the four-transmembrane protein ip39 of euglena forms ... filethe four-transmembrane protein ip39 of...
TRANSCRIPT
The four-transmembrane protein IP39 of Euglena forms strands by
a trimeric unit repeat
Hiroshi Suzuki*, Yasuyuki Ito*, Yuji Yamazaki, Katsuhiko Mineta, Masami Uji, Kazuhiro Abe,
Kazutoshi Tani, Yoshinori Fujiyoshi & Sachiko Tsukita
Supplementary Information
a
bCarbon side
Carbon side
Luminal side
C
Supplementary Figure S1 | Cross-link experiment of solubilized IP39 protein.(a) SDS-PAGE analysis of the fractions during the purification procedure of IP39 from Euglena gracilis, followed by Western blotting probed with anti-IP39 polyclonal antibodies (kindly provided by Dr. T. Suzaki) (lanes 1, 2) or Coomassie Brilliant Blue (CBB) staining (lanes 3, 4). The Western blotting shows the differences in the composition ratios of oligomers before (lane 1, NaOH-treated membrane fraction) and after (lane 2, OG-solubilized supernatant) solubilisation. The sample in lane 3 is identical to that of lane 2, and lane 4 represents the final purified fraction of IP39 after anion-exchange chromatography. (b) A schematic diagram illustrating the locations and topology of a vesicular crystal and carbon films on an electron microscopy grid. The two sandwiching carbon films contact the outer surface of the crystal, causing distortions of the structures. (c) Purified IP39 proteins solubilised in buffer containing 2% OG were treated with glutaraldehyde (GA) at the indicated concentrations at 37ºC for 1 h. As a control, the tetrameric membrane protein, rat aquaporin-4 (rAQP4; ~32 kD)46, purified and solubilized in 2% OG were treated the same way. Both samples were subjected to SDS-PAGE and silver-stained. Bands I, II, III and IV correspond to monomers, dimers, trimers, and tetramers, respectively. The 10-mM GA concentration is sufficient for the rAQP4 to completely form the cross-linked tetramer, but most of the IP39 fraction comprises monomers.
43
CBB stain
I
II
III
1 2
W.B.anti-IP39
(kDa)
I
II
I
II
III
IV
0 0.1 1 10 100 0 0.1 1 10 100 GA (mM)
IP39 rAQP4
65
80
120
25
5040
(kDa)
37
50
75
100
150
ba
c
Luminal side
Carbon-contacting side
Carbon-contacting side
Supplementary Figure S2 | Antibody-bound 2D crystals of IP39.(a) Negatively stained IP39 crystals bound with the IgG of the anti-phosphotyrosine antibody, labelled with 15-nm gold-conjugated secondary antibodies. During the blocking and binding procedures, the crystalline vesicles were subjected to deformation, but the observed gold labelling on the vesicles shows that the antibodies can specifically recognise and bind the antigens on the crystal surface. The black bar represents 200 nm. (b) Negatively stained IP39 crystals with the addition of the Fab fragments of anti-phosphotyrosine antibody. Reconstituted vesicles incubated with Fab without any harsh treatment maintained their size and shape and the crystalline arrays of the proteins were preserved with some decrease in the resolution (see Supplementary Figure S5 and Supplementary Table S1, S2). The black bar represents 500 nm. (c) Schematic diagram illustrating the attachment of the Fab fragments on IP39 molecules in a 2D crystal. The crystal vesicle does not allow the Fab fragments to permeate the lipid bilayer, and thus the epitopes on the carbon-contacting sides are labeled by the antibodies.
L F L I I A S
T S L L
L T F A
S L I
V F I A
P
Q G V A T
K D
W S
W
R T W
V D
W L
K N C
P H
L P Q
V S
F A I
Y/H T C Q E
D N
I D Q
K M S G G
V A
S G V
C R G Y F
A A
L I K F L G L Y
A L F A
A T L M
L F T A V V A
L Y V C T L T F
C I L V
L A F S
S F L M W I V
I I L F A F V L
F V C G
A L F A
S/A V I F V Q T
G K
L W S K P I
A L A
C N Q E A Y
Q P G N
K
I
T L F
P L
C
W G Y
G Y S
A/Q
S F
K P
F I
P H
E E
A E
A A
V P
Y M
P P
P V Y
L E
P Q
P Y
Y
V P
P Y
E Y
A V
P E
V A P
-/S L/V
A P/Y
S/P
Y/P
P Y
A V
A/Q
P A/P
Y A
P/V
P V L
A A
Y G P V G
V K H
P A
T K
G A
M
ExtracellularSide
IntracellularSide
N-terminalC-terminal
Supplementary Figure S3 | Secondary structure and topology map of IP39.Predicted transmembrane topology of the IP39 polypeptide is represented by the strand of amino acids. The transmembrane regions overlaid on the grey band were predicted using the SOSUI server47. Blue and red letters indicate positively- and negatively-charged residues, respectively. Green letters indicate the consensus motif between the PMP-22/EMP/MP20/Claudin superfamily. Magenta letters indicate potential phosphorylation tyrosine sites. Two characters in one oval indicate the amino acid sequences of IP39_alpha (left) and IP39_beta (right). The black triangle indicates the excision point of the first methionine.
