Structure and Importance of the Dimerization Domain in Elongation Factor Ts from Thermus thermophilus^,

Abstract

Elongation factor Ts (EF-Ts) functions as a nucleotide-exchange factor by binding elongation factor Tu (EF-Tu) and accelerating the GDP dissociation from EF-Tu; thus EF-Ts promotes the transition of EF-Tu from the inactive GDP form to the active GTP form. Thermus thermophilus EF-Ts exists as a stable dimer in solution which binds two molecules of EF-Tu to form a (EF-Tu.EF-Ts)2 heterotetramer. Here we report the crystal structure of the dimerization domain of EF-Ts from T. thermophilus refined to 1.7 A resolution. A three-stranded antiparallel beta-sheet from each subunit interacts to form a beta-sandwich that serves as an extensive dimer interface tethered by a disulfide bond. This interface is distinctly different from the predominantly alpha-helical one that stabilizes the EF-Ts dimer from Escherichia coli [Kawashima, T., et al. (1996) Nature 379, 511-518]. To test whether the homodimeric form of T. thermophilus EF-Ts is necessary for catalyzing nucleotide exchange, the present structure was used to design mutational changes within the dimer interface that disrupt the T. thermophilus EF-Ts dimer but not the tertiary structure of the subunits. Surprisingly, EF-Ts monomers created in this manner failed to catalyze nucleotide exchange in EF-Tu, indicating that, in vitro. T. thermophilus EF-Ts functions only as a homodimer.

Three-dimensional Structure of a Hyperthermophilic 5′-Deoxy-5′-methylthioadenosine Phosphorylase from Sulfolobus solfataricus

“…This exposed active site is similar to that seen in the hexameric E. coli PNP structure (7,14) (Fig. 4a) 43 from an adjacent subunit. The SsMTAP monomer can be divided into two structural domains by cutting at the end of strand ␤5.…”

“…The active site of SsMTAP can be divided into three main regions: the phosphate-binding site, the purine-binding site, and the ribose base-binding site. This geometric arrangement of bound substrates is very similar to those seen in structures of both trimeric and hexameric PNPs (7,8,13,14,28 43 from an adjacent monomer. In addition, the 2Ј-and 3Ј-hydroxyl groups of the bound nucleoside substrate contribute hydrogen bonds.…”

“…Recently there have been reports of single or multiple disulfide bonds in intracellular proteins from hyperthermophilic microrganisms (41)(42)(43)(44)(45). In all cases, these disulfide bonds are not present in the homologous mesophilic proteins.…”

Chloroplast Elongation Factor Ts Pro-Protein Is an Evolutionarily Conserved Fusion with the S1 Domain-Containing Plastid-Specific Ribosomal Protein-7

“…The two domains have a 60% identity and 74% similarity to each other. EF-Ts monomers are composed of two domains: a 50-amino acid N-terminal domain that participates in GDP exchange called the ubiquitinassociated (UBA) domain (Kawashima et al, 1996) and a 146-amino acid carboxy domain that participates in the dimerization of EF-Ts (Jiang et al, 1996). As shown in Figure 1C, both features can be identified in each of the PETs EF-Ts domains.…”

“…Homology comparison was done using BLAST 2 sequences (National Center for Biotechnology Information). UBA and dimerization domains were estimated taking into account Thermus thermophilus EF-Ts (Jiang et al, 1996). Multiple sequence alignments were performed using MacVector 7.0 software.…”