Thermostability of Elongation Factor EF-Tu

To study the strategy of thermostabilization of the three-domain EF-Tu proteins we applied a chimerization approach first to examine the contribution of individual domains to the stability of the proteins. For this purpose, six recombinant chimeric forms of EF-Tu, composed of combinations of mesophilic (E. coli, Ec) and thermophilic (B. stearothermophilus, Bst)  EF-Tu domains, were prepared together with isolated recombinant domain 1 of both EF-Tus (Fig. 1). Domain 1 of EF-Tus is also called the G-domain because it binds GDP and GTP and has a GTPase activity. It is the functional module of the protein. Their binding, enzymatic and thermostability parameters were determined and compared with those of the parental EF-Tus.

Fig. 1 Composition of the constructed proteins. E. coli EF-Tu portions are shown in blue and those of B. stearothermophilus are shown in red. (CH = chimera)

Thermostability of all ten proteins was determined both functionally, as the preservation, at increasing temperature, of their functions (GDP/GTP binding and GTPase activity) and structurally, as the preservation, at increasing temperature, of their a-helix content (measured by the CD spectroscopy). 

Guanine-nucleotide binding experiments revealed that both EF-Tus as well as their chimeric forms bind GDP and GTP with a very similar affinity, which is about 100 times stronger for GDP than for GTP. This also applied to the isolated BstG-domain, it possessed affinities for GDP and GTP quite comparable with those of the whole EF-Tu. In contrast, the EcG-domain affinity for GDP and GTP was about 1000 times and 20 times, respectively, lower than were those of EcEF-Tu. These data imply that in BstEF-Tu the high and differential affinity for GDP and GTP appears to be intrinsic to the G-domain itself, whereas in EcEF-Tu the interaction of all three domains is required to establish this phenotype (Table 1).

Table I. Kinetic parameters of nucleotide interaction of E. coli and B. stearothermophilus EF-Tus and G-domains determined at 0°C.

Thermal stabilization of either EF-Tu and their chimeric forms was found to be consistent with a mechanism involving co-operative contributions from all three protein domains. The G-domains were the main determinants of the thermal stabilization, they set up a “basic“ level of thermostability of either EF-Tu. It was below the growth temperature optimum of the respective organism but about 20°C higher with the BstG-domain than with the EcG-domain (Fig. 2) or fully consistent with the growth temperature optimum difference between both bacteria. It appears that the higher thermostability of the BstG-domain as compared to the EcG-domain could be attributed to two distinct structural features of the BstG-domain: (i) an increase, particularly on the G-domain surface, of charged residues at the expense of polar, uncharged residues (CvP bias) (Fig. 3) and (ii) a decrease in the nonpolar solvent-accessible surface area. This hypothesis is being tested experimentally.

Fig. 2 Heat-inactivation profiles of GDP  forms of E. coli (▲) and B. stearothermophilus (▲) EF-Tus in comparison to those of E. coli (♦) and B. stearothermophilus (♦) G-domains.

Fig. 3 Charged amino acid residues in the G-domain of EF-Tu of B. stearothermophilus newly introduced or different from those in the G-domain of EF-Tu of E. coli. This figure was made using DS ViewerPro program.

The thermostabilizing contributions of domains 2+3 of both E.coli and B. stearothermophilus were similar, only a little influenced by their origin. They consisted in further increasing the thermal stability of a-helical regions of the G-domains up to the level of the respective growth temperature optima of either organism. The stabilization effect of Ecdomains 2+3 was on either G-domain in the GTP-bound form considerably lower than in the GDP-bound form. No such difference was observed with Bstdomains 2+3 (Fig. 4).

       
Fig. 4 Comparison of temperature-induced inactivation profiles of GDP and GTP forms of  E. coli and B. stearothermophilus EF-Tus (A) and chimaeric EF-Tus (B). Effect of exchange of domains 2+3.

Analysis of the GTPase activity and GTPase temperature optimum of individual EF-Tu derivatives and the G-domains significantly complemented the results obtained in the GDP- and GTP-binding and CD experiments. The results imply that EcEF-Tu and BstEF-Tu employ a different G-domain and G-domain-domains 2+3 interaction strategies to establish the same phenotype. The findings extend the list of differences in protein synthesis regulatory mechanisms between G-negative and G-positive bacteria.

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