A Crystal Structure of a Dimer of the Antibiotic Ramoplanin Illustrates Membrane Positioning and a Potential Lipid II Docking Interface

A crystal structure of a dimer of the antibiotic ramoplanin illustrates membrane positioning and a potential Lipid II docking interface James B. Hamburgera, Amanda J. Hoertza, Amy Leeb, Rachel J. Senturiaa, Dewey G. McCaffertya,1, and Patrick J. Lollb,1 aDepartment of Chemistry, Duke University, Durham, NC 27708; and bDepartment of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA 19129 Edited by William F. DeGrado, University of Pennsylvania School of Medicine, Philadelphia, PA, and approved July 1, 2009 (received for review April 30, 2009) The glycodepsipeptide antibiotic ramoplanin A2 is in late stage clinical development for the treatment of infections from Gram- positive pathogens, especially those that are resistant to ﬁrst line antibiotics such as vancomycin. Ramoplanin A2 achieves its anti- bacterial effects by interfering with production of the bacterial cell wall; it indirectly inhibits the transglycosylases responsible for peptidoglycan biosynthesis by sequestering their Lipid II substrate. Lipid II recognition and sequestration occur at the interface between the extracellular environment and the bacterial membrane. Therefore, we determined the structure of ramoplanin A2 in an amphipathic environment, using detergents as membrane mimet- ics, to provide the most physiologically relevant structural context for mechanistic and pharmacological studies. We report here the X-ray crystal structure of ramoplanin A2 at a resolution of 1.4 Å. BIOCHEMISTRY This structure reveals that ramoplanin A2 forms an intimate and highly amphipathic dimer and illustrates the potential means by which it interacts with bacterial target membranes. The structure Fig. 1. The chemical structure of Ramoplanin A2. also suggests a mechanism by which ramoplanin A2 recognizes its Lipid II ligand. faecium (VRE) (3, 4) and has recently been in development for the treatment of Clostridium difficile infections. Since the anti- glycolipodepsipeptide ͉ mechanism ͉ vancomycin resistance biotic’s discovery over two decades ago, no clinical or laboratory- generated resistance to ramoplanin A2 has been reported. atients undergoing acute care in hospitals are at significant The ramoplanins are a family of closely related glycolipodep- Prisk for acquiring opportunistic infections. Such infections sipeptide antibiotics secreted by Actinoplanes ATCC 33076 (5, might be developed after invasive surgery or burn trauma, or via 6). At least three forms are known, A1, A2, and A3; these forms long-term i.v. lines, intracranial shunts and indwelling catheters differ in their acyl chain substituents, but possess essentially (1). Individuals immunocompromised due to organ transplan- identical antibiotic activities. Ramoplanins are structurally and tation, AIDS, or cancer chemotherapy are also vulnerable to hospital-acquired (nosocomial) infection. Nosocomial infections functionally related to ramoplanose, which is produced by Ac- increased sharply in the United States between 1980 and the tinoplanes strain U.K.-71,903 and which differs from factor A2 present day, and seven leading pathogen groups have accounted by one mannose unit (7), and to enduracidin A and B, which are for most of this increase: Escherichia coli, Staphylococcus aureus, produced by Streptomyces fungicidicus B5477 (8–10). This paper coagulase-negative staphylococci, streptococci, Enterococcus focuses on the ramoplanin A2 isomer, the most abundant among faecium, Pseudomonas aeruginosa, and Candida albicans. Four of the A1–A3 forms; for simplicity’s sake, we refer to this isomer as these seven pathogens are Gram-positive bacteria, and resis- ramoplanin for the remainder of the manuscript. Ramoplanin tance to commonly used antibiotics has emerged in all of them consists of a 49-member ring containing 17 amino acids, includ- (2). Given the speed with which such antibiotic resistance is ing several nonproteinogenic amino acids (Fig. 1). The macro- spreading, it is not difficult to foresee a time when our most cycle, essential for activity, is formed by a lactone bond con- serious infectious threats may become largely untreatable. necting the 17th residue, 3-chloro-4-hydroxyphenylglycine In the latter part of the twentieth century, the discovery and (Chp), with the hydroxy group of ␤-hydroxy-Asn-2. The di- development of novel antibiotics, compounds that differ signif- icantly from existing therapeutics in structure and/or mechanism, slowed to a virtual halt. Presently, the glycopeptide Author contributions: J.B.H., D.G.M., and P.J.L. designed research; J.B.H., A.J.H., A.L., R.J.S., D.G.M., and P.J.L. performed research; J.B.H., D.G.M., and P.J.L. analyzed data; and J.B.H., antibiotic vancomycin serves as the principle line