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Title: Probing the ATP Site of GRP78 with Nucleotide Triphosphate Analogs

Authors:
; ; ; ; ; ;
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
FOREIGN
OSTI Identifier:
1257482
Resource Type:
Journal Article
Resource Relation:
Journal Name: PLoS ONE; Journal Volume: 11; Journal Issue: 5
Country of Publication:
United States
Language:
ENGLISH

Citation Formats

Hughes, Scott J., Antoshchenko, Tetyana, Chen, Yun, Lu, Hua, Pizarro, Juan C., Park, Hee-Won, and Silman, Israel. Probing the ATP Site of GRP78 with Nucleotide Triphosphate Analogs. United States: N. p., 2016. Web. doi:10.1371/journal.pone.0154862.
Hughes, Scott J., Antoshchenko, Tetyana, Chen, Yun, Lu, Hua, Pizarro, Juan C., Park, Hee-Won, & Silman, Israel. Probing the ATP Site of GRP78 with Nucleotide Triphosphate Analogs. United States. doi:10.1371/journal.pone.0154862.
Hughes, Scott J., Antoshchenko, Tetyana, Chen, Yun, Lu, Hua, Pizarro, Juan C., Park, Hee-Won, and Silman, Israel. 2016. "Probing the ATP Site of GRP78 with Nucleotide Triphosphate Analogs". United States. doi:10.1371/journal.pone.0154862.
@article{osti_1257482,
title = {Probing the ATP Site of GRP78 with Nucleotide Triphosphate Analogs},
author = {Hughes, Scott J. and Antoshchenko, Tetyana and Chen, Yun and Lu, Hua and Pizarro, Juan C. and Park, Hee-Won and Silman, Israel},
abstractNote = {},
doi = {10.1371/journal.pone.0154862},
journal = {PLoS ONE},
number = 5,
volume = 11,
place = {United States},
year = 2016,
month = 9
}
  • GRP78, a member of the ER stress protein family, can relocate to the surface of cancer cells, playing key roles in promoting cell proliferation and metastasis. GRP78 consists of two major functional domains: the ATPase and protein/peptide-binding domains. The protein/peptide-binding domain of cell-surface GRP78 has served as a novel functional receptor for delivering cytotoxic agents (e.g., a apoptosis-inducing peptide or taxol) across the cell membrane. Here, we report our study on the ATPase domain of GRP78 (GRP78 ATPase), whose potential as a transmembrane delivery system of cytotoxic agents (e.g., ATP-based nucleotide triphosphate analogs) remains unexploited. As the binding of ligandsmore » (ATP analogs) to a receptor (GRP78 ATPase) is a pre-requisite for internalization, we determined the binding affinities and modes of GRP78 ATPase for ADP, ATP and several ATP analogs using surface plasmon resonance and x-ray crystallography. The tested ATP analogs contain one of the following modifications: the nitrogen at the adenine ring 7-position to a carbon atom (7-deazaATP), the oxygen at the beta-gamma bridge position to a carbon atom (AMPPCP), or the removal of the 2'-OH group (2'-deoxyATP). We found that 7-deazaATP displays an affinity and a binding mode that resemble those of ATP regardless of magnesium ion (Mg ++) concentration, suggesting that GRP78 is tolerant to modifications at the 7-position. By comparison, AMPPCP's binding affinity was lower than ATP and Mg ++-dependent, as the removal of Mg ++ nearly abolished binding to GRP78 ATPase. The AMPPCP-Mg ++ structure showed evidence for the critical role of Mg ++ in AMPPCP binding affinity, suggesting that while GRP78 is sensitive to modifications at the β-γ bridge position, these can be tolerated in the presence of Mg ++. Furthermore, 2'-deoxyATP's binding affinity was significantly lower than those for all other nucleotides tested, even in the presence of Mg ++. The 2'-deoxyATP structure showed the conformation of the bound nucleotide flipped out of the active site, explaining the low affinity binding to GRP78 and suggesting that the 2'-OH group is essential for the high affinity binding to GRP78. Altogether, our results demonstrate that GRP78 ATPase possesses nucleotide specificity more relaxed than previously anticipated and can tolerate certain modifications to the nucleobase 7-position and, to a lesser extent, the beta-gamma bridging atom, thereby providing a possible atomic mechanism underlying the transmembrane transport of the ATP analogs.« less
  • The plasma membrane of yeasts contains an H+-ATPase similar to the other cation transport ATPases of eukaryotic organisms. This enzyme has been purified and shows H+ transport in reconstituted vesicles. In the presence of Mg2+, formycin triphosphate (FTP) is hydrolyzed by the H+-ATPase and supports H+ transport. When combined with terbium ion, FTP (Tb-FTP) and ATP (Tb-ATP) are no longer hydrolyzed. Competition between Mg-ATP and Tb-FTP for ATP hydrolysis indicates that terbium-associated nucleotides bind to the catalytic site of the H+-ATPase. The fluorescent properties of the Tb-FTP complex were used to study the active site of the H+-ATPase. Fluorescence ofmore » Tb-FTP is greatly enhanced upon binding into the nucleotide site of H+-ATPase with a dissociation constant of 1 microM. Tb-ATP, Tb-ADP, and Tb-ITP are competitive inhibitors of Tb-FTP binding with Ki = 4.5, 5.0, and 6.0 microM, respectively. Binding of Tb-FTP is observed only in the presence of an excess of Tb3+ with an activation constant Ka = 25 microM for Tb3+. Analysis of the data reveals that the sites for Tb-FTP and Tb3+ binding are independent entities. In standard conditions these sites would be occupied by Mg-ATP and Mg2+, respectively. These findings suggest an important regulatory role of divalent cations on the activity of H+-ATPase. Replacement of H/sub 2/O by D/sub 2/O in the medium suggests the existence of two types of nucleotide binding sites differing by the hydration state of the Tb3+ ion in the bound Tb-FTP complex.« less
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