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

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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 antibacterial 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 mimetics, 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). exceptions are Hpg-3, Phe-9, and Chp-17, which are buried The structures of ramoplanin and the related ramoplanose within the interior of the U (i.e., pointing toward the center of have been determined in aqueous solution and shown to be the disk). Burial of these hydrophobic side chains is likely to monomeric (7, 18), which is difficult to reconcile with a 2:1 contribute much of the driving force behind assuming the antibiotic/ligand stoichiometry. Because ramoplanin encounters U-shape, along with steric repulsion between the side chains its natural substrate in the context of the bacterial membrane, arrayed along the convex face of the U. Additional stabilization Walker and coworkers explored its solution structure in meth- is contributed by hydrogen bonds between the phenolic hydroxyl anol, reasoning that a more hydrophobic solvent might mimic the of Chp-17 and the carbonyl oxygen of Hpg-11 and between the dielectric of a membrane–water interface and better reflect the phenolic hydroxyl of Hpg-3 and the main chain amide of Phe-9. environment at the bacterial surface. Indeed, they found that The monomer conformation seen in the crystal structure is ramoplanin dimerizes in methanol (13). The mechanism by very similar to the monomeric solution structure previously which ramoplanin oligomerizes at the membrane–water inter- reported, with an RMS difference in C␣ positions of approxi- face has thus far remained undefined, as have the structural mately 0.8 Å (Fig. S1) (18, 27). The greatest difference between details of ramoplanin’s selective capture of Lipid II. the two structures occurs at Gly-14, where the backbone phi and To provide a membrane-relevant structural context for mech- psi angles differ between the NMR and X-ray structures. Each anistic and pharmacological studies of ramoplanin, we explored of the six structures deposited as the NMR ensemble differ by the use of detergents as membrane mimetics, and were able to approximately 0.1 Å between each other but by approximately use the detergent 1-hexadecyl-trimethyl-ammonium bromide 0.8 Å between either of the crystal chains. The ensemble of NMR (CTAB) to produce highly ordered single crystals of the anti- structures was produced from one of the five structures with the biotic. We report here the x-ray crystal structure of ramoplanin, lowest restraint violations and the RMS deviation between these at a resolution of 1.4 Å. The structure provides a high-resolution five lowest restraint violation structures is 0.7 Å (18). It is not view of the ramoplanin dimer and illustrates a potential means known if the differences seen in the NMR and crystal chains by which ramoplanin interacts with bacterial target membranes. would be more similar if another low restraint violation structure Together with recent structure-activity studies, it also suggests a was chosen for molecular dynamics. In contrast to the moderate mechanism by which ramoplanin recognizes its Lipid II ligand differences between the X-ray and NMR structures, the two (10, 12, 15, 19–25). monomers found in the crystal structure are essentially identical in conformation. Minor differences can be seen in the confor- Results mations of the fatty acid chains, but apart from this, the Quality of the Ramoplanin Structure. SAD phasing coupled with backbones and side chains of the two chains adopt the same density modification led to an extremely high quality experi- structures, with an RMS difference in the positions of C␣ atoms mental electron density map. In this map, positions were defined of only 0.08 Å. unambiguously for all non-carbohydrate atoms of the ramoplanin dimer, as well as for many detergent and solvent molecules. Ramoplanin Dimer Structure. The two monomers in the crystal Refinement of the structure at 1.4 Å resolution was straightfor- asymmetric unit are assembled as an intimate dimer. If each ward and yielded an exceptionally clean and well-defined struc- monomer is considered as a half-disk, as described above, then ture. The exception to this high degree of order is the mannosyl- the dimer is assembled by stacking two half-disks, face-to-face, (1,2)-␣-D-mannose disaccharide attached to Hpg-11, which is with the flat edges of the two half-disks parallel. The second disordered in both monomers. We see diffuse electron density in monomer is related to the first by a 180° rotation, imparting C2

2of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0904686106 Hamburger et al. Downloaded by guest on September 26, 2021 BIOCHEMISTRY

Fig. 2. Architecture of the ramoplanin dimer. A schematic view of the dimer, with one monomer being colored blue and the other gold, is shown in A. The U-shaped character of each monomer and the hemidisk-like shape of the monomers are illustrated in B. C represents the actual structure of the dimer, showing the peptide backbone only. D is a stereoview of the complete dimer, with selected side chains of the blue monomer labeled. An asterisk marks the fatty acid moiety.

