G13A Substitution Affects the Biochemical and Physical Properties of the Elongation Factor 1R. A Reduced Intrinsic GTPase Activity Is Partially Restored by Kirromycin ² Mariorosario Masullo, ‡,§ Piergiuseppe Cantiello, § Barbara de Paola, § Francesca Catanzano, | Paolo Arcari,* ,§ and Vincenzo Bocchini §,⊥ Dipartimento di Scienze Farmacobiologiche, UniVersita ` degli Studi di Catanzaro Magna Graecia, Complesso Ninı ` Barbieri, Roccelletta di Borgia, I-88021 Catanzaro, Italy, Dipartimento di Biochimica e Biotecnologie Mediche, UniVersita ` di Napoli Federico II, and CEINGE, Biotecnologie AVanzate Scarl, Via S. Pansini 5, I-80131 Napoli, Italy ReceiVed July 20, 2001; ReVised Manuscript ReceiVed October 19, 2001 ABSTRACT: The G13A substitution in the G13XXXXGK[T,S] consensus sequence of the elongation factor 1R from the archaeon Sulfolobus solfataricus (SsEF-1R) 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-1R wt), G13ASsEF-1R 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-1R showed an increased affinity for GDP and GTP. Surprisingly, the decreased intrinsic GTPase Na of G13ASsEF-1R can be partially restored by kirromycin, an effect not found for SsEF-1R wt. The temperature inducing a 50% denaturation of G13ASsEF-1R was somewhat lower (-5 °C) than that of SsEF-1R wt, and the decrease in its thermophilicity was slightly more accentuated (-10 °C). These results indicate that the nature of the residue in position 13 is important for the functional and physical properties of SsEF-1R. The elongation factor 1R from the hyperthermophilic archaeon Sulfolobus solfataricus 1 is a GTP-binding protein; thus, it is endowed with a GTPase activity (1-3). Its amino terminal displays the selective homologies of the guanine nucleotide-binding proteins (3). These GTPases are charac- terized by the consensus sequences [G,A]XXXXGK[T,S], DXXG, and NKXD that are involved in the binding of GDP or GTP (4). Furthermore, the three-dimensional (3D) struc- tures of eubacterial EF-Tu (5-9) and EF-G (10, 11) and that of eucarial EF-1R (12) show that the guanine nucleotide- binding domains share similar structural features (13, 14). Interestingly, however, in the sequence motif [G,A]XXXXGK, the first amino acid residue is alanine in EF-2 and EF-G, whereas in EF-1R and EF-Tu, this position is occupied by glycine (4). In the 3D structure of EF-Tu, in either the GDP- or the GTP-bound form, this glycine is not directly involved in the binding of phosphate (14, 15). Therefore, it appears interesting to investigate its role in the functional properties of the translational elongation factor EF-1R from the archaeon S. solfataricus (SsEF-1R). In a previous work, it was found that A26G substitution activated the intrinsic GTPase of SsEF-2 (16). Our results show that G13A re- placement affects the affinity of SsEF-1R for guanine nu- cleotides as well as the ability to hydrolyze GTP and sustain poly(Phe) synthesis. In addition, G13A mutation makes SsEF-1R sensitive to the antibiotic kirromycin, a typical inhibitor of eubacterial but not of archaeal or eucarial protein synthesis. MATERIALS AND METHODS Chemicals, Enzymes, and Buffers. Restriction enzymes, modifying enzymes, labeled compounds, and chemicals were used as already reported (17); standard molecular biology techniques were performed as previously described (18). The following buffers were used: buffer A was 20 mM Tris HCl (pH 7.8), 50 mM KCl, and 10 mM MgCl 2 ; buffer B was 20 mM Tris HCl (pH 7.8), 10 mM MgCl 2 , and 1 mM DTT. Plasmid Construction, Expression, and Purification of the Mutant G13ASsEF-1R. The E8 plasmid containing the gene ² This work was supported by CNR, MURST (Rome, Italy), and the European Community Biotechnology Program (Contract BIO4- CT97-2188). * Corresponding author. Phone: +39 081 7463120. Fax: +39 081 7463653. E-mail: arcari@dbbm.unina.it. ‡ Universita ` deli Studi di Catanzaro Magna Graecia. § Universita ` di Napoli Federico II. | CEINGE, Biotecnolgie Avanzate Scarl. ⊥ Died on June 28, 2001. 1 Abbreviations: EF, elongation factor; Ss, Sulfolobus solfataricus; Ec, Escherichia coli; Tt, Thermus thermophilus; Ta, Thermus aquaticus; G13ASsEF-1R, SsEF-1R carrying the G13A substitution; GTPase Na , intrinsic GTPase of SsEF-1R triggered by 3.6 M NaCl; CD, circular dichroism. 628 Biochemistry 2002, 41, 628-633 10.1021/bi015598h CCC: $22.00 © 2002 American Chemical Society Published on Web 12/14/2001