of defense for D.G.M., and P.J.L. wrote the paper. all major Gram-positive pathogenic infections; this includes The authors declare no conﬂict of interest. infections caused by pathogens resistant to other antibiotics, such as methicillin-resistant S. aureus (MRSA) and cephalos- This article is a PNAS Direct Submission. porin-resistant Streptococcus pneumonia. However, resistance to Data deposition: The atomic coordinates have been deposited in the Cambridge Structural Database, Cambridge Crystallographic Data Centre, Cambridge CB2 1EZ, United Kingdom vancomycin is on the rise, reducing available therapeutic options. (CSD reference no. 729786). Ramoplanin A2 is a promising candidate for treating serious 1To whom correspondence may be addressed. E-mail: [email protected] or Gram-positive infections that is active against both vancomycin- [email protected]. and beta lactam-resistant pathogens (Fig. 1). Ramoplanin A2 is This article contains supporting information online at www.pnas.org/cgi/content/full/ highly effective against MRSA and vancomycin-resistant E. 0904686106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0904686106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 26, 2021 mannose group is attached to the side chain hydroxyl of hy- the expected positions of the sugars, but it is not readily droxyphenylglycine-11 (Hpg-11). interpretable, most likely because multiple conformers are Ramoplanin acts at a late stage in peptidoglycan biosynthesis present. For this reason the dimannosyl carbohydrate has not by sequestering Lipid II, the substrate for the transglycosylase been included in the final refined model. The failure of the and transpeptidase enzymes. Lipid II sequestration precludes sugars to adopt a well-ordered conformation argues against any formation of the mature, fully cross-linked peptidoglycan, lead- critical role for them, and in fact they do not contribute to ing to a mechanically weakened cell wall and bacterial death antimicrobial activity or Lipid II binding (16, 21, 22, 26). Indeed, from osmotic lysis (11–14). Thus, like the glycopeptide antibi- the structurally related depsipeptide antibiotic enduracidin con- otics, ramoplanin inhibits cell wall biosynthesis, but the molec- tains no carbohydrate, yet has comparable activity to that of ular underpinnings are distinct, since the binding epitopes on ramoplanin (9, 12). Hence, it appears that the sugars contribute Lipid II that are recognized by the two types of antibiotics are mainly to solubility and help define the amphipathic nature of largely non-overlapping (3, 4, 10, 15, 16). This differential the molecule. targeting likely explains ramoplanin’s excellent activity against vancomycin-resistant microorganisms. Structure of the Monomer. The crystal asymmetric unit contains Unfortunately, efforts to identify the structure of the ramo- two ramoplanin monomers, arranged in an intimate dimer. Each planin-Lipid II complex in aqueous solution have been ham- monomer adopts an antiparallel beta structure, with the peptide pered by ligand-induced polymerization of the antibiotic-Lipid II backbone forming a miniature beta sheet consisting of two complex, which produces insoluble fibrils that lack antimicrobial extended strands, both of which are connected at either end by activity. However, the use of 20% dimethyl sulfoxide has been tight turns. The two strands are joined by eight carbonyl-to- shown to minimize fibril formation. Employing this solvent amide hydrogen bonds (Table S1). In addition to these backbone mixture, NMR titrations with peptidoglycan precursors were hydrogen bonds, another cross-strand hydrogen bond is formed used to measure intermolecular NOEs and chemical shift per- between the side chains of Thr-5 and Thr-12. turbations, implicating specific residues in Lipid II binding (12). The two-stranded sheet of the monomer is bent into a Determining the stoichiometry with which Lipid II binds ramo- U-shape, assuming (very approximately) the shape of half a disk, planin has been complicated. Early circular dichroism titrations with the two beta strands running along the circumference of the suggested a 1:1 stoichiometry (14). However, this value was disk (Fig. 2). The width of the disk is roughly 9–10 Å, corre- subsequently revised after analysis of the effects of ramoplanin sponding to the two antiparallel strands. The pattern of D- and on the conversion of heptaprenyl Lipid I to heptaprenyl Lipid II L-amino acids in the ramoplanin sequence places the side chains by the transglycosylase/transpeptidase PBP1b; these experi- of most of the residues on the convex side of the sheet (i.e., ments led to an estimate of a 2:1 stoichiometry for the ramo- splayed outward from the circumference of the half-disk); the planin:Lipid II complex (17).

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