symmetry to the dimer. This allows the strands made up of allows us to infer the dimer’s membrane-binding topology, as has residues 9–15 of each monomer to interact in an antiparallel been done for membrane protein-detergent complexes (29). The manner, with a total of six backbone–backbone hydrogen bonds hydrophilic face of the dimer corresponds to the convex portions connecting the two monomers (Table S2). The dimer therefore of the stacked half-disks, while the flat faces represent the contains a single, highly curved, four-stranded antiparallel beta hydrophobic face. The side chains found on the hydrophilic face sheet. of the dimer include Orn-4, Thr-5, Hpg-6, Hpg-7, Hpg-11, In addition to the backbone hydrogen bonding interactions Thr-12, and Hpg-13, while side chains oriented toward the stabilizing the dimer, a hydrogen bond connects the side chains of residue 1 (␤-hydroxy-Asn) of one chain and one conformer of Orn-10 in the other chain. The amine group of the other conformer of Orn-10 approaches the face of Hpg-13 in the opposite monomer, and is well positioned to interact with the ring’s pi electrons. Along with these polar interactions, the dimer is also stabilized by hydrophobic interactions. For example, the aromatic rings of the two Chp residues stack parallel to one another, with a distance of approximately 3.5 Å between the two ring planes. Also, the side chain of Leu-15 in each monomer covers the face of Phe-9 on the opposite monomer. Each monomer buries 30% of its total surface area in the dimer interface (440 Å2 interface area/average monomer surface area of 1,450 Å2); about 61% of the buried area is hydrophobic. The fraction of fully buried atoms (fbu) in the dimer interface is 0.5, a very high value that reflects the intimate packing and high complementarity between the two monomers (Fig. S2) (28). The structure of the ramoplanin dimer is highly amphipathic in nature, a fact that is easily appreciated when examining the distribution of ordered solvent molecules around the dimer (Fig. Fig. 3. The ramoplanin dimer is amphipathic. The dimer is shown in blue and 3). These ordered solvent molecules include water, halide ions, yellow, using an orientation similar to that shown in Fig. 2. Also shown are all and the detergent 1-hexadecyl-trimethyl-ammonium bromide of the ordered waters (red), chloride ions (magenta), and detergent molecules (green) seen in the crystal structure. The distribution of solvent molecules (CTAB). Their distribution around the dimer is strikingly asym- clearly illustrates that one face of the dimer is hydrophilic, whereas the metric, with virtually all water molecules and ions clustering opposite face is hydrophobic. The dotted line shows the boundary between around the hydrophilic face of the dimer, whereas all of the these two faces and is likely to represent the position of the membrane detergent molecules lay on the opposite, hydrophobic face. This interface when ramoplanin interacts with the bacterial target membrane.

