Download Atomic model of human Cystic Fibrosis Transmembrane

Atomic model of human Cystic Fibrosis Transmembrane conductance Regulator (CFTR): membrane-spanning
domains and coupling interfaces.
Jean-Paul Mornon, Pierre Lehn, Isabelle Callebaut
Supplementary Data
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Supplementary data 1: Comparison of the
crystal structure of the isolated human
CFTR NBD1 and of the Sav1866-based
model of the human CFTR NBD1, within the
context of the whole domain assembly.
The crystal structure of the isolated human CFTR
NBD1 (pdb 2bbo) and our Sav1866-based model of
the human CFTR NBD1 (in complex with MSD1
(light blue), MSD2 and NBD2 (light red) assembly)
were superimposed, showing no fundamental
differences in the core and associated regular
secondary structures. However, local differences
that can be observed principally concern surface
regions and the orientation of the ABC-specific α
sub-domain relative to the α/β core.
Surface topologies in the isolated NBD1 crystal
structure may be slightly different relative to what
happen in the NBD1 structure within the context
of the whole domain assembly, due to missing
contacts with neighboring domains (NBD2 and
MSD1:MSD2). This can be illustrated for instance
by the region encompassing the Walker B motif
and the beginning of the red helix, for which an
additional α-helix turn is observed in the
NBD1:NBD2 heterodimer model relative to the NBD1 crystal structure. This slight local difference is of functional importance as it allows to build an
appropriate NBD1:NBD2 interface, including the ATP-binding sites. Moreover, in the NBD1 crystal structure, contact artifacts may exist between side
chains, which are no longer allowed in the whole domain structure, as illustrated for instance for the NBD1:MSD2 interface (Figure 6; contacts observed
between W496 and F508).
Finally, one can note that the slight rotation that can be observed in the ABC α-specific subdomain relative to the α/β core in the Sav1866-based model
as compared yo the NBD1 crystal structure allows appropriate contacts with the MSD intracellular loops.
Note that the regulatory insertion (RI), which is not seen in the crystal structure, has been modeled in our Sav1866-based model using the template of the
regulatory insertion found in another crystal structure (see Supplementary data 3).
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Supplementary data 2: Alignment of the human CFTR MSDs and NBDs sequences with those of Sav1866.
The observed secondary structures, as deduced from the Sav1866 experimental structure (pdb 2HYD) are reported on top of the Sav1866 sequences. Identities
are indicated as white letters in black boxes, similarities are boxed. The conserved motifs are indicated, as well as the position of F508. Blue stars indicate the
one amino acid insertion in the Sav1866 sequence relative to that of CFTR, leading to a larger loop in the region including F508 (see also Figure 6). In MsbA,
this loop is two amino acids longer than in CFTR. Note that a large insertion, called regulatory insertion, exists between the two first β strands of CFTR
NBD1, and that a hinge, larger than that observed in Sav1866, links CFTR MSD2 to NBD2. The sequence of the CFTR R domain, which follows NBD1, is
not indicated in the Figure.
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Supplementary data 3: Modeling of the NBD1 regulatory insertion.
The modeling of the NBD1 regulatory insertion has been performed using as template the experimental 3D structure of the isolated mouse CFTR NBD1 ([2],
pdb 1r0z; at left), for which only six amino acids at the centre of the loop (dotted line) are not seen and which thus directly allows to completely model this
long insertion. The regulatory insertion starts with a typical amphipatic helix, which is centered on the hydrophobic cluster 10011001 (mouse CFTR amino
acid sequence FGELLEKV), highly typical of helices [3] and which, in the isolated NBD1, is in close contact with the external hydrophobic region of the αc1-2
helix (M469 region). The insertion ends with a second helix which interacts with the first one.
When superimposed with the present model, the 1r0z structure interpenetrates the NBD2 domain. However, as this loop can easily move around its ends [1],
we pivoted it mainly around the positions E402 and P439 to allow a direct contact of its hydrophobic face (human CFTR FGELFEKA) with the hydrophobic
FVLV strand (1337-1340) preceding the NBD2 ABC signature. This strand is less hydrophobic in Sav1866 (TEVG (465-468)), because of the absence in this
structure of a segment existing in CFTR NBD1. Such a reorientation of the loop keeps the residue W401 in right place, in contact with ATP in the NBD1
ATP-binding site. Moreover, it may be noted that the αc1-2 helix, which was the contact point of the regulatory insertion in the isolated NBD1, appears to be, in
the full-length CFTR structure, in contact with ICL4 (W1063 region).
On the right, the regulatory insertion built by Serohijos et al. [4] on the basis of a loop database search is depicted in the same orientation. Here, the
hydrophobic face of the first helix is oriented towards the solvent.
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Supplementary data 4: HCA alignment of the human CFTR MSDs with those of S. aureus Sav1866 and E. coli MsbA .
The sequences are shown on a duplicated α-helical net, in which the hydrophobic amino acids (V, I, L, F, M, Y, W) are indicated with a contour. These
residues form hydrophobic clusters, which have been shown to mainly correspond to the internal faces of regular secondary structures. Special symbols are
indicated in the inset. Guidelines for using the HCA method are given in [5]. Transmembrane helices, as observed in the Sav1866 structure, are boxed.
Sequence identities are indicated in red/orange/pink/yellow, whereas similar shapes in the hydrophobic clusters are shown in green.
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Supplementary data 5: Details of the interactions between CFTR ICL4 and the NBD1 F508 region.
Remarkably, the plane of F508 O, G509 O, S511 OG and that of the R1070 guanidinium group are nearly superimposable and the unique H-bond
potential of arginine is almost satisfied with four contacts depicted in yellow (R1070 NE - F508 O (3.0 Å); R1070 NH2 – F508 O (2.9 Å); R1070
NH2 – G509 O (3.3 Å); R1070 NH1 –S511 OG (2.8 Å)). Additional contacts occur between R1070 NH1 and NH2 and I507 O, R1070 being also
in close contact with Y1073.
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Supplementary data 6: Effects of the V510D mutation analyzed by using the CFTR ΔF508 model.
As shown on the right, V510D leads to the addition of a novel bridge (between NH1 R1070 and D510) to the basic state of the ΔF508 model (only one
hydrogen bond between the R1070 NH2 and I507 O, as shown on the left) and this may explain the partial restoration of protein maturation promoted by the
V510D mutation [6].
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References
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