IP39_alphaIP39_betaEMP-3PMP-22EMP-2EMP-1Claudin-3Claudin-4Claudin-1Claudin-2MP20
K T L F L I S A I T S L L A F T L S L I A I F V W K D T W D L W K C I A V S Q 63
M K G Y F A A T Q V F I V S G C V F A F L A L V F A F L . I I G K K A L A 128
L Y V C T L T F L A F S C I L V S F L MW I Q K Y S Y G W I C A V V A T F L A F L A M L L F 195
K I 263
K T L F L I S A I T S L L A F T L S L I A I F V W K D T W D L W K C I A V S Q 63
M K G Y F A A T Q V F I V A G C V F A F L A L V F A F L . I I G K K A L A 128
L Y V C T L T F L A F S C I L V S F L MW I Q K Y S Y G W I C A V V A T F L A F L A M L L F 195
K I 264
M S L L L L V V S A L H I L I L I L L F V A T L WW T L S N L W Y C T W N D T 49
V S G W L K A V Q V L M V L S L I L C C L S F I L F M F . Q L Y T G G L F 97
Y A T G L C Q L C T S V A V F T G A L I Y A E G F G Y C F A L A W V A F P L A L V S G I I Y 156
I H 163
M L L L L L S I I V L H V A V L V L L F V S T I W I V G A D L W Q C S T S S G 48
S S E W L Q S V Q A T M I L S I I F S I L S L F L F F C . Q L F T G G R F 96
Y I T G I F Q I L A G L C V M S A A A I Y T E Y Y G F A Y I L A W V A F P L A L L S G V I Y 153
V I 160
M L V L L A F I I A F H I T S A A L L F I A T V WW V G F D V W R C T N T N C 48
F Q S T L Q A V Q A T M I L S T I L C C I A F F I F V L . Q L F R G E R F 95
V L T S I I Q L M S C L C V M I A A S I Y T D G Y G Y S Y I L A W V A F A C T F I S G M M Y 160
L I 167
M L V L L A G I F V V H I A T V I M L F V S T I W L V S S G L W K C T N S C S 50
Y A D A L K T V Q A F M I L S I I F C V I A L L V F V F . Q L F T G N R F 95
F L S G A T T L V C W L C I L V G V S I Y T N Y H G Y S Y I L G W I C F C F S F I I G V L Y 151
L V 157
S M G L E I T G T A L A V L G W L G T I V C C A W R V S G T W G L WM C V V S T G 59
L A Q D L Q A A R A L I V V A I L L A A F G L L V A L V G C V Q D A K I T 117
I V A G V L F L L A A L L T L V P V S W S A R R M G A G L Y V G W A A A A L Q L L G G A L L 180
C C 220
S M G L Q V M G I A L A V L G W L A V M L C C A W R V T G T W G L WM C V V S T G 60
L A Q D L Q A A R A L V I I S I I V A A L G V L L S V V G C L E D A K T M 118
I V A G V V F L L A G L M V I V P V S W T A Q R M G A S L Y V G W A A S G L L L L G G G L L 181
C C 209
N A G L Q L L G F I L A F L G W I G A I V S T A W R I Y G T Y G L WM C V S S T G 60
L N S T L Q A T R A L M V V G I L L G V I A I F V A T V G C L E D Q R M A 119
V I G G A I F L L A G L A I L V A T A W Y G Q Y F G Q A L F T G W A A A S L C L L G G A L L 182
C C 211
S L G L Q L V G Y I L G L L G L L G T L V A M L W K T S G T S G L WM C A T S T G 60
L G A D I Q A A Q A MM V T S S A I S S L A C I I S V V G F C Q E A R V A 118
V A G G V F F I L G G L L G F I P V A W N L R F I G E A L Y L G I I S S L F S L I A G I I L 181
C F 230
M Y S F M G G G L F C A W V G T I L L V V A T A WM Q Y G . H G L W R C L G K C Y 52
T E A Y W N A T R A F M I L S S L C A T S G I I M G I V A F A Q Q T S R P 98
F S A G I M F F A S T F F V L L A L A I Y T V W F S W S Y I L G W V A L L M T F F A G I Y M 158
C A 173
CC
CC
CCCC
IP39_alphaIP39_betaEMP-3PMP-22EMP-2EMP-1Claudin-3Claudin-4Claudin-1Claudin-2MP20
IP39_alphaIP39_betaEMP-3PMP-22EMP-2EMP-1Claudin-3Claudin-4Claudin-1Claudin-2MP20
IP39_alphaIP39_betaEMP-3PMP-22EMP-2EMP-1Claudin-3Claudin-4Claudin-1Claudin-2MP20
