular and intracellular misfolded protein deposits. The brain deposits of these
misfolded proteins contain amyloid fibrils characterized by a fiber core composed of β strands running
0022-2836/© 2014 Elsevier Ltd. All rights reserved.
perpendicular to the axis of the fibers, the product
of a complex assembly pathway [1,2]. A variety of
therapies targeting specific protein aggregation
pathways, such as those for amyloid-β, the microtubule-associated protein tau and pre-synaptic
protein α-synuclein, are in all stages of drug development, and these drug candidates include vaccines,
monoclonal antibodies (MAbs), antisense oligonucleotides and small molecules [3–6]. Recent studies
J. Mol. Biol. (2014) 426, 2500–2519
2501
G3P remodels amyloid fibers
(a)
(b)
8x106
Alexa488 FI (A.U.)
Response units (A.U.)
1000
750
500
250
0
6
4x106
2x106
0
4000
8000
12000
10-10
Time (s)
(c)
6x106
10-9
M13 (M)
10-8
(d)
5x104
400
4x104
ThT Fl (A.U.)
ThT Fl (A.U.)
500
3x104
300
200
100
2x104
0
0
1
2
3
4
5
6
480
Time (days)
(e)
500
520
540
560
Wavelength (nm)
Cellulose acetate membrane Nitrocellulose membrane
fAβ+6.5x1013/ml M13
fAβ+1.3x1013/ml M13
fAβ+1.3x1012/ml M13
fAβ+1.3x1011/ml M13
fAβ only
[T0] fAβ+1.3x1013/ml M13
Aβ monomer only
100 67
44 30 20
100 67 44
30 20
Aβ (ng/well)
Fig. 1. M13 binds to and remodels Aβ fibers (fAβ). (a) SPR binding profiles show M13 binding to immobilized fAβ (black
line; estimated KD ~ 3.6 nM) but not Aβ monomers (red line). (b) Concentration-dependent binding of M13 to fAβ
measured by co-sedimentation of Alexa488-M13 with fAβ (estimated KD ~ 7 nM). (c) Prolonged incubation at 37 °C of
aggregated Aβ (2.5 μM) with M13 (1 × 10 12 phage/ml) reduces ThT binding fluorescence at 450 nm (red line). Aβ fibers
incubated under identical conditions show no reduction in ThT binding (black line). (d) M13‐mediated remodeling of
fAβ monitored by ThT binding fluorescence spectra. fAβ (2.5 μM) was pre‐incubated with (red continuous line) or without
(black continuous line) 1 × 10 12 M13 phage/ml for 3 days at 37 °C, and the fluorescence spectra were recorded in
the presence of 5‐fold excess ThT. Fluorescence spectra of buffer alone (grey broken line) and the fAβ‐M13 mixture
without any pre‐incubation (red broken line) are plotted for comparison. (e) Concentration-dependent remodeling activity
of M13 monitored by the CA filter retention assay. Aggregated Aβ (2.5 μM) was co‐incubated with varying concentrations
of M13 for 3 days at 37 °C. Samples were serially diluted and spotted on a CA membrane, washed under vacuum and
probed with MAb 6E10. A nitrocellulose membrane blot, which non-selectively retains total Aβ from each reaction, is
shown as a control.
show two or more species of misfolded protein can
underlie neurodegenerative diseases, which potentially complicates understanding disease patho-
physiology and proving single target (i.e., single
misfolded protein) approaches [7,8]. For example,
brains from AD patients contain two cardinal protein
2502
G3P remodels amyloid fibers
deposits, extraneuronal Aβ plaques and neurofibrillary tangles of tau, and frequently a third deposit of
aggregated α-synuclein, normally associated with
Parkinson's disease [9]. Because a combination of
these three neuropathologies could contribute to
disease progression, approaches that can target
amyloid deposition generically could offer a therapeutic advantage over single target approaches.
A serendipitous discovery showed that filamentous bacteriophage M13 can mediate disruption of
amyloid assemblies consisting of the non-amyloid
component of amyloid plaque (NAC), a hydrophobic
region (residues 65–95) of human α-synuclein [10,11].
The NAC peptide shares sequence homology to the
nucleating sequences of several aggregates, including
Aβ and the mammalian prion sequence [12–14]. Here,
we confirm and extend this finding by characterizing
the activities of M13 on multiple amyloid targets. We
show that highly purified preparations of native M13
potently and broadly mediate binding to and disruption
of a variety of misfolded protein assemblies, including
Aβ, α-synuclein, tau and yeast prion Sup35. Characterization of amyloid fiber binding and remodeling
indicates that the bacteriophage minor capsid protein,
gene 3 protein (g3p), is critical for this activity. We
functionally define the two N-terminal domains of g3p
that facilitate binding and disruption of amyloids as a
general amyloid interaction motif (GAIM).
Results
Filamentous bacteriophage M13 mediates amyloid
remodeling activity through binding interactions
To characterize the interactions between M13 and
misfolded protein aggregates, we used fAβ as a
model aggregate. Surface plasmon resonance
(SPR) binding assays show that highly purified
preparations of native M13 potently bind immobilized
fAβ but not Aβ monomers (Fig. 1a). The apparent
equilibrium dissociation constant (KD) calculated
from SPR binding studies is 3.6 nM (Table 1), a KD
that is similar to fAβ binding of anti-N-terminal Aβ
Table 1. SPR kinetic parameters of commercial
antibodies, M13, GAIM-based molecules binding to fAβ
Analyte
fAβ binding parameter
kon (M−1 s−1)
M13
3D6 [16]
6E10a [15]
G3P
Ig-G3P
2.6
6.6
2. 1
1.5
1.7
×
×
×
×
×
3
10
104
105
103
104
koff (s−1)
9.2
2
2.9
2.4
1.6
×
×
×
×
×
−6
10
10−4
10−4
10−4
10−4
KD (nM)
3.6
3
1.4
150
9.4
a
Aβ1–40 fibers were used to calculate the binding parameters
for 6E10.
MAbs, such as 3D6 and 6E10 [15,16]. However,
unlike these MAbs, the fAβ binding association (kon)
and dissociation (koff) rates of M13 binding differ
significantly. M13 binding to fAβ is ~ 100-fold slower
and effectively irreversible (koff = 9.2 × 10 − 6 /s)
when compared to the kinetics determined for the
anti-Aβ MAbs (Table 1).
We took advantage of the differential sedimentation
properties of fAβ and M13 to develop a quantitative
solution binding assay (Fig. S1a). Fluorescently
labeled M13 (Alexa488-M13) sediments at low
centrifugal forces (7500g) only when in a complex
with fAβ, while unbound Alexa488-M13 remains in the
supernatant. Alexa488-M13 sedimentation depends
on the concentration of fAβ present in the reaction
(Fig. S1b) suggesting first-order binding kinetics. The
apparent KD estimated by quantifying the pelleted
Alexa488-M13:fAβ complex (KD = 7 nM) is similar to
that calculated using SPR assays of M13 binding to
immobilized fAβ (Fig. 1b).
To investigate whether M13 binding to fAβ alters
the conformation of the fibers, we utilized both
thioflavin T (ThT) binding [17] and cellulose acetate
(CA) filter retention assays [18,19]. ThT binds cross-β
sheet fibers to produce characteristic high fluorescence emission maxima around 485 nm, but ThT
binds poorly to non-fibrillar aggregates and monomeric
peptides [20,21]. Based on the fluorescence intensity,
ThT binding to the M13:fAβ complex was unchanged
at 0, 4 or 12 h, which shows that M13 binding does not
alter the fiber conformation for incubation times ≤ 24 h
(Fig. 1c). However, longer incubation times (2–5 days)
of the M13:fAβ complex lead to significant loss of ThT
fluorescence, indicating M13-mediated structural
changes to the amyloid fold (Fig. 1c and d).
We used a CA filter retention assay [19] to measure
loss of amyloid conformation (remodeling) mediated by
M13. Unlike nitrocellulose membranes that retain
equally all forms of Aβ (monomers, oligomers and
fibrillar aggregates), CA membranes with 0.2 μM pores
retain amyloid fibers, which are quantitatively detected
by anti-N-terminal Aβ MAbs, such as 6E10. Non-fibrillar
aggregates and peptides flow through the membrane.
CA retention assays show that M13 potently remodels
Aβ fiber structure in a concentration-dependent manner (EC50 = 1 to 5 × 10 12 phage particles per milliliter
or 1.7–8.5 nM particle concentration), consistent with
the apparent M13:fAβ binding KD (Table 1). The
reductions in CA membrane retention of M13-treated
fAβ occurs only after 24 h (Fig. S2a), consistent with
the ThT amyloid dissociation assay data (Fig. 1c). By
comparison with fAβ quantification at each time point
on nitrocellulose (Fig. 1e), we show that the apparent
activity measured by CA retention is not due to M13
interference with 6E10 binding.
