Translocation. XV. Characteristics of and structural requirements for the interaction of 24,25-dihydrofusidic acid with ribosome.elongation factor G complexes

Abstract

Fusidic acid inhibits polypeptide chain elongation by binding to the ribosome - elongation factor-G - GDP complex and thereby preventing its dissociation. The experiments reported here quantitate the interaction of the antibiotic [3H]-24,25-dihydrofusidic acid, an active analog of fusidic acid, with the ribosome - elongation factor-G - GDP comples. All components of the complex are essential for [3H]-24,25-dihydrofusidic acid binding. The stoichiometry of the interaction is ca. 1:1, and the Ka apparent, as determined by equilibrium dialysis, is 2.6 times 10-6 M-minus 1. It is further shown that GTP and GDP are equally effective in forming complexes to which the antibiotic may bind, whereas GMP and beta,gamma-methyleneguanosine triphosphate will not form complexes to which the antibiotic may bind. In order to examine the structural basis of the mode of antibiotic action shown by fusidic acid, we have considered two activities of 21 structural analogs of this antibiotic: ability to bind to the aforementioned ternary complex and ability to stabilize this complex. The comparative binding capability of the analogs were extablished through competition experiments with [3H]-24,25-dihydrofusidic acid. The data obtained from these experiments can be summarized as follows. (1) The C17-20 double bond of fusidic acid appears to be critical for both binding and complex stabilization activities. (2) A carboxyl group in the vicinity of the C20 carbon is also essential for both activities. (3) Modifications of other functional groups in the molecule can lead to significantly decreased stabilization of the ternary ribosome complex and/or ability to compete with [3H]-24,25-dihydrofusidic acid for binding to the complex, but do not demonstrate absolute structural requirements for either activity.

Fusidic Acid Targets Elongation Factor G in Several Stages of Translocation on the Bacterial Ribosome

“…Some translation-targeting drugs lock the ribosome in long lived states via binding to translation factors. Among these is the steroid-like antibiotic fusidic acid (FA), 3 which stalls the ribosome in complex with EF-G and GDP (3). FA has low affinity to EF-G off the ribosome but forms a strong complex with ribosome-bound EF-G (3,4).…”

“…Among these is the steroid-like antibiotic fusidic acid (FA), 3 which stalls the ribosome in complex with EF-G and GDP (3). FA has low affinity to EF-G off the ribosome but forms a strong complex with ribosome-bound EF-G (3,4). EF-G plays double roles in bacterial protein synthesis by both catalyzing mRNA translocation during the peptide elongation cycle and, aided by ribosome recycling factor, splitting the post-termination ribosome into subunits (5) for a new round of initiation of mRNA translation (6,7).…”

“…In experiments with naked ribosomes, it was shown that FA allows for a first round of GTP hydrolysis on ribosome-bound EF-G (12) and then traps the ribosome in a complex with GDP-bound EF-G from which FA dissociates slowly (3). The K 50% value for FA inhibition of multiple rounds of GTP hydrolysis as catalyzed by the naked ribosome was estimated as 1 M (4, 13).…”

Mutations in the G-domain of Elongation Factor G fromThermus thermophilus Affect Both Its Interaction with GTP and Fusidic Acid

“…Fusidic Acid Binding-The studies of the EF-G function in the presence of FA has resulted in the conclusion that EF-G interaction with the ribosomes and consequent GTP hydrolysis may generate or uncover the FA binding site (31). Moreover, the addition of the ribosomes to EF-G and a GTP analog decreases the dissociation constant of the analog from the complex, which was found to be 59 nM (32).…”

“…FA binds with EF-G and retains it on the ribosome after GTP hydrolysis (23,31,32). For this reason, it was suggested that FA binds to EF-G in its intermediate conformation just after GTP hydrolysis (11).…”