Alteration of the Cystic Fibrosis Transmembrane Conductance Regulator Folding Pathway

doi:10.1074/jbc.271.13.7261

Alteration of the Cystic Fibrosis Transmembrane Conductance Regulator Folding Pathway

EFFECTS OF THE ΔF508 MUTATION ON THE THERMODYNAMIC STABILITY AND FOLDING YIELD OF NBD1 (*)

Bao-He Qu and
Philip J. Thomas^(§)

From the Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9040

^§To whom correspondence should be addressed:
Dept. of Physiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9040.

Abstract

The cellular phenotype of the most common cystic fibrosis-causing mutation, deletion of phenylalanine 508 (ΔF508) in the amino-terminal nucleotide binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR), is the inability of the mutant protein to fold and transit to the apical membrane of affected epithelial cells. Expressed NBD1s were purified and folded in vitro into soluble monomers capable of binding nucleotide. Here we report that the ΔF508 mutation has little effect on the thermodynamic stability of the folded NBD1. The ΔG⁰ is 15.5 kJ/mol for the wild type NBD1 and 14.4 kJ/mol for NBD1ΔF. In contrast, the mutation significantly reduces the folding yield at a variety of temperatures, indicating that Phe-508 makes crucial contacts during the folding process, but plays little role in stabilization of the native state. Under conditions that approximate the efficiency of maturation in vivo, the rate off-pathway is significantly increased by the disease causing mutation. These results establish a molecular mechanism for most cases of cystic fibrosis and provide insight into the complex processes by which primary sequence encodes the three-dimensional structure.

Footnotes

↵* This work was supported by Grant I-1284 from the Welch Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

CFTR

cystic fibrosis transmembrane conductance regulator

TNP-ATP

2′(3′)-O-(2,4,6-trinitrophenyl)adenosine 5′-triphosphate

GdnHCl

guanidine hydrochloride

PAGE

polyacrylamide gel electrophoresis

Tricine

N-tris(hydroxymethyl)methylglycine.
- Received December 15, 1995.
- Revision received January 26, 1996.