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MOLECULAR MODELS OF THE OPEN AND CLOSED STATES OF THE
WHOLE CFTR PROTEIN
Jean-Paul Mornona, Pierre Lehnb, Isabelle Callebauta
Supplementary Data
1
Table 1:
Comparison of the amino acid content of R "linkers" from some ABC proteins, in the
increasing order of the percentage in strong hydrophobic amino acids (V, I, L, F, M, Y, W).
l indicates the length of the considered segment (in amino acids). The absolute numbers (nH)
and percentages (%H) in hydrophobic amino acids (V, I, L, F, M, Y, W) are given in the
following columns, as well as the absolute number (nL) of loop-forming amino acids (P, G,
D, N, S) and the ratio between the number of hydrophobic and loop-forming amino acids (R
= nH/nL).
The CFTR R1 sub-domain, the CFTR R1-R2 and the CFTR R2 sub-domain have values
between those encountered for typical disordered regions (e.g. the R region observed in the
crystal structure of MDR/P-gp (Aller et al, 2009)) and for canonical, folded globular domains
(e.g. the NBD1 and NBD2 domains, which have 33 % of strong hydrophobic amino acids). R1
is here considered to start at aa 646 (after the helix 8 observed in the crystal structure of
Glcv, see Figure S6) and R2 to end at amino acid 840, before the so-called N-helix, see Figure
S4). The two last lines of the table refer to the R domains of ABCA1, the phosphorylation of
which also regulating the protein function (Roosbeek et al, 2004).
Protein
l
nH
%H
nL
R=nH/nL approximative
limits
UniProt
CG7627
MRP6
MRP5
SUR1
MRP1
MRP4
MDR1
(P-gp)
MRP7
ABCCB
Yor1p
MRP3
MDR3
MRP9
MRP2
ABCCB
CFTR R1
+R1-R2
link
CFTR R2
CFTR Nter
CFTR
NBD1
CFTR
NBD2
ABCA1 R1
ABCA1 R2
58
75
52
63
86
60
63
6
8
8
10
14
10
11
10
11
15
16
16
17
17
22
29
19
13
27
27
22
0.27
0.28
0.42
0.77
0.52
0.37
0.50 (1)
671-728
854-928
791-842
933-995
871-956
634-693
631-693
Q9VLN6_DROME
MRP6_HUMAN
MRP5_HUMAN
ABCC8_HUMAN
MRP1_HUMAN
MRP4_HUMAN
MDR1_HUMAN
50
50
60
84
60
70
93
80
129
9
9
11
16
13
16
21
19
36
18
18
18
19 (2)
22
23
23
24
28
11
12
15
25
15
31
31
20
36
0.82
0.75
0.73
0.64
0.87
0.76
0.68
0.95
1.00
821-870
736-785
811-870
854-937
634-693
705-774
867-959
658-737
646-725
MRP7_HUMAN
ABCCB_HUMAN
YOR1_YEAST
MRP3_HUMAN
MDR3_HUMAN
MRP9_HUMAN
MRP2_HUMAN
ABCBB_HUMAN
CFTR_HUMAN
65
62
17
21
26
34
16
17
1.06
1.24
776-841
1-62
CFTR_HUMAN
CFTR_HUMAN
245
73
30
54
1.35
386-645
CFTR_HUMAN
245
79
32
57
1.39
1150-1446 (3)
CFTR_HUMAN
195
49
25 (4)
60
0.80
1132-1326
ABCA1_HUMAN
115
37
32
35
1.06
2147-2261
ABCA1_HUMAN
(1) In the 3D structure, 55 consecutive amino acids are not visible, covering a distance of 48 Å.
(2) 27 % on three clusters within the first 60 amino acids.
(3) unstructured regions have been omitted for calculation.
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(4) 38 % on five clusters covering 80 amino acids.
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Figure S1: Modeling of the inward-facing conformation of CFTR (closed channel).
The passage from the MSD configuration found in the MsbA closed apo model to a "tight"
closed apo conformation, likely representing the closed form of the CFTR channel, is
illustrated here. This occurs through a small rigid body rotation of the two MSDs around the
ICLs pivots.
