The cystic fibrosis transmembrane conductance regulator

Nucleotide binding to a synthetic peptide segment from the second predicted nucleotide binding fold

Young Hee Ko, Philip J. Thomas, Peter L. Pedersen

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

Previous studies from this laboratory with a 67-amino acid synthetic peptide (P-67) demonstrated directly that the first predicted nucleotide binding fold of the cystic fibrosis transmembrane conductance regulator (CFTR) binds ATP (Thomas, P. J., Shenbagamurthi, P., Ysern, S., and Pedersen, P. L. (1991) Science 251, 555-557). Although mutational analysis within the predicted second nucleotide binding fold indicates that this domain may be functionally important also, direct evidence for nucleotide binding is lacking. Here, we report the design, chemical synthesis, and purification of a 51-amino acid segment (P-51) of the second predicted nucleotide binding fold of CFTR and demonstrate that this peptide binds ATP. P-51 consists of amino acid residues from glutamic acid 1228 through threonine 1278 and contains a motif, GX4GKS, very similar or identical to that found in many nucleotide-binding proteins. The freshly dissolved peptide moves predominantly as a single species upon molecular sieve chromatography and readily binds ATP without eliciting its hydrolysis. P-51 also readily binds the fluorescent ATP analogs TNP-ATP (2′(3′)-0-(2,4,6-trinitrophenyl)-adenosine-5′-triphosphate) and TNP-ADP but exhibits much less capacity to bind TNP-AMP. ATP displaces TNP-ATP with a Kd (ATP) of 0.46 mM. In the presence of the denaturant urea, P-51 loses most of its binding capacity indicating that structure is important for binding. Consistent with this conclusion, circular dichroism spectroscopy revealed that P-51 has significant secondary structure. Elements of such structure calculated from deconvolution of the circular dichroism spectra compare favorably with those predicted from the program of Chou, P. Y., and Fasman, G. D. (1977) J. Mol. Biol. 115, 135-175. These experiments provide the first direct evidence that the second predicted nucleotide binding fold of CFTR binds ATP and define a 51-amino acid segment within the ∼150-amino acid fold critical for this function. They also indicate that the β and γ phosphate groups of ATP may be important for binding and that the 51-amino acid region studied is not sufficient to catalyze ATP hydrolysis. Finally, as seven different mutations within P-51 are known to cause cystic fibrosis, these studies will be important in future efforts to understand the molecular basis of the disease.

Original languageEnglish (US)
Pages (from-to)14584-14588
Number of pages5
JournalJournal of Biological Chemistry
Volume269
Issue number20
StatePublished - May 20 1994

Fingerprint

Cystic Fibrosis Transmembrane Conductance Regulator
Nucleotides
Adenosine Triphosphate
Peptides
Amino Acids
Adenosine
Hydrolysis
Circular Dichroism
Circular dichroism spectroscopy
Molecular sieves
Deconvolution
Threonine
Chromatography
Purification
Urea
Glutamic Acid
Carrier Proteins
Cystic Fibrosis
Phosphates
Gel Chromatography

ASJC Scopus subject areas

  • Biochemistry

Cite this

The cystic fibrosis transmembrane conductance regulator : Nucleotide binding to a synthetic peptide segment from the second predicted nucleotide binding fold. / Ko, Young Hee; Thomas, Philip J.; Pedersen, Peter L.

In: Journal of Biological Chemistry, Vol. 269, No. 20, 20.05.1994, p. 14584-14588.

Research output: Contribution to journalArticle

@article{58900610d2f3497c92b99f88c341b1fc,
title = "The cystic fibrosis transmembrane conductance regulator: Nucleotide binding to a synthetic peptide segment from the second predicted nucleotide binding fold",
abstract = "Previous studies from this laboratory with a 67-amino acid synthetic peptide (P-67) demonstrated directly that the first predicted nucleotide binding fold of the cystic fibrosis transmembrane conductance regulator (CFTR) binds ATP (Thomas, P. J., Shenbagamurthi, P., Ysern, S., and Pedersen, P. L. (1991) Science 251, 555-557). Although mutational analysis within the predicted second nucleotide binding fold indicates that this domain may be functionally important also, direct evidence for nucleotide binding is lacking. Here, we report the design, chemical synthesis, and purification of a 51-amino acid segment (P-51) of the second predicted nucleotide binding fold of CFTR and demonstrate that this peptide binds ATP. P-51 consists of amino acid residues from glutamic acid 1228 through threonine 1278 and contains a motif, GX4GKS, very similar or identical to that found in many nucleotide-binding proteins. The freshly dissolved peptide moves predominantly as a single species upon molecular sieve chromatography and readily binds ATP without eliciting its hydrolysis. P-51 also readily binds the fluorescent ATP analogs TNP-ATP (2′(3′)-0-(2,4,6-trinitrophenyl)-adenosine-5′-triphosphate) and TNP-ADP but exhibits much less capacity to bind TNP-AMP. ATP displaces TNP-ATP with a Kd (ATP) of 0.46 mM. In the presence of the denaturant urea, P-51 loses most of its binding capacity indicating that structure is important for binding. Consistent with this conclusion, circular dichroism spectroscopy revealed that P-51 has significant secondary structure. Elements of such structure calculated from deconvolution of the circular dichroism spectra compare favorably with those predicted from the program of Chou, P. Y., and Fasman, G. D. (1977) J. Mol. Biol. 115, 135-175. These experiments provide the first direct evidence that the second predicted nucleotide binding fold of CFTR binds ATP and define a 51-amino acid segment within the ∼150-amino acid fold critical for this function. They also indicate that the β and γ phosphate groups of ATP may be important for binding and that the 51-amino acid region studied is not sufficient to catalyze ATP hydrolysis. Finally, as seven different mutations within P-51 are known to cause cystic fibrosis, these studies will be important in future efforts to understand the molecular basis of the disease.",
author = "Ko, {Young Hee} and Thomas, {Philip J.} and Pedersen, {Peter L.}",
year = "1994",
month = "5",
day = "20",
language = "English (US)",
volume = "269",
pages = "14584--14588",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "20",

