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Shane Atwell,
Christie G Brouillette, Kris Conners,
Spencer Emtage,
Tarun Gheyi,
William B Guggino,
Jorg Hendle,
John F Hunt,
Hal A Lewis,
Frances Lu,
Irina I Protasevich,
Logan A Rodgers,
Rich Romero,
Stephen R Wasserman,
Patricia C Weber,
Diana Wetmore,
Feiyu F Zhang,
Xun Zhao
[show abstract]
[hide abstract]
ABSTRACT: Upon removal of the regulatory insert (RI), the first nucleotide binding domain (NBD1) of human cystic fibrosis transmembrane conductance regulator (CFTR) can be heterologously expressed and purified in a form that remains stable without solubilizing mutations, stabilizing agents or the regulatory extension (RE). This protein, NBD1 387-646(Delta405-436), crystallizes as a homodimer with a head-to-tail association equivalent to the active conformation observed for NBDs from symmetric ATP transporters. The 1.7-A resolution X-ray structure shows how ATP occupies the signature LSGGQ half-site in CFTR NBD1. The DeltaF508 version of this protein also crystallizes as a homodimer and differs from the wild-type structure only in the vicinity of the disease-causing F508 deletion. A slightly longer construct crystallizes as a monomer. Comparisons of the homodimer structure with this and previously published monomeric structures show that the main effect of ATP binding at the signature site is to order the residues immediately preceding the signature sequence, residues 542-547, in a conformation compatible with nucleotide binding. These residues likely interact with a transmembrane domain intracellular loop in the full-length CFTR channel. The experiments described here show that removing the RI from NBD1 converts it into a well-behaved protein amenable to biophysical studies yielding deeper insights into CFTR function.
Protein Engineering Design and Selection 02/2010; 23(5):375-84. · 2.94 Impact Factor
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Hal A Lewis,
Xun Zhao,
Chi Wang,
J Michael Sauder,
Isabelle Rooney,
Brian W Noland,
Don Lorimer,
Margaret C Kearins, Kris Conners,
Brad Condon,
Peter C Maloney,
William B Guggino,
John F Hunt,
Spencer Emtage
[show abstract]
[hide abstract]
ABSTRACT: 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.
Journal of Biological Chemistry 02/2005; 280(2):1346-53. · 4.77 Impact Factor
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Hal A. Lewis,
Xun Zhao,
Chi Wang,
J. Michael Sauder,
Isabelle Rooney,
Brian W. Noland,
Don Lorimer,
Margaret C. Kearins, Kris Conners,
Brad Condon,
Peter C. Maloney,
William B. Guggino,
John F. Hunt,
Spencer Emtage
[show abstract]
[hide abstract]
ABSTRACT: Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator (CFTR), commonly the deletion
of residue Phe-508 (ΔF508) 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 ΔF508 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 ΔF508 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 ΔF508 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 ΔF508 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.
Journal of Biological Chemistry 01/2005; 280(2):1346-1353. · 4.77 Impact Factor
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Hal A. Lewis,
Xun Zhao,
Chi Wang,
J. Michael Sauder,
Isabelle Rooney,
Brian W. Noland,
Don Lorimer,
Margaret C. Kearins, Kris Conners,
Brad Condon,
Peter C. Maloney,
William B. Guggino,
John F. Hunt,
Spencer Emtage
Journal of Biological Chemistry. 11/2004;
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Hal A Lewis,
Sean G Buchanan,
Stephen K Burley, Kris Conners,
Mark Dickey,
Michael Dorwart,
Richard Fowler,
Xia Gao,
William B Guggino,
Wayne A Hendrickson, [......],
Kai W Post,
Kanagalaghatta R Rajashankar,
Marc E Rutter,
J Michael Sauder,
Stephanie Shriver,
Patrick H Thibodeau,
Philip J Thomas,
Marie Zhang,
Xun Zhao,
Spencer Emtage
[show abstract]
[hide abstract]
ABSTRACT: Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that functions as a chloride channel. Nucleotide-binding domain 1 (NBD1), one of two ABC domains in CFTR, also contains sites for the predominant CF-causing mutation and, potentially, for regulatory phosphorylation. We have determined crystal structures for mouse NBD1 in unliganded, ADP- and ATP-bound states, with and without phosphorylation. This NBD1 differs from typical ABC domains in having added regulatory segments, a foreshortened subdomain interconnection, and an unusual nucleotide conformation. Moreover, isolated NBD1 has undetectable ATPase activity and its structure is essentially the same independent of ligand state. Phe508, which is commonly deleted in CF, is exposed at a putative NBD1-transmembrane interface. Our results are consistent with a CFTR mechanism, whereby channel gating occurs through ATP binding in an NBD1-NBD2 nucleotide sandwich that forms upon displacement of NBD1 regulatory segments.
The EMBO Journal 02/2004; 23(2):282-93. · 9.20 Impact Factor