To more directly measure M13-mediated fAβ
remodeling, we measured the fiber X-ray diffraction
pattern of the M13:fAβ complex at the end of 24 and
48 h. Since amyloid fibers and M13 have distinct
G3P remodels amyloid fibers
diffraction patterns, changes in conformation of either
structure can be detected differentially. These diffraction studies show that the amyloid fibers, but not the
2503
M13 filaments, gradually lose their total β sheet content
over 48 h, an effect consistent with fiber remodeling
(Fig. S2b and c).
Fig. 2. M13 remodels a variety of amyloid fibers with unrelated misfolded protein subunits. (a) CA filter retention assays
showing remodeling of ftau (2.5 μM), fAS (2.5 μM), fMTBR (2.5 μM) and fNM (2.5 μM) in the presence of 1 × 10 13 phage/
ml. The tau, α-synuclein, myc-tagged MTBR and his-tagged Sup35NM fibers retained on the membrane were detected
using tau5 (anti-Tau, 1:10,000 dilution), 4D6 (anti-α-synuclein, 1:10,000), anti-myc (9B11, 1:10,000) and MMS-156P
(anti-his tag, 1:15,000) antibodies, respectively. Monomeric proteins were directly diluted from a urea (8 M) to prevent
oligomerization and non-specific binding. Control nitrocellulose filter blots of each corresponding remodeling reaction are
shown.(b) Detergent solubility of fNM treated with and without 1 × 10 13 phage/ml for 3 days at 37 °C. Samples were
incubated with SDS (1–6%) for 6 h and centrifuged at 30,000 rpm. The fluorescence in the supernatant was used to
calculate soluble Sup35NM release. (c) SDS solubility of α-synuclein fibers treated with and without 1 × 10 13 phage/ml for
3 days at 37 °C. Samples were incubated with SDS (1–6%) for 1 h and then centrifuged at 30,000 rpm. The fluorescence
in the supernatant was used to calculate soluble α-synuclein released. (d–f) A time course study showing increased
solubility of fluorescently labeled α-synuclein fibers after co-incubation with M13. The phage slowly remodels the fibers to a
non-amyloid conformer with concomitant increasing sensitivity to SDS solubilization. (g) Solubility of fAβ fibers treated with
1 × 10 13 phage/ml in 2%, 3% and 4% RIPA buffer without SDS. fAβ:M13 complexes were incubated with 2–4% RIPA
buffer without SDS for 30 min and centrifuged at 40,000 rpm for 10 min. The supernatants (soluble fraction) were spotted
on a nitrocellulose membrane and quantitated using MAb 6E10 for detection and a fluorescent secondary antibody
quantified by densitometry.
2504
G3P remodels amyloid fibers
Fig. 2 (continued).
Taken together, these studies directed to measure
amyloid fiber binding and dissociation show that
M13 potently binds fAβ and disrupts the amyloid
conformation. The binding event is completed in
about 3–4 h, while amyloid fiber remodeling takes
place more slowly over several days.
M13 recognizes the canonical amyloid
conformation
We tested for M13-mediated amyloid fiber remodeling activity with three non-Aβ fiber assemblies, all
with unrelated primary sequences and a broad range
of monomer molecular masses (15–440 kDa). The
three proteins, tau, α-synuclein and the yeast Sup35
prion domain, NM, have different lengths of disordered regions and aggregate by either hydrophobic
(tau and α-synuclein) or glutamine-asparagine-rich
sequences (Sup35NM). Regardless of the protein
species, SPR binding, assembly inhibition and CA
filter retention assays show that all three amyloid fibers
are targets for M13-mediated binding and remodeling
(Fig. 2a and Table 2) suggesting that interaction with
the phage is conformation dependent and is not protein
primary sequence dependent.
To determine if amyloid remodeling following M13
binding produces low molecular weight (b 100 kDa)
products, we filtered M13-bound aggregates (incubated for 3 days) through size-exclusion membranes
with four different molecular mass cutoffs (10, 30, 50
and 100 kDa). No detectable Aβ passed through the
membranes, suggesting that phage treatment does
not release low molecular weight aggregates, oligomers or monomers (data not shown).
2505
G3P remodels amyloid fibers
Table 2. Comparison of activities of GAIM-based molecules with α-synuclein, tau and Aβ assemblies
GAIM
construct
M13
G3P
Ig-G3P
Binding
Assembly inhibition
KD (nM)
fAβ
3.6
150
9.4
ftau/fMTBR
ND
ND
63/10.4
Remodeling
IC50 (nM)
fAS
6
360
49
Aβ
~3
20–100
50
Since M13 binding appears to gradually alter β
sheet content, we measured the differential detergent
solubility of amyloids with or without M13 co-incubation
to uncover alteration of the fiber conformation. Figure
2b and c show M13-mediated changes in solubility of
fluorescently labeled Sup35NM and α-synuclein fibers,
respectively. Sup35NM monomers were co-assembled with 10% fluorescently labeled cy5-labeled
Sup35NM monomers. Likewise, α-synuclein was
co-assembled with 10% Alexa488-labeled α-synuclein
monomers. Under these conditions, the assembly
kinetics of the unlabeled and the 10% labeled proteins
were identical (data not shown). For both fibers, M13
treatment significantly increases detergent solubility.
Further, this increase in fiber solubility is time
dependent (Fig. 2d–f). Similar solubility studies with
fAβ were not performed due to higher inherent
detergent solubility of these fibers to SDS. Instead,
we performed detergent solubility assays of fAβ with
SDS-free radioimmunoprecipitation assay (RIPA)
buffer (containing 0.5% sodium deoxycholate and
1% NP-40). Quantification of solubilized Aβ shows
that M13 treatment significantly increases fAβ sensitivity to detergents (Fig. 2g).
Capsid protein g3p mediates M13:Aβ interactions
To determine whether hydrophobic interactions
could be involved in M13 binding to amyloid fibers,
we measured the effect of temperature changes for
M13-mediated fAβ binding and for fiber remodeling.
Pre-heating M13 prior to activity tests shows that
M13 binding to and remodeling of fAβ increases with
increasing temperature (Fig. 3a and b), suggesting
that hydrophobic interactions are critical. Importantly,
the temperature approximating the melting temperature (Tm) that promotes separation of the two
N-terminal domains (N1 and N2) of the minor capsid
protein g3p (Tm = 48.1 °C) [22] is within the temperature transition (Fig. 3a) for greater Aβ fiber binding
and disaggregation activities.
To investigate more directly the role of g3p for
amyloid targeting, we measured the amyloid binding
activities of phage treated to disable g3p function.
The first M13 variant was prepared by incubating
phage at 95 °C for 10 min, which denatures g3p but
does not affect the filament architecture (Fig. S3a)
[22–24]. The second g3p-disabled variant is a
tau/MTBR
10
ND
ND
EC50 (nM)
AS
10–20
100–150
ND
fAβ
1.7–8.5
N2500
40–100
ftau/fMTBR
1–10
N 2000
150
fAS
1–10
ND
ND
protease-treated preparation of M13 with selectively
cleaved g3pN1N2 domains (described in Fig. S3b).
Consistent with g3p having a central role for M13:fAβ
interactions, denaturation or proteolysis of g3p
significantly reduces both M13 binding to fAβ
(Fig. 3c) and fAβ remodeling activity (Fig. 3d).
To directly demonstrate g3p targeting of amyloid,
we expressed and purified a functionally active
recombinant soluble g3p fragment (called G3P)
containing g3p N1 and N2 domains and the glycinerich hinge sequence connecting them [23,24]. G3P
competes with M13 for binding to fAβ, as measured by
the binding competition assay (Table 1 and Fig. 3e).
G3P also shows potent fAβ binding by SPR (Table 1
and Fig. S8c). To determine if G3P could block Aβ
aggregation, we co-incubated G3P with Aβ under fiber
assembly conditions. Based on ThT binding and
circular dichroism analyses, G3P effectively blocks
fiber assembly with 50% inhibitory concentration
(IC50) = 20–100 nM (Fig. 3f and Fig. S4).
Valence affects G3P potency for amyloid
interactions
Compared to M13, G3P shows reduced potency
for fAβ binding and fiber remodeling (Table 2 and 3).
