Characterization of a Ras mutant with identical GDP- and GTP-bound structures

Bradley Ford; Sean Boykevisch; Chen Zhao; Simone Kunzelmann; Dafna Bar-Sagi; Christian Herrmann; Nicolas Nassar

doi:10.1021/bi901479b

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Characterization of a Ras mutant with identical GDP- and GTP-bound structures

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Characterization of a Ras mutant with identical GDP- and GTP-bound structures

Bradley Ford, Sean Boykevisch, Chen Zhao, Simone Kunzelmann, Dafna Bar-Sagi, Christian Herrmann and Nicolas Nassar

Biochemistry (Easton), Vol.48(48), pp.11449-11457

12/08/2009

DOI: 10.1021/bi901479b

PMCID: PMC4160238

PMID: 19883123

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Abstract

We previously characterized the G60A mutant of Ras and showed that the switch regions of the GTP- but not the GDP-bound form of this mutant adopt an ‘open conformation’ similar to that seen in nucleotide free Ras. Here, we mutate Lys147 of the conserved 145 SAK 147 motif in the G60A background and characterize the resulting double mutant (DM). We show that RasDM is the first structure of a Ras protein with identical GDP- and GTP-bound structure. Both structures adopt the open conformation of the active form of RasG60A. The increase in the accessible surface area of the nucleotide is consistent with a four-fold increase in its dissociation rate. Stopped flow experiments show no major difference in the two-step kinetics of GDP or GTP association to wild type, G60A, or RasDM. Addition of Sos fails to accelerate nucleotide exchange. Overexpression of the G60A or the double mutant of Ras in COS-1 cells fails to activate Erk and shows a strong dominant negative effect. Our data suggest that flexibility at position 60 is required for proper Sos-catalyzed nucleotide exchange and that structural information is somehow shared among the switch regions and the different nucleotide binding motifs.

X-ray crystallography

site directed mutagenesis

Ras

guanine nucleotide exchange

Details

Title: Subtitle: Characterization of a Ras mutant with identical GDP- and GTP-bound structures
Creators: Bradley Ford - Department of Physiology and Biophysics, Stony Brook University, Stony Brook, NY 11794-8661
Sean Boykevisch - Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794-5222 - USA
Chen Zhao - Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794-5222 - USA
Simone Kunzelmann - Ruhr-Universität Bochum, Physikalische Chemie I, Fakultät für Chemie, Universitätsstraße 150, 44780 Bochum – Germany
Dafna Bar-Sagi - Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794-5222 - USA
Christian Herrmann - Ruhr-Universität Bochum, Physikalische Chemie I, Fakultät für Chemie, Universitätsstraße 150, 44780 Bochum – Germany
Nicolas Nassar - Department of Physiology and Biophysics, Stony Brook University, Stony Brook, NY 11794-8661
Resource Type: Journal article
Publication Details: Biochemistry (Easton), Vol.48(48), pp.11449-11457
DOI: 10.1021/bi901479b
PMID: 19883123
PMCID: PMC4160238
NLM abbreviation: Biochemistry
ISSN: 0006-2960
eISSN: 1520-4995
Language: English
Date published: 12/08/2009
Academic Unit: Pathology
Record Identifier: 9984047700902771

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