Hamburger et al. PNAS Early Edition ͉ 3of6 Downloaded by guest on September 26, 2021 hydrophobic face include Phe-9, Leu-15, and Ala-16, as well as chemical shift changes within this region (12). Several intermo- ramoplanin’s cis,trans-8-methyloctadieneoyl chain. The side lecular NOEs were observed, defining the antibiotic–ligand chains of Asn-1, ␤-hydroxy-Asn-2, Thr-8, and Orn-10 occupy interaction interface and suggesting that residues 3 through 10 interfacial positions midway between the hydrophobic and hy- interact with the MurNAc carbohydrate and pyrophosphate drophilic faces. The carbohydrate moieties that are attached to moieties of the ligand (Fig. S4). Alanine scanning of ramoplanin Hpg-11 are found in the center of the hydrophilic face of the confirmed that some of the residues between 3–10 play critical dimer. roles in antimicrobial activity and identified the critical residues as Orn-10, Hpg-3, Hpg-7, and Orn-4 (listed in order of decreas- Crystal Contacts. Each ramoplanin dimer packs against three ing importance) (23). It is interesting that some of the important other dimers in the crystal lattice. Remarkably, none of these residues observed in the SAR studies are the same residues that contacts represent direct ramoplanin–ramoplanin interactions, show perturbed chemical shifts in the NMR interaction studies, and no atoms in any two dimers approach to within van der which were performed in 20% DMSO. This may indicate that Waals distance of each other. All of the crystal contacts are these residues are capable of interacting with peptidoglycan mediated by bridging detergent molecules and (in a few cases) by precursors in both the monomeric and dimeric form. bridging chloride ions, underscoring the importance of the The structure of the ramoplanin dimer can be used to explain detergent for crystallogenesis (Fig. S3). No specific contacts are some of the results that have been observed in SAR studies. For made between the detergent head groups and the ramoplanin example, mutation of Orn-10 to alanine decreases ramoplanin’s molecules; this, in conjunction with the observation that crystals minimum inhibitory concentration (MIC) by 540-fold (23). can be obtained in the presence of different detergents with Orn-10 is situated at the interface between the hydrophilic and various head groups, suggests that it is the detergent’s amphi- hydrophobic regions of the dimer structure. This positions the pathic character, rather than its cationic nature, that is the key ornithine side chain perfectly to capture the pyrophosphate of determinant for crystallization. Lipid II, since insertion of Lipid II’s undecaprenyl chain into the membrane outer leaflet will place the pyrophosphate moiety at Discussion the membrane interface. The most critical recognition element Ramoplanin and Lipid II-Binding Antibiotics. Ramoplanin is one of of Lipid II is the pyrophosphate (15), and mutating Orn-10 is a growing number of clinically important peptide and depsipep- expected to disrupt the ionic interaction driving that recognition. tide antibiotics that capture Lipid II, the substrate for the It is interesting that the residue 10 position within enduracidins transglycosylase and transpeptidase enzymes responsible for cell A and B is not occupied by an ornithine, but rather by the rare wall biosynthesis. Ramoplanins A1–A3 (and the structurally and cationic amino acid enduracididine, which should also be capa- functionally related enduracidins) share a common target with ble of forming a salt bridge with the Lipid II pyrophosphate (10). glycopeptide antibiotics (vancomycin, teicoplanin), lantibiotics The requirement for a positive charge at position 10 is further (nisin, mersacidin, actagardine, epidermin), and other cyclic illustrated by semisynthetic modification experiments that dem- peptides (mannopeptimycins, katanosin B, plusbacin) (9, 10, 16, onstrated that substrate binding and antimicrobial activity can 30–35). There are no significant primary or structural similari- both be preserved after modification of Orn-10, so long as the ties among these molecules, yet a central theme persists. These modified residue retains its cationic character (12). antibiotics all specifically recognize and sequester the pepti- Conversion of Hpg-3 to alanine is another deleterious muta- doglycan precursor Lipid II in the sea of membrane phospho- tion, leading to a 74-fold decrease in the MIC value (23). The lipids that make up the outer leaflet of the bacterial membrane. mutation would eliminate a structurally significant hydrogen Two of these antibiotics, nisin and mersacidin, have been bond between the phenolic hydroxyl of Hpg-3 and the main chain examined structurally within a membrane context. Mersacidin amide of Phe-9; also, burial of the bulky hydrophobic Hpg side forms a tightly associated 1:1 complex with Lipid II, and nisin chain likely provides