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Fusidic acid (FA) and helvolic acid (HA) belong to a small family of naturally occurring steroidal antibiotics known as fusidanes. FA was studied for its ability to alter the biochemical properties supported by elongation factor 2 isolated from the archaeon Sulfolobus solfataricus (SsEF-2). Both poly(Phe) synthesis and ribosome-dependent GTPase (GTPase(r)) were progressively impaired by increasing concentrations of FA up to 1 mM, whereas no effect was measured in the intrinsic GTPase of SsEF-2 triggered by ethylene glycol in the presence of barium chloride (GTPase(g)). The highest antibiotic concentration caused inhibition of either poly(Phe) synthesis or GTPase(r) only slightly above 50%. A greater response of SsEF-2 was observed when HA was used instead of FA. HA caused even a weak impairment of GTPase(g). A mutated form of SsEF-2 carrying the L452R substitution exhibited an increased sensitivity to fusidane inhibition in either poly(Phe) synthesis or GTPase(r). Furthermore, both FA and HA were able to cause impairment of GTPase(g). The antibiotic concentrations leading to 50% inhibition (IC(50)) indicate that increased fusidane responsiveness due to the use of HA or the L452R amino acid replacement is mutually independent. However, their combined effect decreased the IC(50) up to 0.1 mM. Despite the difficulties in reaching complete inhibition of the translocation process in S. solfataricus, these findings suggest that fusidane sensibility is partially maintained in the archaeon S. solfataricus. Therefore, it is likely that SsEF-2 harbors the structural requirements for forming complexes with fusidane antibiotics. This hypothesis is further evidenced by the observed low level of impairment of GTPase(g), a finding suggesting a weak direct interaction between the archaeal factor and fusidanes even in the absence of the ribosome. However, the ribosome remains essential for the sensitivity of SsEF-2 toward fusidane antibiotics.

Valine 114 in the D(109)AAILVVA sequence of elongation factor 1alpha from the archaeon Sulfolobus solfataricus (SsEF-1alpha) was substituted with an acidic (V114E), basic (V114K), or cavity-forming (V114A) residue, and the effects on the biochemical properties of the factor were investigated. This sequence is well-conserved among most of eukaryal and eubacterial counterparts, and in the three-dimensional structure of SsEF-1alpha, V114 is located in a hydrophobic pocket near the first GDP-binding consensus sequence G(13)XXXXGK[T,S] [Vitagliano, L., Masullo, M., Sica, F., Zagari, A., and Bocchini, V. (2001) EMBO J. 20, 5305-5311]. These mutants displayed functions absent in the wild-type factor. In fact, although they exhibited a rate in poly(Phe) incorporation almost identical to that of SsEF-1alpha, V114K and V114A exhibited an affinity for GDP and GTP higher and a capability to bind heterologous aa-tRNA stronger than that elicited by SsEF-1alpha but similar to that of eubacterial EF-Tu. V114E instead displayed not only a weaker binding capability for aa-tRNA but also a lower affinity for GDP. The intrinsic GTPase activity of V114E was drastically reduced compared to those of SsEF-1alpha, V114K, and V114A. Interestingly, the decreased intrinsic GTPase activity of V114E was partially restored by kirromycin, an effect already observed for the G13A mutant of SsEF-1alpha [Masullo, M., Cantiello, P., de Paola, B., Catanzano, F., Arcari, P., and Bocchini, V. (2002) Biochemistry 41, 628-633]. Finally, the V114A substitution showed only a marginal effect on both the thermostability and thermophilicity of SsEF-1alpha, whereas V114K and V114E replacements strongly destabilized the molecule.

The G13A substitution in the G13XXXXGK[T,S] consensus sequence of the elongation factor 1 alpha from the archaeon Sulfolobus solfataricus (SsEF-1 alpha) was introduced in order to study the reasons for selective differences found in the homologous consensus element AXXXXGK[T,S] of the other elongation factor EF-2 or EF-G. In a previous work, it was shown that the main effect of the A26G mutation was the activation of the intrinsic GTPase of SsEF-2 [De Vendittis, E., Adinolfi, B. S., Amatruda, M. R., Raimo, G., Masullo, M., and Bocchini, V. (1994) Eur. J. Biochem. 262, 600-605]. In this work, we found that, compared to the wild-type factor (SsEF-1 alpha wt), G13ASsEF-1 alpha shows (i) a reduced rate of [(3)H]Phe polymerization that was probably due to its reduced ability to form a ternary complex with heterologous aa-tRNA and (ii) a reduced intrinsic GTPase activity that was stimulated by high concentrations of NaCl (GTPase(Na)) [Masullo, M., De Vendittis, E., and Bocchini, V. (1994) J. Biol. Chem. 269, 20376-20379]. In addition, G13ASsEF-1 alpha showed an increased affinity for GDP and GTP. Surprisingly, the decreased intrinsic GTPase(Na) of G13ASsEF-1 alpha can be partially restored by kirromycin, an effect not found for SsEF-1 alpha wt. The temperature inducing a 50% denaturation of G13ASsEF-1 alpha was somewhat lower (-5 degrees C) than that of SsEF-1 alpha wt, and the decrease in its thermophilicity was slightly more accentuated (-10 degrees C). These results indicate that the nature of the residue in position 13 is important for the functional and physical properties of SsEF-1 alpha.