Supplementary Figure S4 | Multiple sequence alignment.The amino acid sequences of genes in PMP-22/EMP/MP20/Claudin superfamily were aligned by Clustal W48.IP39_alpha from Euglena gracilis (GenBank ID: AB167379.1), IP39_beta from Euglena gracilis (GenBank ID: AB167380.1), PMP-22 from Homo sapiens (RefSeq ID: NP_696997), EMP-1 from Homo sapiens (RefSeq ID: NP_001414), EMP-2 from Homo sapiens (RefSeq ID: NP_001415), EMP-3 from Homo sapiens (RefSeq ID: NP_001416), MP20 from Bos taurus (RefSeq ID: NP_776527), Claudin-1 from Homo sapiens (RefSeq ID: NP_066924), Claudin-2 from Homo sapiens (RefSeq ID: NP_065117), Claudin-3 from Homo sapiens (RefSeq ID: NP_001297) and Claudin-4 from Homo sapiens (RefSeq ID: NP_001296). Residues with higher similarity are highlighted in the darker grey. The conserved W-LW-C-C motif sequences are highlighted in yellow. Cylinders above the sequences indicate putative transmembrane regions of IP39 predicted by SOSUI47. The aligned sequences were drawn using ALINE49.
IP39 IP39+Fab
4
4
4
4
4
4
4
4
4
4
4
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3
3
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4
2
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1
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1
4
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2
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22
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1
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1 1
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1
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3
1
1
4
44
2
2
2
1
1
4
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2
2
33
4
4
3
3
3
4
4
4
a*
b*
33
2 233 33 22
332
2
2
2
3
3
33
3
3
1
1
2
2
3
3
4
4
3
3
4
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2
2
1
1
1
1
3
3
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1
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4
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2
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2
2
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1
1
3
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2
2
1
1
4
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3
33
5 Å
7 Å
20 Å
a*
b*
Tilt axis
b
c
d f
e
a 0º 45º
0.008 0.024 0.041 0.057 0.073 0.090 0.106 0.123-2000
2000
0
4000
-180
0
180
90
-90
Phase
Amplitude
lattice line (1,2)
0.139
z*, Å-1
-2000
2000
0
4000
-180
0
180
90
-90
lattice line (4,2)
0.008 0.024 0.041 0.057 0.073 0.090 0.106 0.123 0.139
Phase
Amplitude
z*, Å-1
0.003 0.014 0.024 0.035 0.045 0.056 0.066 0.077-400
600
0
800
-180
0
180
90
-90
lattice line (1,2)
0.087
400
200
-200
0.098
Phase
Amplitude
z*, Å-1
0.003 0.014 0.024 0.035 0.045 0.056 0.066 0.077
0
1000
-180
0
180
90
-90
lattice line (4,2)
0.087 0.098
Phase
Amplitude
z*, Å-1
Supplementary Figure S5 | Analysis of the 2D crystals of IP39 without/with Fab.(a, b) IQ-plots38 calculated from representative non-tilted (a) and 45º-tilted (b) images of a frozen-hydrated IP39 crystal taken by cryo-electron microscopy. Because the 21 symmetry along a* was broken due to the mechanical contact of the 2D crystals with the carbon support films, the diffraction pattern gave a broken mirror symmetry along a* and reflections with an odd index on the a* axis appeared in (a). (c-f) Representative lattice lines from the 3D data sets of the IP39 crystals without (c,d) or with (e,f) the addition of the Fab fragments. The phase and amplitude data are shown in the upper and lower panels of each figure, respectively. The indices of these lattice lines are indicated in the lower panels.
44
1
32
4
4
4
13
4
2
3
2
214
2
2
4
2
4
1
4
4
1
44
34
24
4
3
3
3
4
5 Å
7 Å
20 Å
Supplementary Figure S6 | Diffraction anisotropy of the IP39 crystal.Averaged F/sigma values are plotted for the two unit cell axes of the 2D crystal (black and red for a* and b*, respectively). The maximum indices of H and K were determined to be 21 and 5, respectively (see Materials and Methods). From the plot, the averaged F/sigma of the reflections used is above 2.2 (shown as a grey dotted line). Thus, the effective resolution is 8.3, 11.9, and 8.3 Å resolution along the a*-, b*-, and c*-axis, respectively.
