In this study, the structure of the 70S-Tet(O) complex in the presence of GDPNP has allowed us to visualize the details of binding between the 70S ribosome and Tet(O). In the GDPNP-bound form Tet(O) was earlier shown biochemically to promote release of Tc from the 70S ribosome 20,22
, and the elucidation of the current structure therefore provides direct functional insights into the mechanisms of Tet(O)-mediated Tc resistance.
On the 30S subunit side, Tet(O) is positioned close to the site where Tc has been found in the X-ray structure. The 30S subunit-Tet(O) contact sites we have identified in the present study can be divided into two categories: (1) those which lead to a clash with the space for the binding of Tc via the 507-loop and (2) those which disrupt the structure of the 16S rRNA around the Tc-binding site via the 465-loop toward nucleotide 1209 in 16S rRNA. As in EF-G, the GTP-binding site in Tet(O) is located at the GTPase-associated center. These contacts between the Tet(O) and the ribosome seem to collectively play the role of preventing or reversing the binding of Tc to the ribosome
The most direct effect of Tet(O) binding in preventing Tc from binding to the 30S subunit seems to be the result of a competition between residues 507–509 of Tet(O) and Tc for the same space. When Tet(O) enters into the Tc-bound ribosome, the 507-loop cannot be settled into the ribosome complex because Tc already occupies the close vicinity of nucleotide 1054 and forms multiple hydrogen bonds with the ribosome. The competition for the same site guarantees that Tet(O) and Tc cannot coexist in the ribosomal complex. In addition, binding of Tet(O) disrupts the ribosome structure and reshapes the geometry of the backbone where Tc is anchored (). With this disrupted backbone structure, the nucleotides involved in binding with Tc are reoriented, and thus, Tc loses its bonds with the ribosome. Interestingly, the binding of Tc does not change the backbone shape from its shape in the Tc-free ribosome.
The question arises is by which molecular mechanism Tc inhibits normal translation in the ribosome. One may ask why a Tc-bound ribosome does not accept an entering EF-Tu-bound aminoacyl-tRNA complex, but does accept Tet(O) even though the EF-Tu-aminoacyl-tRNA complex forms a shape highly similar to that of Tet(O). Our current study provides some insights to answer this question. If an aminoacyl-tRNA bound with EF-Tu enters into the ribosome, its anticodon loop must reach the codon site. In the presence of Tc, a primary portion of the space for the anticodon loop is already occupied by Tc () which causes a decisive rejection of the aminoacyl-tRNA from the ribosome before codon-anticodon recognition can take place. In contrast, Tet(O) enters the ribosome with less demand for space in that region than the anticodon loop of the tRNA; the available space provides an opportunity for Tet(O) to be admitted to the factor binding site, important for subsequence GTP hydrolysis.
Tet(O), a GTPase, possesses a structure very similar to that of ribosomal GTPase, elongation factor G. The structural similarity suggests an analogy of their GTP-hydrolysis-induced conformational changes which enable the two ribosomal proteins to perform their respective biological functions. The structural effects of the EF-G-associated GTP hydrolysis on the ribosome have been extensively studied 23–25
. It is known that GTP hydrolysis induces substantial conformational changes in the ribosome. The ribosome’s effect on the conformation of EF-G is substantial, as well, causing domain IV to be reoriented relative to the other domains, as shown by cryo-EM 24
and X-ray structures 12
of the ribosome bound with EF-G in the presence of fusidic acid. The antibiotic fusidic acid traps EF-G in a conformational intermediate between the GTP and GDP forms. In this translocational complex, a contact observed between the 507-loop of EF-G and the P-site tRNA seems to be essential for the translocation of tRNA based on the significant conformational flexibility of this loop 12
. This flexibility allows the loop to participate in the major dynamic motion of the entire domain IV as the GTP hydrolysis takes place. Thus, we see this loop as a functionally required structural element in EF-G. The structural similarity of Tet(O) to EF-G suggests structural flexibility in the equivalently positioned loops of Tet(O). Accordingly, we predict that GTP hydrolysis in Tet(O) results in extensive conformational changes in the distal loops of domain IV, particularly in the three flexible loops. By combining structural and mutational analyses, the present study provides structural insights into how the three loops in domain IV (see ) might cooperate to expel Tc from the ribosome: the 465-loop is responsible for distorting the backbone shape at nucleotides 1051–1054 of 16S rRNA, which weakens or abolishes the binding of Tc at this site with the RNA; the 507-loop in the middle of these three directly pushes Tc out of the ribosome; and the 438-loop along with nucleotide 966 and 1196 should form a corridor allowing Tc to exit.
After completion of this work, a cryo-EM reconstruction of a 70S-Tet(M) complex was published 26
. Our results agree with the results by Wilson and coworkers in all essential details, as expected based on the high sequence homology between Tet(O) and Tet(M).