The elongation factor Tu was isolated from a psychrophilic eubacterial Antarctic Moraxella strain (MoEF-Tu) and its molecular and functional properties were determined. It catalyzed the synthesis of poly(Phe) and bound specifically guanine nucleotides with an affinity for GDP about 12-fold higher than that for GTP. The affinity toward guanine nucleotides was lower than that of other eubacterial EF-Tu. The intrinsic GTPase activity of MoEF-Tu was hardly detectable but was accelerated by 2 orders of magnitude in the presence of the antibiotic kirromycin (GTPase(k)). Such a property resembled Escherichia coli EF-Tu (EcEF-Tu) even though the affinity of MoEF-Tu for the antibiotic was lower. MoEF-Tu showed a thermophilicity higher than that of EcEF-Tu; its temperature for half-denaturation was 44 degrees C. The MoEF-Tu encoding gene corresponding to E. coli tufA was cloned and sequenced. The translated protein had a calculated molecular weight of 43 288 and contained the GTP-binding sequence motifs. Concerning its primary structure, MoEF-Tu showed sequence identity with E. coli and Thermus thermophilus EF-Tu equal to 84% and 74%, respectively, while the identity with EF-1 alpha from the archaeon Sulfolobus solfataricus was equal to 32%.

A recombinant chimeric elongation factor containing the region of EF-1 alpha from Sulfolobus solfataricus harboring the site for GDP and GTP binding and GTP hydrolysis (SsG) and domains M and C of Escherichia coli EF-Tu (EcMC) was studied. SsG-EcMC did not sustain poly(Phe) synthesis in either S. solfataricus or E. coli assay system. This was probably due to the inability of the chimera to interact with aa-tRNA. The three-dimensional modeling of SsG-EcMC indicated only small structural differences compared to the Thermus aquaticus EF-Tu in the ternary complex with aa-tRNA and GppNHp, which did not account for the observed inability to interact with aa-tRNA. The addition of the nucleotide exchange factor SsEF-1 beta was not required for poly(Phe) synthesis since the chimera was already able to exchange [(3)H]GDP for GTP at very high rate even at 0 degrees C. Compared to that of SsEF-1 alpha, the affinity of the chimera for guanine nucleotides was increased and the k(cat) of the intrinsic GTPase was 2-fold higher. The heat stability of SsG-EcMC was 3 and 13 degrees C lower than that displayed by SsG and SsEF-1alpha, respectively, but 30 degrees C higher than that of EcEF-Tu. This pattern remained almost the same if the melting curves of the proteins being investigated were considered instead. The chimeric elongation factor was more thermophilic than SsG and SsEF-1 alpha up to 70 degrees C; at higher temperatures, inactivation occurred.

Guanidine-induced denaturation of Sulfolobus solfataricus beta-glycosidase expressed in Escherichia coli, Sbetagly, was investigated at pH 6.5 and 25 degreesC by means of circular dichroism and fluorescence measurements. The process proved reversible when the protein concentration was lower than 0.01 mg mL-1. Moreover, the transition curves determined by fluorescence did not coincide with those determined by circular dichroism, and the GuHCl concentration corresponding at half-completion of the transition increased on raising the protein concentration in the range 0.001-0.1 mg mL-1. Gel filtration chromatography experiments showed that, in the range 2-4 M GuHCl, there was an equilibrium among tetrameric, dimeric, and monomeric species. These findings, unequivocally, indicated that the guanidine-induced denaturation of Sbetagly was not a two-state transition with concomitant unfolding and dissociation of the four subunits. A mechanism involving a dimeric intermediate species was proposed and was able to fit the experimental fluorescence intensity transition profiles, allowing the estimation of the total denaturation Gibbs energy change at 25 degreesC and pH 6.5. This figure, when normalized for the number of residues, showed that, at room temperature, Sbetagly has a stability similar to that of mesophilic proteins.