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rapidcrimsonMécanique

22 févr. 2014 (il y a 3 années et 4 mois)

53 vue(s)

Supplemental Information

Table
S
1

Crystallographic data and refinement statistics for TTR
-
52 (M5).


Native

Pt Derivative

Data collection



Space group

C2

C2

Cell dimensions




a
,
b
,
c

(Å)

70.32, 33.37, 49.26

70.6
9
,

33.3
3
, 49.5
2



(

)

90.0,

106.8,

90.0

90.0,

107.1,

90.0

Resolution (Å)

50.00
J
2.01(2.08
J
2.01)
*

50.00
J
2.43(2.52
J
2.43)
*

R
merge

0.084 (0.330)

0.135 (0.395)

I
/

I

21.4

(7.9)

14.3

(6.2)

Completeness (%)

99.0

(97.6)

96.5 (91.5)

Redundancy

9.9

(9.9)

7.5

(6.6)




Refinement



Resolution (Å)

33.7
J
2.01


No. reflections/free

7377/
736


R
work

/
R
free

0.17
6
/0.2
23


No. atoms




Protein

89
5



Ligand/ion

0



Water

1
12


B
J
factors




Protein

20.17



Ligand/ion

0



Water

26.47


R.m.s. deviations




Bond lengths (Å)

0.07



Bond angles (

)

1.
107


*Values in parentheses are for the highest
-
resolution shell.






Figure S1.

The electron density maps of
β
-
strands S3, S4 and
β
-
turn T1 in TTR
-
52 (M5).
The blue net represents the 2F
o
-
F
c

electron density map contoured at 1.5
σ.
The cartoon
structure is colored in green, as in Figure 1
B
. Residues are shown as stick models and
hydrogen bonds are shown as black dotted lines. The secondary elements and residues
forming hydrogen bonds are labe
led.

Figure
S
2.

Gel filtration
chromatography
result
s for

wild
-
type TTR
-
52 protein
.
TTR
-
52
-
F
lag
, purified from
the
culture medium of 293T cells transfected
with

pCMV
-
TTR
-
52
-
Flag, was applied to
a
Superdex 75

10/300 GL column (
GE Healthcare, USA
) and gave

a
n

elution
peak at

11
-
11.5 m
l
with a molecular weight of about
35
k
D
a
, the dimer size of
TTR
-
52
. This peak
was confirmed
to be
TTR
-
52
-
F
lag

by Western
blotting
using Anti
-
F
lag

antibody (
TTR
-
52 purified from 293T cells has a molecular weight of about 17 kDa
d
ue to

glycosylation

at N103, data not shown).

Figure
S
3.

S
equence alignment of TTR
-
52 and
PKC
α
-
C2
. The secondary
structural elements are shown
in
consistent

with that in Figure 1C.
R
esidues
involved

in c
a
tion binding
in

TTR
-
52 and PKC
α
-
C2

are highlighted by black
arrows and triangles, respectively.

Although the secondary structure elements
of the two proteins are similarly located in this sequence alignment figure,
they locate differently in the structures.

Figure
S
4.

Affinity
-
purified TTR
-
52
-
mCherry
-
Flag binds to PtdSer on a
membrane lipid strip, while
multiple
-
point mutations D
36
A
/E38A
,
R100A/D101A, D36A/E38A/R100A/D101A

retain the
PtdSer binding
ability of TTR
-
52
.
m
Cherry
-
Flag was used as control in the presence and
absence of EDTA. A
mou
nts of purified proteins used in lipid binding are
shown by immunoblotting (bottom panel).

The PtdSer binding assay was
performed as
described

(
Wang et al. 2010
)
.

Figure
S
5.

B
inding of wild
-
type TTR
-
52 and
its
muta
n
t
s

to the p
lasma
membrane
in yeast
.

Wild
-
type TTR
-
52
-
mC
herry

and the
D36A/E38A/R100A/D101A mutant are shown to bind

to
the
plasma
membrane of wild
-
type yeast, but
single
point muta
nts

D51A, D80A,
and

N85A showed
only
weak membrane
labeling. Scale bars,

m.

Figure
S
6.

Superpos
ition

of the
L2 and L3

of TTR
-
52 (M5, green
) and
the
cation binding loop of
PKC
α
-
C2

(grey).
Since the residues in L2

of

TTR
-
52
were mutated to six consecutive alanines, we built a model to show wild
-
type struct
ure

(cyan)

of TTR
-
52 in this figure.
The crucial residues for cation
binding are shown in stick and labeled. The calcium ions in PKC
α
-
C2

are
shown in sphere. For clarity, only aspartic acids important for calcium
binding in PKC
α
-
C2

are shown and labeled with prime.