As shown for M13, G3P binds Aβ fibers over
monomers, but the fiber binding (K D = 100–
150 nM) is 25- to 40-fold less potent than the intact
phage. G3P did not effectively remodel Aβ fibers in the
3-day remodeling assay, even at concentrations as
high as 2.5 μM (Fig. 4). These quantitative and
qualitative differences between G3P and native
phage are addressed below.
Because g3p is oligomeric (n = 3–5) at the phage
tip, the potency differences between M13 and G3P
for binding and remodeling activities could be due to
valence effects. To address this possibility, we created
two multivalent G3P molecules: (1) dimeric G3P made
from an immunoglobulin (Ig) Fc-G3P genetic fusion
and (2) a multivalent G3P conjugate made using
streptavidin (SA) complexed with biotin-G3P. Ig-G3P
was expressed from mammalian cells and purified
using protein A chromatography. The SA-G3P multimer was prepared by biotinylation of G3P. The
G3P-biotin conjugate was reacted with SA to form
G3P multimers (n = 2–4), which were purified by
size-exclusion chromatography. Both multimeric
2506
G3P remodels amyloid fibers
(a)
(b)
Bound M13 Fl (A.U.)
1.2x104
200 100
9.0x103
50
25
12.5 Aβ(ng/well)
fAβ only
open
6.0x103
closed
3.0x103
0
15
30
45
60
Temperature (°C)
(c)
Alexa488-M13 competed (%)
Alexa488-M13 competed (%)
(d)
100
100
200
80
100
50
25
12.5 Aβ (ng/well)
fAβ only
60
+ArgC treated M13
40
+ M13
20
0
(e)
0
10
00
00
13
M
3P
G
0
G3P [nM]
no
co
m
pe
tit
o
r
0
25
10
20
50
10
0
40
75
10
60
1
80
100
0.
1
fAβ assemblyinhibition(%)
(f)
Fig. 3. M13 g3p is necessary and sufficient for amyloid targeting by M13. (a) Binding of M13 to 10 μM fAβ at different
temperatures monitored by the co‐sedimentation assay. The data were fitted to a sigmoidal curve and the estimated Tm is
46 ± 3 °C. The cartoons of g3pN1 and N2 domains show temperature dependency of interdomain unfolding. (b) Remodeling
activity of M13 is temperature dependent. Aggregated Aβ (2.5 μM) was co‐incubated with M13 (1 × 10 13 phage/ml) for 3 days
at 4 °C and 37 °C and the loss of fiber measured by the CA membrane retention assay. (c) Binding competition of various M13
variants monitored by the fluorescent co-sedimentation assay (see also Fig. S1c). Alexa488‐M13 (5 × 10 11 phage/ml) was
co‐incubated for 1 h with 15‐fold excess of preparations of M13 that had been heat inactivated for 10 min at 95 °C, treated with
endoproteinase ArgC, or native phage was pre‐incubated at either 37 °C or 50 °C, respectively. Binding competition was
measured after centrifugation (7500g) to pellet M13:fAβ complexes, and the loss of fluorescence from the pellets was used to
calculate % binding competition. (d) fAβ remodeling activity of M13 is dependent on intact g3p. Aggregated Aβ (2.5 μM) was
co‐incubated with endoproteinase ArgC‐treated M13 (1 × 10 13 phage/ml) for 3 days at 37 °C and measured for loss of
amyloid in the CA filter retention assay. (e) Binding competition of G3P monitored by the fluorescence co-sedimentation
assay. Alexa488‐M13 (5 × 10 11 phage/ml) was co-incubated with 50-fold excess of soluble G3P or unlabeled M13 followed
by measurement of Alexa488 fluorescence in the co-sedimentation binding assay. (f) G3P inhibits Aβ assembly in a
concentration-dependent manner. Plot showing inhibition of fiber assembly monitored by ThT binding fluorescence.
2507
G3P remodels amyloid fibers
Table 3. Valence of GAIM and activity
Molecule
M13
G3P
Ig-G3P
Valence
(G3P)
fAβ binding (SPR),
KD (nM)
fAβ binding (co-sedimentation),
KD (nM) (estimate)
fAβ remodeling,
EC50 (nM)
Aβ assembly inhibition,
IC50 (nM)
3–5
1
2
3.6
150
9.4
7
250
ND
1.7–8.5
N2500
40–100
~3
20–100
50
constructs were tested for fAβ remodeling. The Ig-G3P
and SA-G3P constructs exhibited relatively potent
amyloid fiber remodeling activities compared to G3P
(Fig. 4), suggesting that g3p valence plays a role in
amyloid fiber binding and remodeling activities.
To directly demonstrate that the G3P dimer
created by Ig-G3P exhibits higher avidity to amyloid
fibers than G3P monomers, we performed SPR
binding and CA filter retention experiments. Table 3
shows that Ig-G3P has significantly increased
binding (Ig-G3P apparent KD = 9.4 nM) and fiber
remodeling (EC50 = 40–100 nM) activities compared
to the G3P monomer (G3P binding KD = 150 nM;
EC50 N 2500 nM). Taken together, the binding and
fiber dissociation assays of both native phage and the
G3P multimers indicate that multimerization of G3P
increases amyloid targeting activities.
To show that the Ig-G3P fusion, like the phage, can
potently bind amyloids assembled from unrelated
proteins, we measured binding to assembled fibers of
tau [both full-length tau and 4-repeat tau (MTBR;
C-terminally myc-tagged microtubule binding region
of tau protein, residues 243–375)] and of full-length
200 100
50
25
12.5
α-synuclein. Ig-G3P potently binds tau fibers (apparent KD = 63 nM for full-length tau fibers and apparent
KD = 10.4 nM for MTBR fibers) and α-synuclein fibers
(apparent KD = 49 nM) (Table 2), which supports
that M13 generic amyloid fiber binding is mediated by
g3p.
Ig-G3P does not bind non-amyloid protein
aggregates and blocks binding of
amyloid-specific polythiophenes
To examine the targeting specificity of G3P, we
carried out Ig-G3P binding studies with casein,
elastin, gelatin, bovine and human collagen, bovine
serum albumin (BSA) and monomeric α-synuclein.
This group includes natively disordered proteins,
denatured proteins, glycine-proline-rich proteins and
proteins with exposed hydrophobic surfaces. Ig-G3P
does not avidly bind to any of these test proteins over
a broad concentration range (0–225 nM), consistent
with the hypothesis that g3p specifically recognizes
proteins with an amyloid conformation (Fig. 5a and b
and Fig. S5).
Aβ (ng/well)
fAβ only
+2.5µM G3P
G3P
(monomer)
+ 0.5µMG3P
+ 800nM Ig-G3P
+ 8nM Ig-G3P
Ig-G3P
(dimer)
+ 2.5µM SA-G3P
+ 0.36µM SA (no G3P)
+ 0.5µM SA-G3P
SA-G3P
(multimer)
+ 0.072µM SA (no G3P)
Fig. 4. G3P valence is important for Aβ fiber remodeling. CA filter retention assay shows lack of remodeling effect of
monomeric G3P after 3 days of co-incubation with fAβ at 37 °C. Multimeric G3P (Ig-G3P dimer; SA-G3P, n = 2–4) shows
a dose-dependent remodeling of fAβ under identical conditions.
2508
G3P remodels amyloid fibers
(a)
(b)
140
Fluorescence Intensity
(A.U.)
(c)
Relative absorbance
at 450nm (%)
120
100
80
60
40
120
100
80
60
40
20
20
0
0
(d)
35000
100
30000
fAβ
25000
20000
15000
10000
Aβ monomer
5000
Fluorescence (%)
Relative absorbance
at 450nm (%)
140
80
60
40
20
0
0
540
580
620
660
Emission Wavelength (nm)
0
1
2
3
4
Ig-G3P Concentration [μM]
Fig. 5. G3P binding is specific for amyloid aggregates. (a) Binding of Ig-G3P to various non-amyloid proteins measured
by ELISA assays. Ig-G3P (200 nM) binding to the various substrates is normalized to Ig-G3P binding to fAβ. (b) Ig-G3P
(100 nM) binds specifically to fAS and shows no binding to AS monomers. (c) Fluorescence emission spectra of PTMI
(0.5 μg/ml) co-incubated with either fAβ (2.5 μM) or Aβ monomers (2.5 μM). (d) Competition between PTMI and Ig-G3P for
fAβ binding. The fluorescence intensity of PTMI decreases with increasing concentrations of Ig-G3P competitor. Different
concentrations of Ig-G3P were added to 2.5 μM fAβ and incubated for 30 min. PTMI was then added to the complexes
and fluorescence spectra recorded. The intensity at 590 nm was plotted against the concentration of Ig-G3P (black line).