}

TY - JOUR

T1 - The cystic fibrosis transmembrane conductance regulator

T2 - Nucleotide binding to a synthetic peptide segment from the second predicted nucleotide binding fold

AU - Ko, Young Hee

AU - Thomas, Philip J.

AU - Pedersen, Peter L.

PY - 1994/5/20

Y1 - 1994/5/20

N2 - Previous studies from this laboratory with a 67-amino acid synthetic peptide (P-67) demonstrated directly that the first predicted nucleotide binding fold of the cystic fibrosis transmembrane conductance regulator (CFTR) binds ATP (Thomas, P. J., Shenbagamurthi, P., Ysern, S., and Pedersen, P. L. (1991) Science 251, 555-557). Although mutational analysis within the predicted second nucleotide binding fold indicates that this domain may be functionally important also, direct evidence for nucleotide binding is lacking. Here, we report the design, chemical synthesis, and purification of a 51-amino acid segment (P-51) of the second predicted nucleotide binding fold of CFTR and demonstrate that this peptide binds ATP. P-51 consists of amino acid residues from glutamic acid 1228 through threonine 1278 and contains a motif, GX4GKS, very similar or identical to that found in many nucleotide-binding proteins. The freshly dissolved peptide moves predominantly as a single species upon molecular sieve chromatography and readily binds ATP without eliciting its hydrolysis. P-51 also readily binds the fluorescent ATP analogs TNP-ATP (2′(3′)-0-(2,4,6-trinitrophenyl)-adenosine-5′-triphosphate) and TNP-ADP but exhibits much less capacity to bind TNP-AMP. ATP displaces TNP-ATP with a Kd (ATP) of 0.46 mM. In the presence of the denaturant urea, P-51 loses most of its binding capacity indicating that structure is important for binding. Consistent with this conclusion, circular dichroism spectroscopy revealed that P-51 has significant secondary structure. Elements of such structure calculated from deconvolution of the circular dichroism spectra compare favorably with those predicted from the program of Chou, P. Y., and Fasman, G. D. (1977) J. Mol. Biol. 115, 135-175. These experiments provide the first direct evidence that the second predicted nucleotide binding fold of CFTR binds ATP and define a 51-amino acid segment within the ∼150-amino acid fold critical for this function. They also indicate that the β and γ phosphate groups of ATP may be important for binding and that the 51-amino acid region studied is not sufficient to catalyze ATP hydrolysis. Finally, as seven different mutations within P-51 are known to cause cystic fibrosis, these studies will be important in future efforts to understand the molecular basis of the disease.

AB - Previous studies from this laboratory with a 67-amino acid synthetic peptide (P-67) demonstrated directly that the first predicted nucleotide binding fold of the cystic fibrosis transmembrane conductance regulator (CFTR) binds ATP (Thomas, P. J., Shenbagamurthi, P., Ysern, S., and Pedersen, P. L. (1991) Science 251, 555-557). Although mutational analysis within the predicted second nucleotide binding fold indicates that this domain may be functionally important also, direct evidence for nucleotide binding is lacking. Here, we report the design, chemical synthesis, and purification of a 51-amino acid segment (P-51) of the second predicted nucleotide binding fold of CFTR and demonstrate that this peptide binds ATP. P-51 consists of amino acid residues from glutamic acid 1228 through threonine 1278 and contains a motif, GX4GKS, very similar or identical to that found in many nucleotide-binding proteins. The freshly dissolved peptide moves predominantly as a single species upon molecular sieve chromatography and readily binds ATP without eliciting its hydrolysis. P-51 also readily binds the fluorescent ATP analogs TNP-ATP (2′(3′)-0-(2,4,6-trinitrophenyl)-adenosine-5′-triphosphate) and TNP-ADP but exhibits much less capacity to bind TNP-AMP. ATP displaces TNP-ATP with a Kd (ATP) of 0.46 mM. In the presence of the denaturant urea, P-51 loses most of its binding capacity indicating that structure is important for binding. Consistent with this conclusion, circular dichroism spectroscopy revealed that P-51 has significant secondary structure. Elements of such structure calculated from deconvolution of the circular dichroism spectra compare favorably with those predicted from the program of Chou, P. Y., and Fasman, G. D. (1977) J. Mol. Biol. 115, 135-175. These experiments provide the first direct evidence that the second predicted nucleotide binding fold of CFTR binds ATP and define a 51-amino acid segment within the ∼150-amino acid fold critical for this function. They also indicate that the β and γ phosphate groups of ATP may be important for binding and that the 51-amino acid region studied is not sufficient to catalyze ATP hydrolysis. Finally, as seven different mutations within P-51 are known to cause cystic fibrosis, these studies will be important in future efforts to understand the molecular basis of the disease.

UR - http://www.scopus.com/inward/record.url?scp=0028360128&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0028360128&partnerID=8YFLogxK

M3 - Article

VL - 269

SP - 14584

EP - 14588

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 20

ER -