The initial proposal of changes In conformation (Hall, A., Self, A., 1986) during nucleotide exchange {upper panel) suggest that an activated receptor (R*) binds to a resting Ras»GDP*Mg^* complex (1), which results In the loss of bound Mg^* (2). This complex now takes on a new open conformation, which can freely exchange with GTP (3). The binding of Mg^^ to the R*— Ras»GTP complex results In a conformational change of Ras which has high affinity for the Mg^^ and GTP, but low affinity for R* (4). The final complex Is able to Interact with effector molecules and undergo GTP hydrolysis.
Further functional and structural Information has enabled a more refined model (lower
panel), where the GDP«bound state of Ras binds to an activated GEF. The GEF causes
a destabilisation of the low affinity Mg^* binding coordination, allowing Mg^* release followed by nucleotide release. The GEF Is then able to stabilise Ras In a nucleotlde- free form which represents a cation free conformation and Is the structural transition between the low affinity Mg^* form of Ras and the high affinity binding form as Ras. On Mg** and GTP bjjglng with Ras, Ras takes on a high affinity conformation, which causes the release of the GEF, allowing Ras to Interact with effectors and undergo GTP hydrolysis.
It Is clear that this form of Interaction with GEFs Is a feature of the entire small G-proteIn subfamilies, such as Rho, Ras and Rab.
1 Ras*GDP«Mg^^ + R’ [R*— Ras*GDP»Mg21
[R*— Ras*GDP*Mg^^] R*— Ras*GDP + Mg2+
R*— Ras*GDP GTP. [R*—Ras'GTP] + GDP
4 [R*—Ras‘ GTP] Ras*«GTP«Mg
Figure 10a The simple model of GEF-mediated Nucleotide Exchange
+ R*
Low Affinity
Mg“* State
Ras»Mg^*»GDP GEF'Ras'GDP GEF'Ras GDPHigh Affinity
Mg'* State
Ras»Mg'*»GTP à à GEF GEF f GTP f GEF»Ras»GTPNucieotide
Free State
Figure 10b The refined model of GEF-mediated Nucleotide Exchange
acts as a GEF for the a-subunit). The GEF domains are all composed of a-helices, but their number and arrangement are all different (the Dbl domain contains 11 a-helices in a flattened bundle whereas the Cdc25 domain of Sos consists of 10 helices), however the mechanisms by which they induce nucleotide exchange do show some similarity.
Once again Ras subfamily proteins provide a model which can be contrasted to other members of the family, including the Rho subfamily.
The Ras-Sos complex (Corbalan-Garcia, S., et al 1998) shows a large area of interaction between Sos and the P Loop, Switch I, Switch II and a short area downstream of Switch II. The interaction between Switch II and Sos causes the Switch II region to become ordered in comparison to Ras •GDP’s Switch II structure. Sos forces part of its Helix H between the main body of the protein and Switch I, which pushes it away from Switch II and the active site (P Loop). A Leu, Glu and Ala residue enter the nucleotide and Mg^'^ binding sites, salt bridges are made with Ras residues involved in nucleotide binding, and part of Switch II is restructured. The result is that the active site is no longer compatible with binding nucleotide and its dissociation is encouraged from the exposed active site. The structure of Dbl (Liu, X., et al 1998; Soisson, S., et a l 1998) suggests that it uses a similar mechanism, by thrusting a conserved Rac/Rho binding helix in-between Switch I and the remainder of the protein, although the specific residue interactions are different. The PH domain and linker segments of Dbl have also been shown to act as part of the interface between Dbl and the G-protein, and that the presence of the PH domain increases the overall GEF activity of the protein.
Although the changes in the active site caused by the action of a GEF result in an inability to bind nucleotide, the disruption of the Mg^'^ coordination coupled with a stabilising of the nucleotide and Mg^"^ free state may act as the key elements to accelerating exchange.
The structure of the Mg^'^ binding site within the G Domain have already been described, but briefly, in the GDP-bound state of Ras Mg^"^ is coordinated by the P-phosphate of GDP, a conserved Ser/Thr residue and indirectly with other residues via four HgO molecules. The Mg^"^ found associated with the nucleotide in Rho subfamily members is coordinated by two Thr residues directly, the p-phosphate and three, rather than four, water molecules (Figure 7). The Mg^^ ion is essential for both normal nucleotide binding and hydrolysis, and mutations of the conserved Ser/Thr 17, Asp 57 and T35 (in Ras) (John,]., et al 1993) show increased rates of nucleotide release in both GDP and GTPyS forms, and no longer inhibit nucleotide dissociation by high concentrations of Mg^^, suggesting that the Mg^"^ is important in both the GDP and GTP bound state. However, studies with Dbl show that GDP dissociation does occur in the absence of Mg^'^ (Zhang, B., et a l 2000), suggesting that displacement ofMg^"^ is only part of the effect RhoGEFs have on Rho proteins.
Early studies on Ras proteins (Self, A. and Hall, A., 1986) showed that nucleotide exchange in the absence of Mg^^ was around 40-fold slower than in the presence of Mg^"^. The presence of Mg^^ also increased the binding affinity for GTP as compared to GDP by 10-fold. These data suggested that Ras existed in at least two conformations: closed, where exchange occurred very slowly, and open allowing free exchange of nucleotides, yet having
a greater affinity for GTP. The model proposed by this study can be seen in Figure 10.
More recent studies on the protein Rab5 (Pan, J., et al 1996) and the recent Mg^^ free structure(Shimizu, T., et a l 2000) have shown that Mg^^ binding affects both nucleotide dissociation and small G-protein conformation, suggesting that Mg^'^’s cff-
inhibits GDP dissociation by a chemical affinity to the nucleotide and by imposing structural constraints on the protein molecule. Furthermore, the action of GEF in promoting nucleotide exchange may well be upon the Mg^"^ rather than the nucleotide directly. The Mg^^ may not only impose chemical restraints on the bound nucleotide, but also it could help promote distinct nucleotide-bound structural states, which would suggest the presence of a nucleotide-free state which would lack Mg^^ and gain its stabilised conformation from association with a GEF molecule (Figure !(?). The distinct states of bound nucleotide, GDP- bound, nucleotide-free and GTP'yS-bound forms have been observed through tryptophan fluorescence (Simon, I., etal 1996) in Rab 5 and Rab7, supporting this model.
Once the GEF-nucleotide free state is formed, the binding of GTP into the small G-protein GtP
is more likely than binding of GDP since not only isj[in 30 fold excess in the cell, but the affinity of GTP-bound complexes for Mg^"^ are at least 10 fold greater than that of the GDP- bound form (Simon, I., et a l 1996; John, J. et a l 1993; Self, A. and Hall, A. 1986). The inability of certain Rab mutants to adopt the GTP-bound form suggest that correct Mg^"^ coordination is required to promote further structural rearrangements t) reach the fully GTP-bound state.