GEF-mediated GDP/GTP exchange by monomeric GTPases: A regulatory role for Mg2+?

Kinetics of Interaction between ADP-ribosylation Factor-1 (Arf1) and the Sec7 Domain of Arno Guanine Nucleotide Exchange Factor, Modulation by Allosteric Factors, and the Uncompetitive Inhibitor Brefeldin A

“…Unfortunately, in contrast with the study of Lenzen et al (44) based on immobilized Ras and flowing Cdc25 Mm285 , it appears that the immobilization of Arf1 could interfere with the formation of a high affinity complex and cause a 10-fold decrease of the association rate constant (2.66 ϫ 10 Table 6). The absence of free Mg 2ϩ decreases the GDP and GTP binding affinity by destabilizing the coordination of the nucleotide binding site and promotes the formation of the binary G protein-GEF complex (10,12). In terms of kinetic parameters of binding, our data revealed that Mg 2ϩ ion potentiates by a factor of 2 the counteracting allosteric effect of GDP on the binding of Arf1 to immobilized Sec7 domain, as the absence of ions causes a ϳ2-fold reduction of the association rate constant (Tables 4 and 5).…”

“…Moreover, crystallographic and biological studies revealed that nucleotide binding on G proteins presents a highly conserved coordination of Mg 2ϩ within the site, and disrupting the Mg 2ϩ coordination (for instance by a GEF) promotes a nucleotidefree state of the G proteins (10). The cation strengthens the nucleotide binding and reduces its dissociation rate, thus weakening binding of the GEF to the G protein (11)(12)(13).…”

“…As shown in Table 2, a decrease of the experimental temperature from 25 to 10°C conducts to a progressive loss of in the SR value. Whereas SPR experiments performed at pH 7 or in the presence of 300 mM NaCl revealed that initial conditions conferred the highest association rate (k a on of free Mg 2ϩ on kinetic parameters of the interaction between [⌬17]Arf1 and the Sec7 domain, as the removal of the cation accelerates by a factor of 20 the spontaneous nucleotide departure, giving the binary G protein-GEF complex (10,36).…”

Alternative Splicing of Rac1 Generates Rac1b, a Self-activating GTPase

“…p50 ion is in turquoise. C, structural comparison of the P-loop and switch I regions of Rac1b⅐GppNHp (red), Rac1⅐GppNHp (brown) (17), and the nucleotide-free Rac1 in complex with the DH-PH domain of Tiam1 (green) (42 fulfill their regulatory function in the cell. This and the fact that Rac1b is, without external stimuli, GTP-bound in COS7 cells ( Fig.…”

A disappearing act performed by magnesium: the nucleotide exchange mechanism of Ran GTPase by quantum mechanics/molecular mechanics studies

“…In attempting to understand the exchange process adopted by Ran, it is worth considering the previous studies regarding nucleotide exchange in other Ras superfamily members. Particularly, destabilization of magnesium coordination was strongly implied in the literature which was deemed as a determining factor in the exchange process [13][14][15]. Despite this, there appears to be significant diversity in the mechanistic action.…”