The cystic fibrosis transmembrane conductance regulator (CFTR) contains two nucleotide-binding domains (NBDs) or ATP-binding cassettes (ABCs) that characterize a large family of membrane transporters. Although the three-dimensional structures of these domains from several ABC proteins have been determined, this is not the case for CFTR, and hence the domains are defined simply on the basis of sequence alignment. The functional C-terminal boundary of NBD1 of CFTR was located by analysis of chloride channel function [Chan, Csanady, Seto-Young, Nairn and Gadsby (2000) J. Gen. Physiol. 116, 163–180]. However, the boundary between the C-terminal end of NBD2 and sequences further downstream in the whole protein, that are important for its cellular localization and endocytotic turnover, has not been defined. We have now done this by assaying the influence of progressive C-terminal truncations on photolabelling of NBD2 by 8-azido-ATP, which reflects hydrolysis, as well as binding, at that domain, and on NBD2-dependent channel gating itself. The boundary defined in this way is between residues 1420 and 1424, which corresponds to the final β-strand in aligned NBDs whose structures have been determined. Utilization of this information should facilitate the generation of monodisperse NBD2 polypeptides for structural analysis, which until now has not been possible. The established boundary includes within NBD2 a hydrophobic patch of four residues (1413–1416) previously shown to be essential for CFTR maturation and stability [Gentzsch and Riordan (2001) J. Biol. Chem. 276, 1291–1298]. This hydrophobic cluster is conserved in most ABC proteins, and on alignment with ones of known structure constitutes the penultimate β-strand of the domain which is likely to participate in essential structure-stabilizing β-sheet formation.
Skip Nav Destination
Article navigation
September 2002
- PDF Icon PDF LinkFront Matter
Research Article|
September 01 2002
Functional analysis of the C-terminal boundary of the second nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator and structural implications
Martina GENTZSCH;
Martina GENTZSCH
Mayo Foundation and Mayo Clinic Scottsdale, S.C. Johnson Medical Research Center, 13400 E. Shea Blvd., Scottsdale, AZ 85259, U.S.A.
Search for other works by this author on:
Andrei ALEKSANDROV;
Andrei ALEKSANDROV
Mayo Foundation and Mayo Clinic Scottsdale, S.C. Johnson Medical Research Center, 13400 E. Shea Blvd., Scottsdale, AZ 85259, U.S.A.
Search for other works by this author on:
Luba ALEKSANDROV;
Luba ALEKSANDROV
Mayo Foundation and Mayo Clinic Scottsdale, S.C. Johnson Medical Research Center, 13400 E. Shea Blvd., Scottsdale, AZ 85259, U.S.A.
Search for other works by this author on:
John R. RIORDAN
John R. RIORDAN
1
Mayo Foundation and Mayo Clinic Scottsdale, S.C. Johnson Medical Research Center, 13400 E. Shea Blvd., Scottsdale, AZ 85259, U.S.A.
1To whom correspondence should be addressed (e-mail riordan@mayo.edu).
Search for other works by this author on:
Publisher: Portland Press Ltd
Received:
April 02 2002
Revision Received:
May 16 2002
Accepted:
May 20 2002
Accepted Manuscript online:
May 20 2002
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2002
2002
Biochem J (2002) 366 (2): 541–548.
Article history
Received:
April 02 2002
Revision Received:
May 16 2002
Accepted:
May 20 2002
Accepted Manuscript online:
May 20 2002
Citation
Martina GENTZSCH, Andrei ALEKSANDROV, Luba ALEKSANDROV, John R. RIORDAN; Functional analysis of the C-terminal boundary of the second nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator and structural implications. Biochem J 1 September 2002; 366 (2): 541–548. doi: https://doi.org/10.1042/bj20020511
Download citation file:
Sign in
Don't already have an account? Register
Sign in to your personal account
You could not be signed in. Please check your email address / username and password and try again.
Captcha Validation Error. Please try again.