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Figure S2: Superimposition of the MsbA open apo conformer with the tight conformation
of the CFTR inward-facing model, in the vicinity of the channel entrance (extracellular
side) and showing the transmembrane helices. The transmembrane helices of the MsbA
open apo conformer are depicted in grey and are labeled TM1 to TM12, whereas those of the
tight conformer (colored) are labeled TM1t to TM12t. One can note the good overall
correspondence between the two conformers, noticeably for helices TM6 and TM12 at the
center.
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Figure S3: Detailed views of the CFTR chloride channel in the “tight”, inward-facing
conformation. A) Side view (left) and top view (right); B) Stereo views from the top. The
orientations are similar to those shown in Figure 3. The potential chloride translocation
pathway is symbolized by colored spheres (the colors ranging from blue to red while
moving from the intracellular side to the extracellular side).
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Figure S4 (next page): The R2 domain: Sequence alignment deduced from the HCA
comparison of the C-terminal part of the CFTR R domain (here named the R2 domain),
preceding the second membrane-spanning domain (starting at transmembrane helix
TM7), with the N-terminal tail of the same protein, preceding the first membranespanning domain (MSD1, starting at transmembrane helix TM1).
The CFTR sequences of four species are shown: Homo sapiens (h, genbank identifier (gi):
147744553), Salmo salar (s, gi:12746235), Danio rerio (d, gi:117380075), Fundulus heteroclitus (f,
gi:3015540), illustrating the presently known most divergent CFTR sequences. At top are
presented a few N-terminal sequences of other ABC proteins, sharing significant similarities
with the CFTR N-terminal sequence (hABCCB: Homo sapiens ABCC11, gi:74762666; hMRP9:
Homo sapiens MRP9, gi:161788999; hMRP5: Homo sapiens MRP5, gi:8928547; dGD10651:
Drosophila simulans GD10651, gi:195581868, hMRP4: Homo sapiens MRP4, gi:206729914).
Between the CFTR N-terminal and R2 sequences are shown the N-terminal sequence of
Saccharomyces cerevisiae YOR1 (yYOR1, gi:1730876) and the R sequence of Homo sapiens MRP3
(hMRP3: gi:6920069), which provide intermediate links. The alanine content of the region
aligned with the N-ter helix is in good agreement with such a secondary structure.
Conserved hydrophobic amino acids (V, I, L, F, M, Y, W) are colored dark green, and
residues that can substitute them (A, C, T) are colored light green. Loop-forming amino acids
(P, G, D, N, S) are colored yellow. Conserved aromatic residues (Y, W, F) are colored purple,
conserved basic and acidic residues are colored blue and pink, respectively. The secondary
structures predicted using HCA for the associated hydrophobic clusters (1 stands for V, I, L,
F, M, Y, W; 0 for other residues (Eudes et al, 2007)) are indicated above and below the CFTR
N-terminal and R2 sequence, respectively. Black stars indicate the phosphorylation sites. The
amino acid ranges are shown at the end of the sequences. Below the alignment are shown the
secondary structure propensities estimated for the R2 domain, based on NMR experimental
data, for the non-phosphorylated and phosphorylated forms of CFTR (based on the data
presented in Figure 2 of the Baker’s article (Baker et al, 2007)).
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Figure S5: 3D structures of the regulatory domains (TOBE domains) of three ABC
proteins. From left to right: the Pyrococcus horikoshii multiple sugar binding transport ATPbinding protein (pdb 2d62), the Archaeoglobus fulgidus molybdate/tungstate ABC
transporter ModC (pdb 3d31) and the Sulfolobus solfataricus glucose transporter (pdb 1oxs).
Strands constituting the "Greek-Key" motifs are labeled according to Figure S6. The fifth
strand of each TOBE domain is depicted in pink. The N- and C-termini of the regulatory
domains are indicated. The WO4 groups, which bind to the ModC regulatory domains and
allow a trans-inhibition of the molybdate/tungstate ABC transporter, are shown.
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Figure S6: The R1 domain: Top) Sequence alignment deduced from the HCA comparison of the N-terminal part of the CFTR R domain (here
named the R1 domain) with the regulatory domains found in some bacterial ABC transporters immediately after their NBDs (TOBE domains).
