GTPases oscillate between their GTP- and GDP-bound states via regulated cycles of GTP hydrolysis and exchange of GDP for GTP. The interaction between the GTPase and the G nucleotide is mediated by switch 1 and 2 (sw 1 and sw 2) regions of the GTPase domain. The nature of the bound nucleotide is believed to be the main factor regulating the functional state of the protein, regulating its on / off modes: off in the apo and GDP-bound state, and on in the GTP-bound state.

We used isothermal titration calorimetry to investigate the GTPase cycle of a number of GTPases involved in translation (trGTPases): IF2, EF-G, and eRF3. For these we determined K_d and thus Gibbs energy (ΔG⁰), enthalpy (ΔH⁰), entropy (ΔS⁰) and change in heat capacity (ΔC_p= d(ΔH)/dT) of the interaction between the protein and the G nucleotides. The last parameter (ΔC_p) is directly proportional to change in the solvent accessible area of the protein and thus reflects the extent of the structural rearrangement, bridging the gap between the physical chemistry and structural biology descriptions of the system.

Our ΔC_p data suggest that in the case of EF-G, GTP binding promotes ordering of the sw 1 and sw 2 regions, but GDP does not (1) (Fig. 1) as indicated by the 250 cal·mol^-1·K^-1 ΔC_p difference in GDP and GTP binding. In the IF2 case, in addition to the GTP-mediated ordering of the sw 1 and sw 2 regions that cause the 290 cal·mol^-1·K^-1 difference between GDP and GTP binding, both GTP and GDP promote a large-scale rearrangement in the protein, suggested by the large ΔC_p that binding of either nucleotide causes (Fig. 1). Finally, in the eRF3 case, binding of eRF1 is necessary for GTP binding (2), which promotes a large-scale rearrangement of the eRF1:eRF3 complex (Fig. 1).

The results presented here demonstrate that despite the common functional cycle, different trGTPases do have significant differences in the way they are regulated by G nucleotides.


Figure 1: Enthalpy of binding of GDP (empty circles) and GTP (filled circles) to trGTPases as a function of the temperature (°C) at pH 7.5.

References and Footnotes

1. Hauryliuk, V. et al. Proc Natl Acad Sci USA 105, 15678-15683 (2008).
2. Hauryliuk, V. et al. Biochimie 88, 747-757 (2006).