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PDBsum entry 1xmi
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Membrane protein, hydrolase
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PDB id
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1xmi
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References listed in PDB file
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Key reference
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Title
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Impact of the deltaf508 mutation in first nucleotide-Binding domain of human cystic fibrosis transmembrane conductance regulator on domain folding and structure.
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Authors
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H.A.Lewis,
X.Zhao,
C.Wang,
J.M.Sauder,
I.Rooney,
B.W.Noland,
D.Lorimer,
M.C.Kearins,
K.Conners,
B.Condon,
P.C.Maloney,
W.B.Guggino,
J.F.Hunt,
S.Emtage.
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Ref.
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J Biol Chem, 2005,
280,
1346-1353.
[DOI no: ]
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PubMed id
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Abstract
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Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane
conductance regulator (CFTR), commonly the deletion of residue Phe-508
(DeltaF508) in the first nucleotide-binding domain (NBD1), which results in a
severe reduction in the population of functional channels at the epithelial cell
surface. Previous studies employing incomplete NBD1 domains have attributed this
to aberrant folding of DeltaF508 NBD1. We report structural and biophysical
studies on complete human NBD1 domains, which fail to demonstrate significant
changes of in vitro stability or folding kinetics in the presence or absence of
the DeltaF508 mutation. Crystal structures show minimal changes in protein
conformation but substantial changes in local surface topography at the site of
the mutation, which is located in the region of NBD1 believed to interact with
the first membrane spanning domain of CFTR. These results raise the possibility
that the primary effect of DeltaF508 is a disruption of proper interdomain
interactions at this site in CFTR rather than interference with the folding of
NBD1. Interestingly, increases in the stability of NBD1 constructs are observed
upon introduction of second-site mutations that suppress the trafficking defect
caused by the DeltaF508 mutation, suggesting that these suppressors might
function indirectly by improving the folding efficiency of NBD1 in the context
of the full-length protein. The human NBD1 structures also solidify the
understanding of CFTR regulation by showing that its two protein segments that
can be phosphorylated both adopt multiple conformations that modulate access to
the ATPase active site and functional interdomain interfaces.
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Figure 1.
FIG. 1. Comparison of NBD1 structures. A, sequence
alignment of human and mouse NBD1 with NBD domains from other
ABC transporters. Blue background,  -strands; pink,  -helices; purple, 3[10]
helices; gray, absence of density in the electron density map.
Numbering of the secondary structure elements for CFTR NBD1 is
indicated in shaded blocks in the top row. Bold blue indicates
residues with high sequence conservation in ABC domains, while
bold red indicates residues that have been mutated in forms of
hNBD1. Protein Data Bank ID codes are indicated in parentheses.
B, stereo pair of superimposed worm diagrams of NBD1 from CFTR.
Regions with conformational differences are shown in cyan for
hNBD1-2b-F508A (molecule E), blue for hNBD1-7a-  F508,
and gold for mNBD1 (molecule B). Bound ATP is shown in wire
frame representation employing the same colors. The figure was
made using Spock (31).
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Figure 2.
FIG. 2. Local structure at the site of Phe-508 in NBD1 of
CFTR. A, stereo image of conformation of Phe-508 loop region in
mNBD1 (gold), hNBD1-2b-F508A (cyan), and hNBD1-7a- F508
(blue). B and C, worm diagrams of hNBD1-2b-F508A (B) and
hNBD1-7a- F508 (C). Residues
507-510 in B are modeled from the mNBD1 structure. The position
of Phe-508 is shown in green. Positions of residues 507 and 509
are shown in gold. Helices in are red, -strands are in blue. D
and E, surface properties of hNBD1-2b-F508A (D) and hNBD1-7a-
F508 (E) in same
orientations as in B and C. Residues 507-510 in hNBD1-2b-F508A
structure have been replaced with those from the mNBD1 structure
to provide an image representative of the wild-type human
protein. Residues are colored to indicate hydrophobic (green),
negatively charged (red), positively charge (blue), and neutral
(white) side chains. The "F " label indicates the side chain of
Phe-508, and "V " indicates the side chain of Val-510. White
worms indicate the position of the L-loop from BtuCD (residues
217-227 from PDB id 117v:A) after least squares alignment of the
ABC subdomain from its NBD
with that from hNBD1. The structural differences visible at the
right side of these images derives from a change in the
conformation of the helix 4C-helix 5 loop and is likely a
results of variation in packing contacts between the two crystal
structures. The figure was made using Spock (31).
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2005,
280,
1346-1353)
copyright 2005.
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