The sequences of six of these proteins are shown, distributed into two subfamilies. The similarities between amino acids are reported as in
Figure S4, and phosphorylation sites are indicated by black stars. Grey stars indicate the C-terminal amino acid. The position of the regulatory
extension (RE), observed in the NBD1 crystal structure, is indicated. Bottom) Secondary structure propensities estimated for the R1 domain,
based on NMR experimental data, for the non-phosphorylated and phosphorylated forms of CFTR (based on the data presented in Figure 2 of
the Baker’s article (Baker et al, 2007)).
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Figure S7: Comparison of the HCA plots of the human CFTR R domain (aa 610 to 885) and of the yeast Yor1p corresponding region (aa 775
to 925).
The sequence is shown on a duplicated alpha-helical net, and the strong hydrophobic amino acids (V I L F M Y W) are contoured: these form
hydrophobic clusters, which mainly correspond to regular secondary structures. Guidelines to the use of the method can be found in (Callebaut
et al, 1997; Gaboriaud et al, 1987). The predicted secondary structures and domain limits are reported above the human CFTR plot. Green colors
highlights conservation of hydrophobic amino acids (V I L F M Y W), whereas orange is used for correspondence between hydrophobic amino
acids (V I L F M Y W) and amino acids (A C T), which can substitute them in a context-dependent manner. Sequence identities are colored pink.
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Figure S8: A complement to Figure 6, showing the NBD1:NBD2 assembly from bottom (on
the R1 sub-domain side) and from top (from the interface with membranes).
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Figure S9: Putative dimeric architecture of CFTR (closed, inward-facing conformation): an
alternative solution to that proposed in Figure 5. The view is shown in a similar orientation
as in Figure 5. The interface between each monomer is built of the opposite and symmetric
long faces, as in Figure 5 (compare the bottom views, where CFTR is seen from the
extracellular side). Main contacts are between TM4 and TM4, 2 of NBD2 and R2.
However, in this alternative arrangement, R1 does not contact R1 (in the context of the
proposed location of this domain).
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Figure S10: Possible concerted movements of the NBD1/NBD2 domains and the R1 and R2 parts of the regulatory domain R, between the
inward-facing conformation (closed state) at left and the outward-facing conformation (open state, in which the bound nucleotides are shown).
The NBD1 (blue) being fixed, the NBD2 domain rubs, while moving, the R2 and R1 segments and may interact more tightly with
phosphorylable segments (S660, S737, S795 and S813 are labeled in red to recall their importance (Hegedűs et al, 2009)) in the channel openstate through specific basic patches. To that aim, R1 (in white) may rotate around its N- and C-terminal parts toward a position (in yellow) close
to the NBD2 domain (in orange). See Fig. S11 for the details of amino acids in the R1 region for the outward-facing conformation.
Red stars indicate the possible area of interaction. One can note that no phosphorylable serine is present after S813 within the R2 region which
provides less contact with NBD2.
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Figure S11: Zoom on the model of the R1 domain (outward-facing conformation), in a position where it begins to interact with the two basic
specific patches including K1429, R1434, R1438 (NBD2) and K1457, K1459, K1461 (C-ter domain).
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Figure S12 (next page): Putative alternative pathway towards the entry of the CFTR
channel pore through the NBD1:NBD2 interface, leading to the bundle of
intracellular loops (outward-facing configuration (Mornon et al, 2008)).
A) Global view with, at the center in the middle distance, the cytoplasmic ends of
ICL1 and ICL2 shown in dark blue and ICL3 and ICL4 shown in red. The R1 domain,
which is likely situated in front of this view, is not shown. The R1 domain, with the
position shown in Figure S10, however does not impede access to this pathway.
B) Closer view, in the same direction as in panel A, but showing only the putative
conduction pathway of the NBD1:NBD2 interface. Anions might be attracted by the
basic residues K606 and R1403 and then transported through a small pore including
G576 and A1374, which are 5.1 Å apart in the model, within the Walker B loops (c4 c4-5 loop). They might leave the NBD1:NBD2 interface at the levels of the Q loops
(Q493 and Q1291, which are 5.6 Å apart from each other). Basic residues are shown
in blue, hydrophobic ones in green and hydrophilic ones in pink. Proline residues are
shown in light yellow. A1374 and G576 are shown as van der Waals surfaces, with
between them a chloride ion.
C) Same view as in Figure 7B, but with an alternative position of E267 at the
foreground, which may weakly interact with K968 in one of its possible location.
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