Characterization of a Ras Mutant with Identical GDP- and GTP-Bound Structures,

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• 作者：Bradley Ford ; Sean Boykevisch ; Chen Zhao ; Simone Kunzelmann ; Dafna Bar-Sagi ; Christian Herrmann ; Nicolas Nassar
• 刊名：Biochemistry
• 出版年：2009
• 出版时间：December 8, 2009
• 年：2009
• 卷：48
• 期：48
• 页码：11449-11457
• 全文大小：942K
• 年卷期：v.48,no.48(December 8, 2009)
• ISSN：1520-4995

文摘

We previously characterized the G60A mutant of Ras and showed that the switch regions of the GTP-bound 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 ¹⁴⁵SAK¹⁴⁷ 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 structures. 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 4-fold increase in its dissociation rate. Stopped-flow experiments show no major difference in the two-step kinetics of association of GDP or GTP with the wild type, G60A, or RasDM. Addition of Sos fails to accelerate nucleotide exchange. Overexpression of the G60A or 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.