Structural Insights into the Cyclic Peptide Antibiotic Thiostrepton via NMR Spectroscopy

A complex cyclic polypeptide antibiotic, thiostrepton, is made by two large macrocyclic rings: a 26-membered ring associated with a thiazoline moiety and a 27-membered ring accompanied by a quinaldic acid unit, a dehydropiperidine ring, and a flexible bis-dehydroalanine tail. The site-specific local structure and nuclear-spin dynamics of this polypeptide antibiotic is determined by using 2D solution NMR ( 1 H– 13 C HSQC, 1 H– 13 C HMBC, 1 H– 1 H COSY, 1 H– 1 H TOCSY, and 1 H– 1 H NOESY) and solid-state NMR methods ( 13 C CP-MAS 2D PASS, 1 H– 13 C HETCOR, and site-specific 13 C spin–lattice relaxation measurements). The combined 1 H– 13 C HETCOR and 1 H– 1 H NOESY data demonstrate that the quinaldic acid residue is positioned in close spatial proximity to multiple structural elements, including the thiazoline and piperidine rings, isoleucine–valine chain, butyrine, thiostreptin residue, dehydroalanine units, threonine, and thiazole. These interactions generate a compact hydrophobic core that stabilizes the conformation of the bis-dehydroalanine tail. The flexible side chains of both macrocyclic rings, together with the tail region, accommodate themselves within the interface of ribosomal protein L11 and the 23S rRNA, thereby perturbing the 70S ribosomal subunit. The dynamics of these flexible segments are quantified by employing NMR relaxometry. Therefore, although functional activity is not examined directly, the integrated solution- and solid-state NMR results deliver site-specific structural information and local nuclear spin dynamics at individual carbon sites of this cyclic polypeptide antibiotic. These characteristics are associated with the functional state of thiostrepton and offer a foundation for the rational design of next-generation polypeptide antibiotics.