As a control, PTMI was first added to 2.5 μM fAβ and then incubated for 30 min with Ig-G3P. The fluorescent intensity of
the fibers complexed with the dye was plotted (red line). Ig-G3P shows no significant binding to PTMI (data not shown).
The binding stoichiometry calculated by SPR
studies between G3P and fAβ monomers is approximately 1:50 (G3P:Aβ monomers) (Table 4). This
calculation was made based on the amount of Aβ
deposited on the biosensor chip and the amount of
captured G3P (Table 4). This suggests that G3P binds
a large epitope along the amyloid fiber that repeats
Table 4. GAIM:Aβ binding stoichiometry calculated based
on SPR binding studies
Analyte
Molecular
mass (kDa)
G3P
G3P (IF1)
Ig-G3P
25
25
110
RU
mol/mm2a
400
1300
830
1.6 × 10−14
5.2 × 10−14
7.45 × 10−15
Aβ monomers:
GAIM ratio
55
17
117
a
Calculation is based on 1000 RU = 1 ng protein per area unit
(mm2) and immobilized fAβ 8.88 × 10−13 mol/mm2.
over several β strands (interstrand distance =
0.48 nm). This kind of binding is likely to be very
d if f e r e nt f r o m s m a l l m ol e c ul e (m o le c u l ar
mass b 500 Da) binding that is limited by interactions
with one or two strands at a time [25,26]. To our
knowledge, the only other example of a molecule that
specifically binds amyloids over such large distances
are the luminescent conjugated polythiophenes
(LCPs), polymers of thiophenesline containing 25–
40 subunits [25,26]. We tested these molecules for
fAβ binding competition with Ig-G3P in a solution-based assay. Figure 5c shows the fluorescence
emission spectra of a specific LCP [polythiophene
methyl imidazole (PTMI)] bound to either Aβ fibers or
monomers. When Ig-G3P is pre-incubated with Aβ
fibers, PTMI binding is blocked (Fig. 5d). Because the
binding competition is highly concentration dependent, PTMI and G3P could share binding sites on fAβ.
G3P remodels amyloid fibers
G3P recognizes Aβ fibers specifically through
hydrophobic core residues
A binding stoichiometry of 1:30 (G3P:Aβ) was
measured using 1H NMR (Fig. S6a), which compares with the G3P:Aβ stoichiometry estimated by
SPR. As expected from the KD value, the complex is in
the tight binding limit at 300 μM Aβ and dissociates in
the slow exchange regime on the NMR timescale. The
effects of G3P binding to Aβ fibers were examined by
measuring its influence on the hydrogen/deuterium
(H/D) exchange behavior of fAβ, using 15N transverse
relaxation optimized spectroscopy (TROSY) NMR
following dissolution of individually quenched samples
in 95% DMSO/5% D2O. The exchange profile of fAβ in
the absence of G3P (Fig. 6a and Fig. S6b) shows that
two stretches of Aβ sequence, residues 17–25 and
31–40, are measurably protected from H/D exchange,
and these peptide sequences span the β strands that
form the core of the assembled fiber. Protection is also
evident for residues 11–12 in the N-terminal region of
fAβ. In total, 18 residues have measurable protection
(Fig. S6b). The observed non-exponential (bimodal)
time dependence of H/D exchange for protected
residues is consistent with the previously proposed
dynamic recycling model [27], where exchange
occurs when Aβ monomers dissociate from the
ends of the fibers. A non-exponential (bimodal) time
dependence of H/D exchange is maintained on
co-incubation of fAβ with G3P, but increased protection for residues 11–12, 17–25 and 31–40 is clearly
visible at longer exchange times (Fig. 6b). On binding
of G3P, no residues in fAβ become unprotected and
no new residues become protected. This is consistent
with H/D exchange still occurring through dynamic
recycling of Aβ monomers but where their dissociation
rate from the fiber ends is decreased. This further
supports the data showing that monomeric G3P does
not remodel amyloid fibers.
The ability to interact with multiple misfolded protein
assemblies, together with the binding regions defined
by H/D exchange studies, suggest that G3P may
recognize β edge strands that are capable of selftemplating [28,29].
Amyloid fibers and TolA-C bind to a common
region of g3p
In the closed state, the N1 and N2 domains of g3p
are fully folded [24] (Fig. 7a). The interface between
the two g3p outer domains is rich in β strands that
self-anneal and prevent the g3pN1 domain from
binding its cognate membrane-associated E.coli
receptor, the C-terminal domain of TolA. Infection
initiates when the g3pN2 domain binds to
a predominantly α helical assembly, the F-pilus,
with very high specificity (KD = 3 pM) [30]. This
g3pN2-F-pilus binding triggers dissociation of N1,
which contains the TolA-C binding site, from N2, and
2509
concomitantly proline 213 in the hinge between N2
and N3 isomerizes from the strained cis conformation to a less strained trans conformation. This prolyl
isomerization acts as a switch to maintain g3pN1N2
in an open and infection-competent state (Fig. 7a)
[31,32]. Incubating either phage or g3p at elevated
temperatures to dissociate N1 and N2 shifts the
equilibrium from the cis (closed) to the trans (open)
proline conformation [22,24].
To determine if fAβ binding might involve either the
TolA or F-pilus binding sites on g3p, we compared
amyloid fiber binding of phage g3p mutants that
affect g3p N1–N2 association. The IIHY mutant has
a stabilized closed-state g3p conformation [Tm (IIHY
g3p) = 61.8 °C, Tm (wild-type g3p) = 48.1 °C]. If
TolA-C binding sites are involved in g3p:fAβ interactions, then the closed IIHY mutant should show
relatively lower potency binding to fAβ. Alternatively,
if the F-pilus binding region is involved in fAβ
binding, then the binding should be unaffected by a
constitutively closed N1N2 conformation. Binding
competition studies show that the IIHY mutant poorly
competes with wild-type phage for fAβ binding
(Fig. 7b). Unlike wild-type phage, increasing the
temperature during the binding reaction to 50 °C
does not increase IIHY-fAβ binding (Fig. 7b). These
data are consistent with direct g3pN1:fAβ
interactions.
We next tested a phage mutant that displays
reduced binding to F-pili and carries a constitutively
open conformation of g3pN1N2, both effects caused
by three g3pN2 alanine substitutions [33,34]. Because the AAA mutations create a more exposed
TolA-C binding region through g3p N1–N2 dissociation, this phage displays less temperature sensitivity for TolA binding. Consistent with its TolA-C
binding behavior, the AAA mutant is not affected
by temperature shifts for fAβ binding when compared to wild-type phage. Taken together, these
mutant phage studies suggest that an open g3p N1–
N2 conformation promotes amyloid binding and that
the TolA-C binding region of g3pN1 is involved in
amyloid fiber recognition.
To confirm and extend the observation that g3pN1
is involved directly in amyloid fiber recognition, we
tested the filamentous phage IF1, which displays a
g3p with N1N2 in a constitutively open conformation
[22], for fAβ binding. Studies with both the intact IF1
phage (Fig. S1d) and G3P derived from IF1 (Fig. 7c)
confirm that the open g3p conformation promotes
greater binding to fAβ.
Since TolA-C is the natural substrate of open state
g3p, we carried out a binding competition study to
determine if soluble TolA-C can prevent Ig-G3P
binding to fAβ. Figure 7d shows Ig-G3P binding to
fAβ without and with TolA-C pre-incubation. Consistent with the g3p-soluble TolA-C binding potency
(KD = 22 μM) [35], TolA interferes with G3P:fAβ
binding with IC50 ~ 15 μM.
2510
G3P remodels amyloid fibers
Fig. 6. Time course NMR H/D
exchange of G3P:fAβ. (a) Profiles of
residues R5, G9, F19, E22, M35 and
V40 complexed with (Δ) or without (□)
G3P. (b) Protection offered by G3P to
the Aβ fiber core residues at the end
of 744 h. The normalized intensities
of fAβ (black lines) and fAβ:G3P (red
lines) are shown at the end of 744 h
of incubation.
2511
G3P remodels amyloid fibers
open
closed
(a)
F-pili binding
F-pili binding
Closed
Open
(c)
1500
Response (R.U.)
