Enacyloxin IIa Pinpoints a Binding Pocket of Elongation Factor Tu for Development of Novel Antibiotics

Abstract

Elongation factor (EF-) Tu⅐GTP is the carrier of aminoacyl-tRNA to the programmed ribosome. Enacyloxin IIa inhibits bacterial protein synthesis by hindering the release of EF-Tu⅐GDP from the ribosome. The crystal structure of the Escherichia coli EF-Tu⅐guanylyl iminodiphosphate (GDPNP)⅐enacyloxin IIa complex at 2.3 Å resolution presented here reveals the location of the antibiotic at the interface of domains 1 and 3. The binding site overlaps that of kirromycin, an antibiotic with a structure that is unrelated to enacyloxin IIa but that also inhibits EF-Tu⅐GDP release. As one of the major differences, the enacyloxin IIa tail borders a hydrophobic pocket that is occupied by the longer tail of kirromycin, explaining the higher binding affinity of the latter. EF-Tu⅐GDPNP⅐enacyloxin IIa shows a disordered effector region that in the PhetRNA Phe ⅐EF-Tu (Thermus aquaticus)⅐GDPNP⅐enacyloxin IIa complex, solved at 3.1 Å resolution, is stabilized by the interaction with tRNA. This work clarifies the structural background of the action of enacyloxin IIa and compares its properties with those of kirromycin, opening new perspectives for structure-guided design of novel antibiotics. Elongation factor (EF)5 Tu is a GTPase that, as all members of this superfamily, cycles between the active GTP-bound and the inactive GDP-bound state. In bacterial protein synthesis EF-Tu is the carrier of aa-tRNA to the mRNA-programmed ribosome (for review see Refs. 1 and 2). The codon-anticodon interaction triggers the hydrolysis of the bound GTP, inducing the release of EF-Tu⅐GDP, the positioning of aa-tRNA into the ribosomal A-site, and peptide bond formation with the P-site-bound peptidyl-tRNA. EF-Tu folds in three distinct domains: the nucleotide-binding domain 1 (residues 1-199 in Escherichia coli (Ec) EF-Tu⅐GDP) shows the classical GTPase mixed ␣/␤ Rossman fold, whereas domains 2 and 3 (residues 209 -299 and 300 -393, respectively) are ␤-barrels. The association of the domains in EF-Tu⅐GDP with reduced interfaces and a central hole becomes more compact in EF-Tu⅐GTP with extensive interfaces and no hole (3-5). EF-Tu is the target of four structurally unrelated families of antibiotic inhibitors of protein synthesis (for review see Ref.2), of which kirromycin and enacyloxin IIa hinder the release of EF-Tu⅐GDP from the ribosome after GTP hydrolysis, thus inhibiting its recycling and peptide bond formation. The high-resolution crystal structure of Thermus thermophilus (Tt) EF-Tu⅐GDP in complex with methylkirromycin (also called aurodox) showed that the antibiotic binds in the interface of domains 1 and 3, inducing a unique, compact EF-Tu-GTP-like conformation, which is the reason for its inhibition of the EF-Tu⅐GDP release (6). These conclusions have been further confirmed in studies of the Phe-tRNA Phe ⅐ EF-Tu⅐GDPNP⅐kirromycin. 6 Specific differences between the enacyloxin IIa and kirromycin complexes with EF-Tu concern various aspects such as the electrophoretic migration on native gel, binding affinity, GTPase activity, different effects of...

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“…Arg5 Arg Arg5 Arg5 Arg5 Arg5 Arg5 A Arg5 Arg5 Arg5 Arg5 A g5 A A A A A A 9 9 9 9 9 9 9 9 9 9 9 9 9 (27) bound to enacyloxin IIA (blue sticks) and a nonhydrolyzable GTP-analog guanosine 5′-(β,γ-imido)triphosphate (GDPNP) (red). (E) Superimposition of E. coli EF-Tu (PDB ID: 1DG1) (26) and P. putida EF-Tu from the PPHD putida :EF-Tu complex; the switch I loop is highlighted (P. putida EF-Tu, blue; E. coli EF-Tu, red).…”

Elongation Factor Tu3 (EF-Tu3) from the Kirromycin Producer Streptomyces ramocissimus Is Resistant to Three Classes of EF-Tu-Specific Inhibitors

“…8a) and might also contribute to the kirromycin-resistant character of EF-Tu3. The binding site of enacyloxin IIa partially overlaps with the kirromycin binding site (28). The strong salt bridge between Lys313 and the antibiotic is no longer possible due to the K313A substitution in EF-Tu3.…”

Facile synthesis of the cyclohexane fragment of enacloxins, a series of antibiotics isolated from Frateuria sp. W-315

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