Fusidic and helvolic acid inhibition of elongation factor 2 from the archaeon Sulfolobus solfataricus.
Fusidic acid (FA) and helvolic acid (HA) belong to a small family of naturally occurring steroidal antibiotics known as fusidanes. FA was studied for its ability to alter the biochemical properties supported by elongation factor 2 isolated from the archaeon Sulfolobus solfataricus (SsEF-2). Both poly(Phe) synthesis and ribosome-dependent GTPase (GTPase(r)) were progressively impaired by increasing concentrations of FA up to 1 mM, whereas no effect was measured in the intrinsic GTPase of SsEF-2 triggered by ethylene glycol in the presence of barium chloride (GTPase(g)). The highest antibiotic concentration caused inhibition of either poly(Phe) synthesis or GTPase(r) only slightly above 50%. A greater response of SsEF-2 was observed when HA was used instead of FA. HA caused even a weak impairment of GTPase(g). A mutated form of SsEF-2 carrying the L452R substitution exhibited an increased sensitivity to fusidane inhibition in either poly(Phe) synthesis or GTPase(r). Furthermore, both FA and HA were able to cause impairment of GTPase(g). The antibiotic concentrations leading to 50% inhibition (IC(50)) indicate that increased fusidane responsiveness due to the use of HA or the L452R amino acid replacement is mutually independent. However, their combined effect decreased the IC(50) up to 0.1 mM. Despite the difficulties in reaching complete inhibition of the translocation process in S. solfataricus, these findings suggest that fusidane sensibility is partially maintained in the archaeon S. solfataricus. Therefore, it is likely that SsEF-2 harbors the structural requirements for forming complexes with fusidane antibiotics. This hypothesis is further evidenced by the observed low level of impairment of GTPase(g), a finding suggesting a weak direct interaction between the archaeal factor and fusidanes even in the absence of the ribosome. However, the ribosome remains essential for the sensitivity of SsEF-2 toward fusidane antibiotics.
De Vendittis
Emmanuele
E
Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli Federico II, via S. Pansini, 5, Italy. devendittis@dbbm.unina.it
De Paola
Barbara
B
Gogliettino
Maria Angela
MA
Adinolfi
Bianca Stella
BS
Fiengo
Antonio
A
Duvold
Tore
T
Bocchini
Vincenzo
V
D019943:Amino Acid Substitution / Q000235:genetics; D019843:Archaeal Proteins / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics; D001120:Arginine / Q000235:genetics; D004352:Drug Resistance, Microbial; D005672:Fusidic Acid / Q000031:analogs & derivatives* / Q000737:chemistry*; D007930:Leucine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D020652:Peptide Elongation Factor 2 / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics; D011500:Protein Synthesis Inhibitors / Q000737:chemistry*; D016944:Sulfolobus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D019843:Archaeal Proteins; D020652:Peptide Elongation Factor 2; D011500:Protein Synthesis Inhibitors; D005672:Fusidic Acid; D001120:Arginine; D007930:Leucine; C011267:helvolic acid