Alexa488-M13 pelleted (%)
(b)
75
50
25
1250
1000
750
500
250
0
0
WT
IIHY
AAA
0
200 400 600 800 1000 1200
Time (s)
Absorbance at 450nm
(d)
0.75
0.50
0.25
0.00
0
3.125
12.5
25
TolA [μM]
Fig. 7. Effect of g3pN1N2 conformation for fAβ binding. (a) A schematic ribbon diagram showing the N1 (golden) and N2
(violet) domains of G3P in the closed and open conformations. The F-pili binding region on g3pN2 is marked and the area
shaded. On the inner side of the two domains (“inverted horseshoe” conformation), smaller and larger shaded regions
represent the TolA-C binding sites in the closed and the open states, respectively. (b) Binding competition study with the
closed g3p carrying IIHY phage variant and open g3p carrying AAA phage variant, respectively. Fluorescently labeled
phage was allowed to bind fAβ pre-incubated with wild type (wt), IIHY or AAA phage at 37 °C or 50 °C. (c) SPR studies
measuring binding of wt-G3P (black) and IF1-G3P (red) to fAβ. (d) Binding competition study showing a
concentration-dependent inhibition of Ig-G3P binding to fAβ in the presence of soluble TolA-C. Ig-G3P (3 nM) was
pre-incubated with increasing concentrations of soluble TolA-C prior to incubation with fAβ (1.6 μM). This estimated
TolA-C IC50 for blocking Ig-G3P binding to fAβ is ~ 15 μM.
Discussion
Our studies demonstrate amyloid interaction activities of the filamentous phage minor capsid protein
g3p, which correlate with the structure function of g3p
described for E. coli infection [34,36,37]. Filamentous phage infection of E. coli requires g3p, a protein
displayed as 3–5 copies at the filament tip. g3p
mediates the earliest infection steps, including recognition of both primary (F-pilus) and secondary (TolA)
receptors required for infection. The two outer domains
of g3p define two alternative functional states represented in Fig. 7a. In the unbound or “closed” state,
the two N-terminal domains of g3p adopt an inverted
horseshoe-like conformation with a hydrophobic
groove running between the two lobes [23]. Binding
2512
of the g3pN2 domain to the host cell F-pilus triggers
g3p N1–N2 interdomain dissociation that exposes the
hydrophobic groove (“open” state). Dissociation of the
N1 domain in turn triggers a cis–trans isomerization of
proline 213 located in the N2–N3 interdomain hinge
region and breaks or destabilizes a number of
hydrogen bonds at the interface of the two N-terminal
domains [32]. These two events allow N1 to bind to a
co-receptor, the C-terminal domain of a periplasmic
protein, TolA, to trigger infection. Crystal structure
of the N1-TolA complex shows that the association
between the g3pN1 and TolA-C domains involves
extensive hydrogen bonding [23,37], similar to β
strand–β strand interactions described for several
naturally self-templating and de novo synthesized
amyloids [38,39]. Based on binding and g3p mutant
analyses, we hypothesize that g3p utilizes TolA-C
binding sequences (including the g3p β4 strand) to
recognize and bind the canonical amyloid fold represented in Aβ fibers. This hypothesis was initially based
on the temperature dependency of M13:fAβ binding
and dissociation, which is predicted by the temperature
dependency of g3p–TolA interactions. The hypothesis
is further supported by direct evidence that g3p is both
necessary and sufficient for amyloid targeting activities,
since destruction of phage-associated g3p drastically
reduces M13 binding and remodeling of fAβ and that
G3P alone can mediate amyloid recognition. Finally,
TolA-C binding competition experiments show that
TolA-C and fAβ may interact with shared binding sites
on g3p.
H/D exchange studies on G3P:fAβ complex clearly
indicate binding to two separate stretches of hydrophobic residues, 17–25 (17-LVFFAEDVG-25) and 31–
40 (31-IIGLMVGGVV-40) are strongly affected by
binding. These stretches form the core β strands in
Aβ fibers [40]. Since most amyloids, including fAβ,
assemble in a nucleation-dependent manner [1,2,41],
only a relatively low concentration of G3P molecules
(1–10%), compared to the concentration of nucleating
monomers, would be required to cap fAβ seeds in a
spontaneous assembly reaction. This is consistent with
the relative concentrations of G3P (20–100 nM) and
Aβ (2500 nM) necessary for effective assembly
inhibition (Table 3).
The biphasic H/D exchange protection of early
(b 50 h) C-terminal region and late (N 200 h) Aβ
middle residues by G3P together with evidence for
binding stoichiometry of 1:30::G3P:Aβ suggest that
G3P binds more than one region on the fiber: (a) β
strands that form edges of the growing amyloid fiber
and (b) exposed hydrophobic patches along the
C-terminal regions of the fiber. Preliminary mutational analysis suggests that, in addition to the TolA
binding g3p β4 strand, other hydrophobic or reactive
edge β strands, such as the β10 strand on the N2
domain (that anneals and protects the TolA-C binding
sequence), might also participate in amyloid recognition. Deletion of the N2 domain or altering interdomain
G3P remodels amyloid fibers
interactions leads to less efficient binding and/or
remodeling (data not shown). Multiple binding events
to different regions of the amyloid might explain both
the unusually potent binding for fAβ and the
conformational rearrangement we measure by ThT
binding, CA filter retention and loss of fibrillar fAβ by
X-ray diffraction methods (Fig. S2b and c). Whether
or not the same types of binding interactions are
found between g3p and tau and/or α-synuclein
aggregates has not been determined, the common
role for hydrophobic edge-to-edge association for
amyloid assembly indicates an analogous mechanism [28,39]. Further NMR studies of the amyloid:
G3P complex should help to identify precisely the
g3p residues involved in binding to amyloids.
The ability to bind and engage a larger conformational motif that spans ~ 30 monomers of Aβ peptide
similar to LCPs is a unique feature of G3P. If the
repeating twist length in an amyloid fiber is between
11.5 and 14.5 nm or about 25–30 Aβ monomers
stacked along the fiber axis, then each G3P molecule
would wrap around an entire helical twist [42]. The
flexible interdomain linker that allows the two domains
to move apart by almost 20 nm during E. coli infection
[43] could permit engaging both the middle region and
the C-terminal sequences along the fiber. Interestingly, LCP molecules, such as polythiophene acetic acid
and PTMI, have been predicted to bind and alter the
structures of amyloids in a similar fashion [44].
The Ig-G3P we created shares several features
with previously described antibodies that have been
reported to bind, remodel or block assembly of various
misfolded proteins. For example, both polyclonal and
monoclonal IgM antibodies that recognize conformational epitopes on amyloid fibers have been
characterized [45,46], and other antibodies have
been shown to selectively interact with fibrillar [47]
and non-fibrillar oligomers [48–50]. However, to our
knowledge, no amyloid targeting agent that is as
potent and as broadly acting as Ig-G3P has been
described. These activities include sequence-independent recognition with high specificity for misfolded amyloid aggregates over native or unfolded
proteins, including polymers and hydrophobic proteins, high binding selectivity of aggregates over
monomer subunits and the ability to inhibit fiber
assembly at an G3P:Aβ ratio (1:30) that implicates
oligomer interactions. In fact, we have performed
binding assays comparing Aβ monomer, oligomer
and fiber binding that show evidence for oligomer
recognition (Fig. S7). Evidence for general amyloid
recognition is based on M13 and Ig-G3P binding data
for amyloid fibers of Aβ, α-synuclein and tau proteins.
Ig-G3P also recognizes other misfolded protein assemblies, including fibers of aggregated transthyretin,
superoxide dismutase 1, the NAC peptide and
huntingtin polyQ, all implicated in neurodegenerative
diseases (data not shown). The NMR H/D exchange
studies suggest that binding between g3p and β strand
2513
G3P remodels amyloid fibers
edges could explain both fiber and oligomer recognition
activities.
Finally, Ig-G3P binding causes structural alteration
of the fibers similar to that described for N-terminal
antibodies [51,52]. Interestingly, g3p appears to
recognize C-terminal and middle regions of the Aβ
sequence, similar to anti-Aβ middle region-specific
or C-terminal region-specific MAbs, respectively.
However, these MAbs, unlike the N-terminal anti-Aβ
MAbs, cannot directly mediate fiber disaggregation
[53]. Naturally occurring autoantibodies, which recognize C-terminal and mid-region Aβ sequences,
have been described, but these antibodies interact only
with oligomer assemblies and have poor recognition of
amyloid fibers [54]. Taken together, g3p appears to
represent a unique type of amyloid targeting motif,
which we call GAIM, which has the potential to serve
as the basis for the creation of novel therapeutics for
neurodegenerative diseases.
Materials and Methods
Low-retention microcentrifuge tubes (Fisher Scientific
02-681-320) were used for all experiments to ensure that
there are no artifacts related to protein binding to the tubes.
All the phage, Ig fusion and recombinant protein preparations were tested to ensure that there was no protease
contamination. Binding and remodeling reactions with or
without protease inhibitor cocktails (complete mini protease
inhibitor tablets 11 836 170 001; Roche Diagnostics, USA)
showed identical binding and remodeling activities. All buffer
samples contained 0.1% NaN3 as a preservative.