0 0.05 0.1 0.15Resolution (1/Å)
1.5
2.5
3.5
|F| /
σ
along a* axisalong b* axisalong c* axis
K=5 H=212.2
b c d
Supplementary Figure S7 | Cross-sections of B-Mol1, 2, and 3.(a) Molecular surfaces of the Mol1-3 density map contoured at 1.2σ and superimposed model helices are indicated by the same colour code as in Figure 6. The dashed lines in the whole structure indicate the positions for each section shown in b-d. (b-d) The cross-sections parallel to the membrane plane are viewed from the intracellular side of the B-strand and coloured according to the contour levels by gradation from blue (1.2σ) to red (4.6σ). The surfaces of the Mol1-3 trimeric units are represented by gray mesh and the model helices are coloured as in a. The right-angled arrows in the boxes indicate the directions of the a- and b-axis.
(b)
(c)
(d)
Mol3Mol2
Mol1
a
ab
ab
ab
180º
Supplementary Figure S8 | Stereo EM density maps of a trimeric unit in strand B.The EM density maps (light gray mesh) of the Mol1-3 in the IP39 crystal are contoured at 1.2σ and shown in stereo. The model helices are superimposed in the maps and indicated by the same colour code as in Fig. 4e. Both upper and lower panels represent views parallel to the membrane plane.
Supplementary Table S1 | Phase residuals of all orthogonal space groups calculated by ALLSPACE44
Space group Phase residual (No.) Phase residual (No.) Target residual based on vs. other spots vs. theoretical statistics statistics taking Friedel (90º random) (45º random) weight into account p1 13.5 (52) 9.6 (52) p2 17.5†(26) 8.7 (52) 19.3 p12_b 26.2 (20) 38.7 (8) 14.6 p12_a 76.5 (18) 6.6 (4) 14.1 p121_b 68.7 (20) 60.2 (8) 14.6 p121_a 14.6†(18) 6.5 (4) 14.1 c12_b 26.2 (20) 38.7 (8) 14.6 c12_a 76.5 (18) 6.6 (4) 14.1 p222 52.4 (64) 9.3 (52) 15.8 p2221_b 20.6‡(64) 10 (52) 15.8 p2221_a 54.8 (64) 10.8 (52) 15.8 p22121 56.4 (64) 37.7 (52) 15.8 c222 52.4 (64) 9.3 (52) 15.8 p4 34.9 (34) 9.4 (52) 17.9 p422 54.8 (76) 9.4 (52) 15.5 p4212 52.1 (76) 37.7 (52) 15.5 †Acceptable ‡Should be considered
Supplementary Table S2 | Electron crystallographic data (IP39)
Two-dimensional crystal
Space group P2
Lattice constants a = 174.3 Å, b = 59.3 Å, c = 200.0 Å (assumed), γ = 90.0°
Used number of images
Approximate tilt angle
0° 4
20° 51
45° 96
60° 159
Total 310
Resolution limit
For map
In membrane plane (Å) 8.3, 11.9 (a*, b* directions)
Normal to membrane plane (Å) 8.3
For merging
In membrane plane (Å) 7.0
Normal to membrane plane (Å) 8.0
Range of underfocus (Å) 8,700 ~ 33,200
Number of observed reflections 33,162
Number of independent reflections 18,946
Overall weighted phase residualsa 39.4
Overall weighted R-factora 0.355
a. Used reflections are better than IQ 7.
Supplementary Table S3 | Electron crystallographic data (IP39+Fab)
Two-dimensional crystal
Space group P2
Lattice constants a = 174.3 Å, b = 59.3 Å, c = 200.0 Å (assumed), γ = 90.0°
Used number of images
Approximate tilt angle
0° 11
20° 29
45° 158
Total 198
Resolution limit
For map
In membrane plane (Å) 10.3, 11.9 (a*, b* directions)
Normal to membrane plane (Å) 14.3
For merging
In membrane plane (Å) 10.0
Normal to membrane plane (Å) 14.3
Range of underfocus (Å) 6,200 ~ 23,000
Number of observed reflections 13,936
Number of independent reflections 5,728
Overall weighted phase residualsa 36.2
Overall weighted R-factora 0.404
a. Used reflections are better than IQ 7.
Supplementary references
46. Hiroaki, Y. et al. Implications of the aquaporin-4 structure on array formation and cell
adhesion. J. Mol. Biol. 355, 628–639 (2006).
47. Hirokawa, T., Boon-Chieng, S. & Mitaku, S. SOSUI: classification and secondary structure
prediction system for membrane proteins. Bioinformatics 14, 378–379 (1998).
48. Thompson, J. D., Higgins, D. G. & Gibson, T. J. CLUSTAL W: improving the sensitivity of
progressive multiple sequence alignment through sequence weighting, position-specific gap
penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680 (1994).
49. Bond, C. S. & Schüttelkopf, A. W. ALINE: a WYSIWYG protein-sequence alignment
editor for publication-quality alignments. Acta Crystallogr. D Biol. Crystallogr. 65, 510–
512 (2009).