Chemical structures of ramoplanin factor A2 (25) and enduracidins A and B (27, 38).

(A Left) ¹H NMR titration spectra of ramoplanin (1) with citronellyl–Lipid intermediate I (5). As a 1:1 stoichiometry is reached (center trace), the intensity of the NMR signals is diminished and subjected to severe line broadening because of the formation of insoluble peptide fibrils. (A Right) ¹H NMR titration spectra of ramoplanin (1) with equimolar P¹-citronellyl-P²-MurNAc pyrophosphate (9) and l-Ala-γ-d-Glu-l-Lys-d-Ala-d-Ala (4) added in trans. At the 1:1:1 stoichiometric titration point (center trace), the NMR spectra of the mixture is the superposition of each individual component. No chemical shift changes or line broadening was observed during the titration, indicating that no interaction between the antibiotic and in trans peptidoglycan fragments occurred. (B) Transmission electron micrograph of the fibrils formed from the complexation of ramoplanin (1) with citronellyl–Lipid I (5). The fibrils formed from the interaction of 1 with 6 and 7, respectively, exhibited similar morphology by transmission electron micrograph (TEM). (C) Part of the aliphatic region of the ¹H NMR spectrum of free ramoplanin (1), free Park's nucleotide (6), and a 1:1 mixture of the two depicting the chemical shift changes that occur on binding. (D) Representative K_d determination plot of the binding of ramoplanin (1) to Park's nucleotide (6) as obtained by ¹H NMR titration. The curve denotes the experimental (o) and calculated (solid line) chemical shifts (δ) of the lactyl ether methyl protons (3a) of 6 as a function of ramoplanin concentration.

Primary and backbone tertiary backbone structures of ramoplanin A2 (green) and two functionally related type-C lantibiotics, mersacidin (purple) and actagardine (blue). Nonstandard residues are denoted as follows: Abu, aminoisobutyric acid; 3-OH Asn, threo-3-hydroxy-l-asparagine; Chp, chlorohydroxyphenylglycine; Dha, dehydroalanine; FA, (2Z,4E)-7-methyl-octadienoic acid; Man, mannose. d-Amino acids are depicted by black circles. Ribbon representations of the tertiary structures were drawn using the program insight ii. Coordinates for ramoplanin (1DSR) and actagardine (1AJ1) NMR structures have been deposited in the Protein Data Bank. NMR coordinates for mersacidin were provided by Dr. Thomas Prasch.

Chemical structures of synthetic and naturally occurring analogues of bacterial peptidoglycan monomers used in this study.

(A) NMR localization of the binding interface between ramoplanin (1) and Park's nucleotide (6) (1:1 molar ratio). Protons that exhibit downfield chemical shifts on binding are colored green; those that shift upfield are depicted in orange. Protons that do not shift on binding are colorless. Intermolecular NOEs are depicted by red arrows. Possible anchoring electrostatic interactions between ramoplanin Orn⁴ and Orn¹⁰ residues and 6 are indicated by blue arrows. NMR experiments further localize the minimum PG structure recognized by ramoplanin to the intact pyrophosphate, the muramyl carbohydrate, and the first two amino acids of the pentapeptide. (B) Surface localization of the peptidoglycan monomer/lipid intermediate binding region of ramoplanin. Residues comprising the binding interface identified by NMR that lie on the same face of the antibiotic are colored yellow and mapped onto (C) the three-dimensional NMR structure of ramoplanin obtained in 20% DMSO (25).