Bacteriophage preparation
Bacteriophage M13 wild type was obtained from Prof. J.
Messing (Rutgers University, New Brunswick, NJ). The
nucleotide sequence is deposited with GenBank (accession number JX412914.1). Phage fd and IF1 variants and
a G3P expression plasmid were gifts from Prof. Franz X.
Schmid (Universität Bayreuth, Germany) [22].
Small-scale (b 1 l) bacteriophage M13, fd mutant and
IF1 stocks were produced as previously described [55] and
modified [10] using polyethylene glycol to concentrate
clarified infection supernatants and with additional purification using cesium chloride (CsCl) buoyant density
equilibrium centrifugation after the polyethylene glycol
step [56], followed by dialysis of the isolated M13 band
against phosphate-buffered saline (PBS) to remove CsCl.
M13 phage were grown by infecting either of two F-pilus
expressing E. coli strains, TG-1 [supE Δ(lac-proAB) hsdR4/F′
traD36 proAB + lacIq (lacZ)ΔM15] [57,58] (Zymo Research, Irvine, CA) or JM109 [supE Δ(lac-proAB) hsdR17
recA1/F′ traD36 proA + prob + lacIq (lacZ)ΔM15] [59],
obtained from the American Type Culture Collection
(ATCC#53323) and from J. Messing, Rutgers University,
New Brunswick, NJ.
Bacteriophage preparations following equilibrium centrifugation and dialysis reproducibly produced ~ 1 × 10 14
phage particles per milliliter determined by optical density
(OD269 nm/OD320 nm) using the following equation to
calculate phage particle titer: [(OD269 nm − OD320 nm) ×
6 × 10 16]/6.4 × 10 3 = phage/ml. †
M13 stocks were measured for plaque-forming units
(PFU) using the TG-1 host, as previously described [60].
The PFU titer assay typically produces a lower titer (0.1–0.3)
compared to the phage particle titer determined by OD due
to variability of the PFU method.
Expression, purification and assembly of amyloid fibers
Full-length tau-441 and tau-MTBR expression, purification
and assembly
Human full-length tau (2N/4R isoform, MAPT NM_005910
from Origene) and the microtubule binding region (residues
243–375) were cloned into the bacterial expression vector
pET21a (+ ) with a C- terminal myc tag. Recombinant fulllength tau protein was expressed in Rosetta™ 2(DE3)
pLysS competent cells (Novagen) and purified as described
by Margittai and Langen [61]. Purified protein was assembled into fibers at 37 °C for 7 days by dissolving 10 μM tau
monomers in 50 mM ammonium acetate buffer (pH 7.0)
containing 5 mM DTT. Aggregation was initiated by adding
2.5 μM low molecular weight heparin. Recombinant
tau-MTBR protein was expressed and purified similar to
full-length tau with minor changes: bacterial pellets containing tau-MTBR were resuspended in lysis buffer [50 mM
4-morpholineethanesulfonic acid (pH 6.5), 250 U/ml
benzonase nuclease, 1 mg/ml lysozyme and 1 complete
protease inhibitor tablet] and incubated on ice for 2 h and
homogenized by sonication. Following ammonium sulfate
precipitation, pellets were resuspended in 50 mM 4-morpholineethanesulfonic acid (pH 6.5), 10 mM NaCl, 0.5 mM
EDTA and 2 mM DTT, at 50 °C for 30–60 min and filtered
through a 0.22-μm filter before being applied onto a HiTrap
SP XL column (GE Healthcare Life Sciences, Piscataway,
NJ). tau-MTBR was eluted with 0.1–0.5 M NaCl and dialyzed
into 100 mM sodium acetate buffer (pH 7). tau-MTBR fibers
were assembled by dissolving 40 μM tau-MTBR in 0.1 M
sodium acetate buffer, 2 mM DTT and 40 μM low-molecular-weight heparin.
α-Synuclein purification and assembly
Full-length α-synuclein (NM_000345; Origene) was
cloned into a pET21a (+) vector and expressed as described
by Hoyer et al. [62]. The purified protein was aggregated
using 2 mM SDS as described by Ahmad et al. [63]. The
fibers were thoroughly dialyzed using a 30-kDa membrane
(Slidelyzer, Pierce) to remove trace amounts of detergent
and lower-molecular-weight α-synuclein species.
Purification and assembly of yeast prion domain NM of Sup35
The first 253 residues of the yeast translation termination protein Sup35 also referred to as NM region of Sup35
was PCR amplified from a genomic DNA preparation
and cloned into a pET21a (+) vector. Cysteine mutants
were prepared as described in Tessier and Lindquist
[64]. The wild type and the cysteine variant proteins were
expressed using Rosetta™ 2(DE3) pLysS competent
cells (Novagen) and purified as described in Tessier and
Lindquist [64]. The urea-methanol pellet containing NM
protein was resuspended and centrifuged at 13,000 rpm for
2514
30 min on a bench top centrifuge. The pellet containing NM
monomers was dissolved in PBS (5 μM final concentration)
and allowed to assemble at room temperature by placing it
on a rotator.
Aβ fiber assembly
Aβ (Bachem H-1368) was dissolved in hexafluoroisopropanol (HFIP), sonicated and incubated at room temperature
for 2–18 h until a clear solution developed. The peptide
solution was dried under vacuum for 2–3 h. The peptide was
aggregated as described by LeVine [20]and Stine et al. [65].
Aβ peptide aggregation using DMSO/HCl:Aβ monomer was
dissolved (200 μg) in 37.5 μl DMSO, sonicated for 1 min
and diluted with 520 μl of 10 mM HCl solution (final Aβ
concentration, 80 μM). Samples were incubated for 3 days at
37 °C (without shaking). Aβ peptide aggregation DMSO/
PBS:Aβ monomer (100 μg) was dissolved in 18 μl DMSO,
sonicated for 1 min and diluted with 382 μl of PBS containing
0.1% NaN3 (final Aβ concentration, 55 μM). Samples were
incubated for 3 days at 37 °C (without shaking). Fiber quality
for each of the abovementioned amyloid proteins was
monitored by transmission electron microscopy and by
performing dye binding studies with ThT and the luminescent
conjugated probe polythiophene acetic acid [26].
SPR binding studies
Aβ fiber binding studies
SPR experiments were performed on a BIACORE2000
or T200 instruments using CM3 sensor chips. Reagents
and sensor chips were procured from GE Health Sciences.
CM3 sensor chips was activated by injecting a 1:1 ratio of
0.4 M EDC and 0.1 M NHS solutions for 7 min at a flow
rate of 20 μl/min. Sonicated fAβ (1 min in water bath
sonicator) diluted 1:1 (40–50 μM final concentration) in
10 mM sodium acetate buffer (pH 5.0) and immobilized at
20 μl/min for 7 min. The remaining activated surface
groups were blocked with 1 M ethanolamine (pH 8.0) for
7 min; in these studies, the fibril immobilization densities
varied from 3000 to 5000 RU. Aβ monomer solution
(1 mg/ml in DMSO) was immobilized using a similar
protocol. M13, G3P and Ig-G3P were used at 25 °C or
pre-incubated at 37 °C for 3 h, followed by 1 h cool down
to 25 °C before injection. Binding was performed at 25 °C
at a flow rate of 3 or 10 μl/min for M13 and G3P, Ig-G3P,
respectively. Binding parameters were calculated using
BIAevaluation software on the BIACORE2000.
Tau fiber binding studies
ftau (10 μM) in HBS-EDTA (10 mM Hepes, 150 mM NaCl
and 3 mM EDTA) at pH 8 was allowed to react with EDC/
NHS activated CM3 sensor chip surface for 7 min at 5 μl/min
followed by ethanolamine blocking. Surface density was
1000 RU under these conditions. Ig-G3P (1 μM) in PBS was
allowed to bind at a flow rate of 5 μl/min for 10 min at 25 °C.
PBS was used as the running buffer.
AS fiber binding studies
α-Synuclein fibers were diluted in 10 mM acetate buffer
to final concentration of 25 μM and the solution pH was
G3P remodels amyloid fibers
adjusted to pH 3.5. Immobilization was performed at flow
rate of 10 μl/min on EDC/NHS activated CM3 sensor chip
followed by ethanolamine blocking. Surface density was
3500 RU under these conditions. Ig-G3P (2 μM) in HBS
was allowed to bind at a flow rate of 5 μl/min for 10 min at
25 °C. HBS was used as the running buffer.