Figure S
7.

Aggregation state of TTR
-
52.

Simulated sedimentation velocity
data and

fit

residuals

of TTR
-
52 and
TTR
-
52 (M5)
-
R63D
/
W65Q
/
Q73A

are
shown in A) and B), respectively
.

For clarity, only every third scan used

to
ana
lyze the data is shown. In the upper
panel
, the individual data points are
represented as
dot
s. The fit to these data using the c(
M
) distribution is shown
as a solid line. The
X
-
axis shows the radius from the center of rotation. The
Y
-
axis shows the magnit
ude of the signal. The lower
panel

of the figure
shows the residuals as a

function of
the
radius, where the residual is equal to
the y
-
value of a given data point

minus the y
-
value of the fitted data point.
C
)
The c(
M
) distribution that best

describes thes
e data.
The left panel shows the
result of TTR
-
52 (M5) and the right panel shows the result of
TTR
-
52 (M5)
-
R63D
/
W65Q
/
Q73A
.

Figure S
8.

Deletion mutant of
ttr
-
52
.

The gene structure of
ttr
-
52

is shown with empty boxes representing exons and thin lines
indicating introns. The arrow pointing away from the boxes indicates the direction of
transcription. The gray bar below the transcript shows the size and position of the deleted
region in the
tm2
078
allele.



Supplemental Experimental Procedures

Plasmid C
onstruction

Standard methods of PCR amplification, cloning and sequencing were used. To construct

the
pET21b
-
TTR
-
52
Δ
N
-
His vector,
ttr
-
52
cDNA without
its
N
-
terminal secretion signal
(21
-
135aa)
was amplified by PCR, and inserted into pET21b (Novagen, Germany)
using
the

Nhe I

and
Xho I

sites.
M
uta
n
t TTR
-
52
Δ
N
-
His alanine block constructs were
generated with a QuickChange mutagenesis kit (Stratagene, USA).
The s
ame
mutagenesis method was used to co
nstruct TTR
-
52 (M5)
-
R63D
/
W65Q
/
Q73A
-
His
using

pET21b
-
TTR
-
52 (M5)
-
His. To generate
GST
-
CED
-
1 F1, GST
-
CED
-
1
Δ
F1, and GST
-
CED
-
1 EMI constructs, truncated fragments of CED
-
1 (shown in Figure
5
) were
amplified by PCR, and inserted into pET41b
(Novagen, Germany)
using the

Spe I

and
Xho I

sites. To construct
GST
-
CED
-
1 F1
Δ
EMI truncation, PCR was performed using
pET41b
-
CED
-
1 F1 as the template. The PCR product was digested with
Pst I

and
Xho I
,
and the 260 bp

fragment was recovered and then inserted into pET41b
-
CED
-
1 F1 using
the same sites.

The muta
n
t forms of
P
hsp
TTR
-
52
-
mCherry constructs (i.e., D36A
/
E38A, R100A
/
D101A,
D51A, L53A, P54A, L55A, D80A, P83D, N85A, R63A
/
W65Q
/
Q73A,
and

I113R
/
L132K)
were
generated

with a
QuickChange mutagenesis kit. To construct
P
hsp
TTR
-
52
-
ΔC
-
mCherry, the fragment of TTR
-
52 ΔC (1
-
130aa) was amplified by PCR and inserted into
P
hsp
mCherry vectors
using the

Nhe I

and
Xho I

sites.

To generate p416GPD
-
TTR
-
52
-
D51A
-
mCherry, p416GPD
-
TTR
-
52
-
D80A
-
mCherry,
and

p416GPD
-
TTR
-
52
-
N85A
-
mCherry, a
QuickChange mutagenesis kit was used with
p416GPD
-
TTR
-
52
-
mCherry
(
Wang et al. 2010
)

as the template.
The s
ame strategy was
applied to generate pCMV
-
TTR
-
52
-
D5
1A
-
mCherry
-
Flag, pCMV
-
TTR
-
52
-
D80A
-
mCherry
-
Flag,
and

pCMV
-
TTR
-
52
-
N85A
-
mCherry
-
Flag
, using

pCMV
-
TTR
-
52
-
mCherry
-
Flag

as the template.

