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Characterization of a Goα Mutant That Binds Xanthine Nucleotides

Yu, Bo and Slepak, Vladlen Z. and Simon, Melvin I. (1997) Characterization of a Goα Mutant That Binds Xanthine Nucleotides. Journal of Biological Chemistry, 272 (29). pp. 18015-18019. ISSN 0021-9258. http://resolver.caltech.edu/CaltechAUTHORS:YUBjbc97

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Abstract

Several GTP binding proteins, including EF-Tu, Ypt1, rab-5, and FtsY, and adenylosuccinate synthetase have been reported to bind xanthine nucleotides when the conserved aspartate residue in the NKXD motif was changed to asparagine. However, the corresponding single Goα mutant protein (D273N) did not bind either xanthine nucleotides or guanine nucleotides. Interestingly, the introduction of a second mutation to generate the Goα subunit D273N/Q205L switched nucleotide binding specificity to xanthine nucleotide. The double mutant protein GoαD273N/Q205L (GoαX) bound xanthine triphosphate, but not guanine triphosphate. Recombinant GoαX (GoαD273N/Q205L) formed heterotrimers with βγ complexes only in the presence of xanthine diphosphate (XDP), and the binding to βγ was inhibited by xanthine triphosphate (XTP). Furthermore, as a result of binding to XTP, the GoαX protein underwent a conformational change similar to that of the activated wild-type Goα. In transfected COS-7 cells, we demonstrate that the interaction between GoαX and βγ occurred only when cell membranes were permeabilized to allow the uptake of xanthine diphosphate. This is the first example of a switch in nucleotide binding specificity from guanine to xanthine nucleotides in a heterotrimeric G protein α subunit.


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Additional Information:©1997 by The American Society for Biochemistry and Molecular Biology, Inc. (Received for publication, March 25, 1997, and in revised form, May 5, 1997) We thank members of the Simon laboratory for helpful discussions and Drs. Lorna Brundage and Tau-Mu Yi for comments on the manuscript. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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Deposited On:21 Dec 2008 02:35
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