M13 Alexa488 labeling
Alexa488 labeling of M13 was carried out by adding
1.6 μl of Alexa488-succinimidyl ester [10 mg/ml stock or
15.5 mM of Alexa488-NHS (A20100; Life Technologies)]
to 1 ml of 1 × 10 14 phage. The samples were incubated in
the dark for an hour with mild vortexing at regular intervals.
The labeling reaction was stopped by adding 50 μl of
0.5 M glycine-NaOH buffer (pH 8.5) and the M13 solution
was extensively dialyzed (10 kDa membrane; Slidelyzer,
Pierce). The labeling efficiency was calculated by estimating the concentrations of bound dye and the g8p coat
protein (by ELISA trap phage quantitation assay).
Quantitative solution binding assay
We pre-incubated 10 μM fAβ with or without Alexa488labeled phage (1 × 10 10 to 5 × 10 13/ml). The mixtures
incubated at 37 °C for 3 h were centrifuged at 7500g for
10 min. The pelleted fluorescent phage was washed in
PBS and the concentration was estimated by measuring
the fluorescence at 520 nm (excitation, 485 nm).
ThT fluorescence
We pre-incubated 2.5 μM fAβ with different concentrations of M13 (1 × 10 10 to 1 × 10 12/ml) at 37 °C for 3 days.
We added 5-fold excess of ThT to each sample at the end of
the incubation and recorded the fluorescence spectra using
a Sapphire II Tecan plate reader. All samples were excited at
450 nm.
CA filter retardation assay
This assay is used to monitor the destabilization or
remodeling of amyloid fibers into non-amyloidogenic or
soluble aggregates. The assays were primarily adapted from
Wanker and Chang and Kuret [18,19]. We pre-incubated
2.5 μM preparations of amyloid fibers (fAβ, ftau, fAS and
fNM) with different concentrations of M13 at 37 °C for
3 days. At the end of the incubation, fibers with and without
phage were diluted and spotted on CA membranes on
vacuum blots. The membranes were extensively washed
with PBS and probed with specific primary antibodies for 1 h.
HRP-conjugated secondary Ab was used to quantitate the
fibrillar aggregates retained on the membrane.
Preparation of ArgC phage
The endoproteinase ArgC (Clostripain, Worthington, catalog #P09870) was used to selectively remove the g3p
domains N1 and N2 from M13. ArgC specifically hydrolyzes
the amide bonds at the carboxylic side of the residue arginine
in a polypeptide. ArgC was activated by adding CaCl2 and
DTT (final concentration CaCl2 = 7.5 mM, DTT = 5 mM).
To 0.5 ml of M13 (1 × 10 14 phage/ml), we added 30 μl of
sequencing grade ArgC (1 mg/ml) and incubated the
2515
G3P remodels amyloid fibers
solution at 37 °C for 2 days. At the end of the incubation, the
enzyme was inactivated by adding 50 mM EDTA. The
phage was then purified over a Superdex 200 size-exclusion chromatography column and concentrated. The
removal of the N1N2 domain was verified by SDS-PAGE,
Western blotting and peptide mapping analysis before
use in disaggregation assays. In addition, mass spectrometry was used to analyze the products of proteolysis,
which confirmed specific cleavage of g3p (data not
shown).
Cloning, expression and purification of human Ig fusion
proteins
The DNA sequence corresponding to the N1N2 domain
of g3p protein was cloned into the pFUSE-hIgG1-Fc2
(Invivogen) vector. FreeStyle MAX 293 Expression
System (Invitrogen) was used for large-scale transient
transfection and protein expression of the fusion constructs. Transfected FreeStyle 293F cells are grown for
7 days before the medium is harvested by centrifugation.
The fusion constructs were purified on HiTrap Protein A
Fast Flow Columns (GE Health Sciences) and eluted in
0.1 M glycine (pH 3.0) as per manufacturer's instructions.
This protein was neutralized with 1 M Tris–HCl (pH 8.0)
and dialyzed against PBS prior to use in binding and
disaggregation assays. The Ig fusions were characterized
for protein quality and activity (Fig. S8a–d).
Assembly inhibition studies
Aβ, tau and α-synuclein assembly inhibition studies
were carried out to calculate the amyloid aggregation
inhibitory concentrations (IC 50). Phage (1 × 10 13 to
1 × 10 9 particles/ml), G3P (2000–0.2 nM) or Ig-G3P
(500–0.2 nM) were serially diluted (10-fold dilutions) into
fiber assembly reactions of Aβ (2.5 μM), tau (2 μM) or
α-synuclein (10 μM), respectively, and incubated at
37 °C for 7 days. Small aliquots of each reaction mix
were removed at the end of 7 days to perform ThT
binding assays. The inhibition concentrations were
calculated based on reduced ThT fluorescence compared to control assembly reactions. Aggregation of tau
and α-synuclein were initiated by adding 0.5 μM heparin
(low molecular weight) or by agitation (850 rpm),
respectively.
Detergent solubility of amyloid–M13 complexes
Sup35NM fibers and α-synuclein fibers were fluorescently labeled as per the manufacturer's protocols and a
mixture of unlabeled monomer and 10% labeled monomer
was assembled into fibers. The fibers were co-incubated
with M13 at indicated concentrations for 1–3 days.
Samples were transferred into varying concentrations of
and incubated at 37 °C for 6 h (Sup35NM) or 1 h
(α-synuclein) centrifugation at 30,000 rpm for 30 min.
Released fluorescence in supernatants was measured
with a Sapphire II Tecan plate reader.
Ig-G3P binding ELISA for non-amyloid proteins
Proteins were immobilized on Maxisorb plates (ThermoFisher) 225 ng/well in PBS containing 0.05% sodium azide
at 37 °C for ~ 16 h before being blocked in Superblock
(ThermoFisher) for 1 h at room temperature. Non-amyloid
proteins were from Sigma (BSA, A3059; gelatin, G9391;
casein, C3400) and prepared as previously described
[45]; elastin (E1625), bovine collagen (C9879), human
collagen (C5483) and Aβ fibers were tip-sonicated for
5 × 30 s before immobilization on plates. Ig-G3P was
added in PBS-Tween (0.05%) at the indicated concentrations and incubated at 37 °C for 1 h. All washes were
performed within PBS-Tween (0.05%). After incubation with
human specific Fc-HRP antibody at 1:10,000 in PBS-Tween
(0.05%) and 10% superblock for 1 h at room temperature,
the signal was developed with TMB solution (Sigma). The
reaction was stopped by the addition of 2 N HCl and
absorbance at 450 nM was read in a Sapphire II Tecan plate
reader.
Ig-G3P binding ELISA for α-synuclein and Aβ
We immobilized 1.6 μM fiber or monomer in PBS
containing 0.05% sodium azide on Maxisorb plates
(ThermoFisher) pre-coated with 1% BSA solution.
Ig-G3P was added in PBS-Tween (0.05%) at the indicated
concentrations and incubated at 37 °C for 1 h. All washes
were performed within PBS-Tween (0.05%). After incubation with human specific Fc-HRP antibody at 1:2500 in
PBS-Tween (0.05%), 1% milk for 1 h at room temperature, the signal was developed with TMB solution (Sigma).
The reaction was stopped by the addition of 2 N HCl and
absorbance at 450 nM was measured with a Sapphire II
Tecan plate reader.
CD analysis
G3P, Ig-G3P and their complexes with Aβ were buffer
exchanged into 20 mm potassium phosphate buffer
(pH 7.4) using an Amicon ultrafiltration setup (3 kDa).
The ellipticity at 205 nm was measured using a 1-mm path
length cuvette. Temperature melting data were recorded by
placing the cuvettes on a peltier heated sample chamber
(Chirascan, Applied Photophysics).
NMR studies
Materials for NMR studies
Unlabeled Aβ (cat. A-1002-2, lot. 5181142T) and
HFIP-treated 15N Aβ (cat. A-1102-2, lot. 12091142N) were
from r-peptide LLC, Georgia, USA. Peptides were treated
with HFIP prior to assembly into fibers. HFIP treatment
involved dissolution of Aβ in 100% HFIP at 1 mg/ml and
overnight fume hood evaporation at 25 °C to 1/8th original
volume, followed by lyophilization. Standard reagents were
purchased from Fisher Scientific, Melford and Sigma. In
solutions where the majority of water was deuterated, pD was
determined using the formula pD = 0.929 × pH(app) + 0.42,
where pH(app) is the apparent uncorrected pH meter reading
[66].
G3P preparation for NMR studies
G3P was buffer exchanged at 4 °C from purification buffer
into d.H2O (0.01% NaN3) with 3 rounds of diafiltration, the
protein concentration determined by UV spectroscopy
(ε = 43,235) and then lyophilized. One-dimensional (1D)
2516
H NMR indicated complete refolding of G3P in PBS buffer
(pH 7.4) (10 mM NaHPO4, 2.0 mM KH2PO4, 2.7 mM KCl,
137 mM NaCl and 1.0 mM EDTA) following lyophilization.