To construct P
ced
-
1
CED
-
1
-
Δ
EMI
-
GFP,
the
ced
-
1

3’ end
fragment (3.9 kb, starting from
120
th

amino acid of CED
-
1, just downstream of
the
EMI domain) was amplified from
template pPD95.67
-
Pced
-
1
-
ced
-
1mg
-
GFP
(
Zhou et al. 2001
;
Cal
lebaut et al. 2003
)
, and
inserted into

vector pPD95.77
-
Pced
-
1
using the

Sma I

and

Kpn I

sites to
give

pPD95.77
-
Pced
-
1
-
CED
-
1 C
. T
his intermediate vector was
then
digested by
Sma I

and
the
PCR
amplified
ced
-
1

5’ end fragment (120 bp cDNA including
the
signal peptide sequence)
was inserted
using the

same site.

To generate P
hsp
SS40
-
EMI
-
GFP,
the
SS40
-
EMI fragment (
ced
-
1

5’ end 357 bp, 119
amino acids including
the
signal peptide) was amplified by PCR and inserted into vector
P
hsp
GFP
using the

Nhe I

site.

T
he construct P
ced
-
1
SS40
-
EMI
-
TM
-
GFP was generate
d

in
3
steps. Firstly,
pPD49.26
-
TM
-
GFP

was
generated

by insertion of TM
-
GFP (TM, CED
-
1 transmembrane domain 909
-
932 amino acids)
(
Wang et al. 2010
)

into pPD49.
26
using the

Nhe I

and
Sac I

sites, and
a

Cla I

site was introduced
after

the
Nhe I

site.
The
SS40
-
EMI fragment was
then
amplified
by PCR and inserted into pPD49.26
-
TM
-
GFP
via the

Nhe I
and

Cla I

sites to
give

pPD49.26
-
SS40
-
EMI
-
TM
-
GFP.
The

ced
-
1

promoter (5.0 kb) was
then
inserted into
pPD49.26
-
SS40
-
EMI
-
TM
-
GFP
via the

BamH I

site
to give

P
ced
-
1
SS40
-
EMI
-
TM
-
GFP.

In order to generate
the
P
ced
-
1
SS40
-
TM
-
GFP construct,
the
ced
-
1

SS40 fragment (120

bp
cDNA, 40 amino acid
s

including
the

secretion signal)

was amplified by PCR and inserted
into pPD49.26
-
TM
-
GFP
via the

Nhe I
and

Cla I

sites to
give

pPD49.26
-
SS40
-
TM
-
GFP
.
The

ced
-
1

promoter was
then
inserted
via the

BamH I

site.

To construct
P
ced
-
1
CED
-
1 F1
Δ
EMI
-
TM
-
GFP,
the
PCR
-
amplified
ced
-
1

EGF2 fragment
(first 2 EGF repeats, 240bp cDNA coding 120
-
199 amino acids of CED
-
1) was inserted
into
pPD49.26
-
SS40
-
TM
-
GFP
via the

Cla I

site, and
the

ced
-
1

promoter was
then
inserted
via the

BamH I

site.

Sedimentation velocity

analysis

A

Beckman XL
-
I
analytical ultracentrifuge (Beckman Coulter, USA) was used.
The
samples were concentrated at about 0.6
-
0.8

mg/ml in phosphate buffer (20

mM
sodium
phosphate at
pH 7.3, 100

mM Na
C
l). E
xperiments

were performed
at 20

C
and were
centrifuged at
6
0,000 rpm
. S
ta
ndard methods

were used as previously described
(
Padrick
et al. 2010
)
. The data was processed by SEDFIT
(
Schuck 2000
)
.

Supplemental Reference

Callebaut I, Mignotte V, Souchet M, Mornon JP. 2003. EMI domains are widespread and reveal the
probable orthologs of the Caenorhabditis elegans CED
-
1 protein.
Biochem Biophys Res Commun

300
: 619
-
623.

Padrick SB, Deka RK, Chuang JL, Wynn RM, Chuang DT, Norgard MV, Rosen MK, Brautigam CA. 2010.
Determination of protein complex stoichiometry through multisignal sedimentation velocity
experiments.
Anal Biochem

407
: 89
-
103.

Schuck P. 2000. Size
-
distribution

analysis of macromolecules by sedimentation velocity ultracentrifugation
and lamm equation modeling.
Biophys J

78
: 1606
-
1619.

Wang X, Li W, Zhao D, Liu B, Shi Y, Chen B, Yang H, Guo P, Geng X, Shang Z et al. 2010. Caenorhabditis
elegans transthyretin
-
like

protein TTR
-
52 mediates recognition of apoptotic cells by the CED
-
1
phagocyte receptor.
Nat Cell Biol

12
: 655
-
664.

Zhou Z, Hartwieg E, Horvitz HR. 2001. CED
-
1 is a transmembrane receptor that mediates cell corpse
engulfment in C. elegans.
Cell

104
: 43
-
56.