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J. Biol. Chem., Vol. 280, Issue 2, 1346-1353, January 14, 2005

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Impact of the F508 Mutation in First Nucleotide-binding Domain of Human Cystic Fibrosis Transmembrane Conductance Regulator on Domain Folding and Structure^*

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¶, and Spencer Emtage

From the Structural GenomiX, Inc., San Diego, California 92121, the ^¶Department of Biological Sciences, Columbia University, New York, New York 10027, and the ^||Department of Physiology, School of Medicine, The Johns Hopkins University, Baltimore, Maryland 21205

Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator (CFTR), commonly the deletion of residue Phe-508 (F508) 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 F508 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 F508 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 F508 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 F508 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.

Received for publication, September 23, 2004 , and in revised form, November 2, 2004.

The atomic coordinates and structure factors (codes 1XMI and 1XMJ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by a research contract from the Cystic Fibrosis Foundation Therapeutics, Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Structural GenomiX, 10505 Roselle St., San Diego, CA 92121. E-mail: hal_lewis{at}stromix.com.

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