Fiber preparation for NMR studies
For each time point, two 1-mg HFIP-treated 15N Aβ vials
were each dissolved in 1 ml of 10 mM HCl (pH 4.0),
immediately sonicated for 10 min and incubated at 37 °C
for 4 days. Upon completion, two 5-μl batch representative samples were removed for electron microscopy
analysis that revealed uniform fiber morphology with
periodic twists and little lateral association. The Aβ fiber
preparations (1 mg/ml) were centrifuged individually at
179,000g (RCFMax) for 30 min at 4 °C to pellet mature
fibers. The pellets were washed twice by addition of 1 ml
of dH2O (0.01% NaN3) and centrifugation repeated. The
supernatant was immediately removed and the pellet was
dried with filter paper strips and lyophilized in the individual
centrifuge tube.
H/D exchange of Aβ fibers
On ice, lyophilized G3P was resuspended to 25 μM in
anhydrous D2O PBS buffer, pD 7.4, (10 mM NaHPO4,
2.0 mM KH2PO 4, 2.7 mM KCl, 137 mM NaCl and
1.0 mM EDTA) and 1.0 ml was added to each Aβ
lyophilized fiber pellet in the two centrifuge tubes. The
two pellets were resuspended, aliquoted into a single
Eppendorf tube and incubated at 37 °C for the allotted
H/D exchange time. fAβ fiber preparations in 500 μl of
95% DMSO/5% D 2 O were measured at a mean
concentration of 300 μM by integration of NMR aliphatic
peak intensity against TSP; thus, it was determined that
the Aβ:G3P stoichiometric ratio was 30:1, which equates
to 75:25 μM in the 2-ml H/D exchange solution.
Following H/D exchange, we centrifuged the preparation
at 13,000g (RCFMax), for 15 min at 4 °C. On ice, the
supernatant was immediately removed, the pellet was
washed by 2.0 ml addition of D2O at pD 4.0 (20 μM HCl)
[pD 4.0 to minimize H/D exchange] and the centrifugation was repeated. The pellet was dried using filter paper
strips and the exchange was quenched by freezing in
liquid nitrogen, followed by lyophilization. The fiber
preparation dead-time was on average 42 min from the
start of centrifugation to flash freezing. The procedure was
repeated identically for the Aβ fiber control with omission
of G3P.
NMR experiments were performed on a Bruker
600-MHz Avance cryoprobe equipped NMR spectrometer
at 25 °C. For NMR analysis, the H/D exchanged fibers
were dissolved in 500 μl of 95% DMSO-d6 and 5% D2O
(adjusted to pD 4.0 with microliter additions of D2-dichloroacetic acid) vortexed for 1 min, briefly centrifuged and
pipetted into an NMR tube that was quickly hand centrifuged.
A series of 6-min TROSY experiments over 70 min were
acquired immediately following sample equilibration, shimming, pulse optimization and performance of a 1D proton
NMR spectrum. The dead-time between DMSO dissolution
and commencement of the TROSY data collection had a
typical value of 14 min.
G3P-Aβ NMR titration 1D H NMR experiments were
performed at 37 °C on a sample containing 300 μM Aβ in
500 μl D2O PBS buffer (10 mM Na2HPO4, 2.0 mM
G3P remodels amyloid fibers
KH2PO4, 2.7 mM KCl, 137 mM NaCl and 0.01% NaN3,
pD 7.4). An initial Aβ fiber spectrum was obtained and the
fiber solution was then mixed with lyophilized G3P (30 μM
final concentration) and left to stand at 25 °C for 15 min to
ensure resuspension before an NMR spectrum was
obtained. A control 1D H NMR spectrum of lyophilized and
re-dissolved 30 μM G3P sample from the same preparation
used above was collected.
X-ray fiber diffraction
Sample preparation for diffraction studies
The aggregated form of Aβ42 was prepared by
incubating the lyophilized peptide (2.5 mg) in 10 mM
HCl solution for 24 h, after which the pH was adjusted to
7.4. The aggregation of Aβ42 was monitored by ThT
assay and transmission electron microscopy (see
above). The aggregated Aβ42 was mixed with M13 in
phosphate buffer solution (PBS) and incubated for 0 and
48 h at 37 °C. Samples were then dialyzed at 4 °C against
water to remove any residual salt and DMSO and were then
lyophilized.
The lyophilized samples for X-ray diffraction included
M13 alone (2.5 × 10 13 phage), aggregated Aβ42 alone
(400 μg), aggregated Aβ42 (400 μg) + M13 (2.5 × 10 13)
at incubation time t = 0 and aggregated Aβ42
(400 μg) + M13 (2.5 × 10 13) at t = 48 h. Each lyophilized
sample was packed into a thin walled glass capillary of
0.7 mm in diameter.
X-ray diffraction measurements
Measurements of X-ray diffraction patterns from the
peptides were conducted at room temperature using the
Oxford Diffraction Xcalibur PX Ultra System (Oxford
Diffraction Ltd., Concord, MA) located in the laboratory of
Dr. Andrew Bohm (Department of Biochemistry, Tufts
University, Boston, MA). The CuK α X-ray beam with
wavelength of 1.542 Å was generated at 45 kV/40 mA
using an Enhance Ultra, which is a sealed tube-based
system incorporating confocal multilayer optics. The
X-ray beam was monochromated and the K β component
was removed by means of the double bounce within
the confocal optic. The X-ray beam was focused to
0.3 mm × 0.3 mm (full width at half-maximum width at
detector position). A two-dimensional Onyx CCD detector
(Oxford Diffraction Inc., Concord, MA) with the pixel size of
121 μm was placed 85 mm from the sample position,
covering the scattering range for Bragg spacings 1.8–
54 Å. The sample-to-detector distance was calibrated
using a spherical ylid crystal (molecular formula,
C10H10SO4) or a cubic alum crystal according to the
information given by manufacturer. Exposure time was
150 s. The observed intensity of powder diffraction was
measured using the angular averaging the chosen image
area using FIT2D.
Linear combination of Aβ and M13 intensities
Series of diffraction patterns from the lyophilized
samples containing Aβ42 and M13 were observed as a
function of incubation time (t) for t = 0 and t = 48 h. The
observed intensity as a function of the radial component of
2517
G3P remodels amyloid fibers
spherical coordinates was decomposed by two basis sets
that were the normalized intensities of lyophilized Aβ42 (IA)
and lyophilized M13 (IM). The total intensity is given by
I T ðR Þ ¼ k A I A ðR Þ þ k M I M ðR Þ;
where k A þ k M ¼ 1:
Note that the intensities contained both peaks and
background and were normalized in the range from R =
0.0460 (1/Å) to 0.4484 (1/Å) so that the area under the
curve was 1. The scale factors refer to the weight content.
The best-fit curve was derived when the relative error
between the observed and calculated curves (R-factor)
was a minimum, where
X
X
R¼
jI T −I obs j= jI obs j
Acknowledgements
We are grateful to Prof. J. Messing (Rutgers
University, New Brunswick, USA) for providing wildtype M13 and to Prof. Franz X. Schmid (Universität
Bayreuth, Germany) for providing fd variants and the
G3P construct and for valuable discussions. We
appreciate the editorial assistance of Ms. Roxanne
Bales.
Appendix A. Supplementary data
Supplementary data to this article can be found
online at http://dx.doi.org/10.1016/j.jmb.2014.04.015.
Received 27 January 2014;
Received in revised form 21 March 2014;
Accepted 14 April 2014
Available online 22 April 2014
Keywords:
amyloid;
amyloid remodeling;
gene 3 protein;
Ig fusion
R.K. and H.T. contributed equally to this work.
†
http://www.biosci.missouri.edu/smithgp/
phagedisplaywebsite/phagedisplaywebsiteindex.html.
Abbreviations used:
AD, Alzheimer's disease; BSA, bovine serum albumin;
MAb, monoclonal antibody; g3p, gene 3 protein; GAIM,
general amyloid interaction motif; LCP, luminescent
conjugated polythiophene; PTMI, polythiophenes methyl
imidazole; PBS, phosphate-buffered saline; ThT, thioflavin
T; CA, cellulose acetate; TROSY, transverse relaxation
optimized spectroscopy; H/D, hydrogen/deuterium; RIPA,
radioimmunoprecipitation assay; PFU, plaque-forming
units; SPR, surface plasmon resonance; SA, streptavidin.
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