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  Nucleotide-binding characteristics of human guanylate-binding protein 1 (hGBP1) and identification of the third GTP-binding motif

Praefcke, G. J. K., Geyer, M., Schwemmle, M., Kalbitzer, H. R., & Herrmann, C. (1999). Nucleotide-binding characteristics of human guanylate-binding protein 1 (hGBP1) and identification of the third GTP-binding motif. Journal of Molecular Biology (London), 292(2), 321-332. doi:10.1006/jmbi.1999.3062.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0024-59A7-6 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0024-59B4-8
Genre: Journal Article
Alternative Title : Nucleotide-binding characteristics of human guanylate-binding protein 1 (hGBP1) and identification of the third GTP-binding motif

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JMolBiol_292_1999_321.pdf (Any fulltext), 264KB
 
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Praefcke, Gerrit J. K., Author
Geyer, Matthias1, Author              
Schwemmle, Martin, Author
Kalbitzer, Hans Robert1, Author              
Herrmann, Christian, Author
Affiliations:
1Emeritus Group Biophysics, Max Planck Institute for Medical Research, Max Planck Society, ou_1497712              

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Free keywords: GTP-binding protein; aluminium fluoride; interferon; titration; kinetics
 Abstract: hGBP1 is a GTPase with antiviral activity encoded by an interferon- activated human gene. Specific binding of hGBP1 to guanine nucleotides has been established although only two classical GTP-binding motifs were found in its primary sequence. The unique position of hGBP1 amongst known GTPases is further demonstrated by the hydrolysis of GTP to GDP and GMP. Although subsequent cleavage of orthophosphates rather than pyrophosphate was demonstrated, GDP coming from bulk solution cannot serve as a substrate. The relation of guanine nucleotide binding and hydrolysis to the antiviral function of hGBP1 is unknown. Here we show similar binding affinities for all three guanine nucleotides and the ability of both products, GDP and GMP, to compete with GTP binding. Fluorimetry and isothermal titration calorimetry were applied to prove that only one nucleotide binding site is present in hGBP1. Furthermore, we identified the third canonical GTP-binding motif and verified its role in nucleotide recognition by mutational analysis. The high guanine nucleotide dissociation rates measured by stopped-flow kinetics are responsible for the weak affinities to hGBP1 when compared to other GTPases like Ras or Gsmall alpha, Greek. By means of fluorescence and NMR spectroscopy it is demonstrated that aluminium fluoride forms a complex with hGBP1 only in the GDP state, presumably mimicking the transition state of GTP hydrolysis. Tentatively, the involvement of a GAP domain in hGBP1 in GTP hydrolysis is suggested. These results will serve as a basis for the determination of the differential biological functions of the three nucleotide states and for the elucidation of the unique mechanism of nucleotide hydrolysis catalysed by hGBP1

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Language(s): eng - English
 Dates: 1999-07-201999-05-071999-07-211999-09-17
 Publication Status: Published in print
 Pages: 12
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Degree: -

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Title: Journal of Molecular Biology (London)
  Other : J Mol Biol
Source Genre: Journal
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Publ. Info: London : Academic Press
Pages: - Volume / Issue: 292 (2) Sequence Number: - Start / End Page: 321 - 332 Identifier: ISSN: 0022-2836
CoNE: https://pure.mpg.de/cone/journals/resource/954922646042