The Crystal Structure of Sulfolobus solfataricus Elongation Factor 1R in Complex with Magnesium and GDP ²,‡ Luigi Vitagliano, &,§ Alessia Ruggiero, | Mariorosario Masullo, ⊥,# Piergiuseppe Cantiello, # Paolo Arcari, #,% and Adriana Zagari *,&,|,% Istituto di Biostrutture e Bioimmagini, CNR, Via Mezzocannone 6, I-80134 Napoli, Italy, Centro interuniVersitario di ricerca sui Peptidi bioattiVi (CIRPEB), Via Mezzocannone 6, I-80134 Napoli, Italy, Dipartimento di Chimica Biologica, Sezione di Biostrutture, UniVersita ` degli Studi di Napoli Federico II, Via Mezzocannone 6, I-80134 Napoli, Italy, Dipartimento di Scienze Farmacobiologiche, UniVersita ` degli Studi di Catanzaro “Magna Graecia”, Roccelletta di Borgia 88021 Catanzaro, Italy, Dipartimento di Biochimica e Biotecnologie Mediche, UniVersita ` degli Studi di Napoli Federico II, Via Pansini 5, I-80131 Napoli, Italy, CEINGE, Biotecnologie AVanzate Scarl, Via Comunale Margherita 482, I-80145 Napoli, Italy ReceiVed December 30, 2003; ReVised Manuscript ReceiVed March 10, 2004 ABSTRACT: Recent studies have shown that elongation factors extracted from archaea/eukarya and from eubacteria exhibit different structural and functional properties. Along this line, it has been demonstrated that, in contrast to EF-Tu, Sulfolobus solfataricus EF-1R in complex with GDP (SsEF-1R‚GDP) does not bind Mg 2+ , when the ion is present in the crystallization medium at moderate concentration (5 mM). To further investigate the role that magnesium plays in the exchange process of EF-1R and to check the ability of SsEF-1R‚GDP to bind the ion, we have determined the crystal structure of SsEF-1R‚GDP in the presence of a nonphysiological concentration (100 mM) of Mg 2+ . The analysis of the coordination of Mg 2+ unveils the structural bases for the marginal role played by the ion in the nucleotide exchange process. Furthermore, nucleotide exchange experiments carried out on a truncated form of SsEF-1R, consisting only of the nucleotide binding domain, demonstrate that the low affinity of SsEF-1R‚GDP for Mg 2+ is due to the local architecture of the active site and does not depend on the presence of the other two domains. Finally, considering the available structures of EF-1R, a detailed mechanism for the nucleotide exchange process has been traced. Notably, this mechanism involves residues such as His14, Arg95, Gln131, and Glu134, which are strictly conserved in all archaea and eukarya EF-1R sequences hitherto reported. Guanine nucleotide binding proteins (GNBP) 1 are enzymes involved in fundamental biological processes (1, 2). The members of this family play crucial roles in cell growth and proliferation, signal transduction, membrane trafficking, and protein biosynthesis. In these enzymes, the transition from the active GTP-bound form to the inactive GDP-bound state is associated with a conformational switch, which activates important cellular processes. Although the magnitude of the conformational change varies among different GNBP, it represents a fundamental step for their function. In GNBP involved in protein biosynthesis, the conforma- tional changes associated with the GTP hydrolysis produces a major rearrangement of the multidomain structure of these enzymes. The most studied translation GNBP are the elongation factors that transport the aminoacyl-tRNA to the ribosome (EF-Tu in bacteria and EF-1R in eukarya and archaea) (3-5). In carrying out their biological functions, these enzymes interact with several cellular components: GTP, GDP, aminoacyl-tRNA, ribosome, and the exchange factors (EF-Ts in eubacteria and EF-1 in eukarya and archaea), which activate these GNBP by favoring the release of the GDP. In the past decade, a number of impressive three- dimensional (3D) structure determinations of EF-Tu have shed light on the structural details of the elongation cycle in eubacteria. These investigations have provided a clear picture of the conformational flexibility of EF-Tu, whose shape is modulated by the interactions with GDP (6-9), GDPNP (10, 11), aminoacyl-tRNA (12-14), EF-Ts (15, 16), and antibiot- ics (17, 18). On the other hand, there is limited structural information available on the translation elongation factors isolated from eukarya and archaea. Only recently, the 3D structures of the ² This work was financially supported by PRIN 2003 (Rome). ‡ The atomic parameters (code 1skq) have been deposited in the Protein Data Bank. * To whom correspondence should be addressed. Telephone: +39- 0812536614. Fax: +39-0812536603. E-mail: zagari@chemistry.unina.it. & Istituto di Biostrutture e Bioimmagini, CNR. § CIRPEB. | Dipartimento di Chimica Biologica, Universita ` degli Studi di Napoli Federico II. ⊥ Universita ` degli Studi di Catanzaro “Magna Graecia”. # Dipartimento di Biochimica e Biotecnologie Mediche, Universita ` degli Studi di Napoli Federico II. % CEINGE, Biotecnologie Avanzate Scarl. 1 Abbreviations: GNBP, guanine nucleotide binding proteins; EF- Tu‚GDP, complex of the elongation factor Tu with GDP; SsEF-1R‚ GDP, complex of Sulfolobus solfataricus EF-1R with GDP; SsEF-1R‚ GDP/Mg 2+ , complex of S. solfataricus EF-1R with GDP and Mg 2+ ; ScEF-1/EF-1R complex of Saccharomyces cereVisiae EF-1R with the exchange factor EF-1; ScEF-1/EF-1R‚GDP, complex of S. cereVisiae EF-1R with EF-1 and GDP; Ss(G)EF-1R, guanine nucleotide binding domain of S. solfataricus EF-1R; ppGpp, guanosine-5′-diphosphate- 3′-diphosphate. 6630 Biochemistry 2004, 43, 6630-6636 10.1021/bi0363331 CCC: $27.50 © 2004 American Chemical Society Published on Web 05/04/2004