an important impetus for ramoplanin to sequesters Lipid II via encapsulating the pyrophosphate of Lipid fold into its convex shape (Fig. 3). Hence, this mutation is II within a hydrogen bond network formed by nisin backbone predicted to fundamentally disrupt the molecule’s structure. In amides (36, 37). The nisin-Lipid II complex organizes into a addition, when ramoplanin forms a complex with Park’s nucle- supramolecular pore-like assembly, engendering a breach in otide, the environment around Hpg-3 is altered, as indicated by membrane integrity and leading to cell death by osmotic lysis chemical shift perturbations and changes in the temperature (38–40). Common to both mersacidin and nisin are structural coefficients of amide deuterium exchange (12). This suggests changes within the antibiotic that are triggered by the encounter that Hpg-3 lies in close proximity to Park’s nucleotide and/or with the membrane environment. The ramoplanin dimer struc- experiences a conformational change upon ligand binding, and ture presented here reveals structural features that are also likely may therefore participate in ligand recognition, in addition to its to reflect conformational changes elicited by a membrane en- structural role. vironment, and highlights both similarities and differences Hpg-7 is found on the hydrophilic face of the molecule, with among the greater family of Lipid II-binding antimicrobials. This its side chain projecting outward. Replacement of this residue by structure, in combination with recent structure-activity and alanine leads to a 53-fold decrease in MIC (23). Its surface biophysical studies, provides insight at the molecular level of the position suggests a role in ligand recognition, rather than roles played by specific residues in Lipid II recognition. structure stabilization. Consistent with this idea, NOEs have been measured between the Hpg-7 sidechain and the muramic Contributions of Different Residues to Lipid II Recognition and Anti- acid moiety in Park’s nucleotide, implying direct interaction (12); biotic Activity. Semisynthetic modification, total synthesis, and a similar interaction is likely to occur during the binding and functional studies of ramoplanin have allowed various investi- sequestration of Lipid II. Positioning of the ligand’s muramic gators to evaluate the contributions of individual amino acids acid group next to Hpg-7 implies that the charged side chain of toward the molecule’s activity (10, 12, 15, 19–25). The results Orn-4 can further stabilize the antibiotic:ligand complex by presented here allow us to rationalize many of these observations interacting with the carboxylate groups on Lipid II. Such a role in terms of the structure. would explain the 44-fold decrease in MIC associated with the A previously reported NMR study implicates residues Hpg-3 ornithine-to-alanine substitution at this position (23) and is also through Orn-10 as playing critical roles in Lipid II recognition. consistent with chemical modification data that demonstrate a NMR titrations of ramoplanin with the Lipid II precursor Park’s requirement for a positively charged side chain at position 4 (16). nucleotide [UDP-MurNAc(pentapeptide)] revealed marked One additional important residue is Leu-15; its conversion to

4of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0904686106 Hamburger et al. Downloaded by guest on September 26, 2021 alanine decreases the MIC by 40-fold (23). Leu-15 lies at the dimer, past Hpg-3, to interact with Orn-4 on the opposite side of dimer interface and packs against the side chain of Phe-9 on the a single ramoplanin chain. Both orientations are plausible and opposing chain. Changing Phe-9 to an alanine only decreases the both agree with binding studies that measured NOEs between MIC by 8.6-fold, suggesting that disruption of this interaction is the Lipid II MurNAc sugar and Hpg-6 and between the lactate not the principle consequence of replacing Leu-15. This residue group attached to the MurNAc and Hpg-7 (12). lies at the hydrophobic/hydrophilic interface of the ramoplanin The C2 symmetry of the dimer suggests that two Lipid II dimer, and its role in stabilizing the ramoplanin–membrane binding sites are possible for each dimer, implying a 1:1 stoichi- interaction may be more important than its packing interaction ometry for the complex. However, more complex binding modes with Phe-9. are possible. For example, two ramoplanin dimers could approach a single Lipid II molecule from opposite directions, with Ramoplanin’s Interactions with the Membrane. The amphipathic each dimer contributing one Orn-10 side chain to a salt bridge nature of the ramoplanin dimer clearly establishes its membrane with the doubly negatively charged pyrophosphate of the Lipid orientation and highlights the importance of the N-acyl chain in II molecule. Such a ligand-bridged dimer–dimer interaction is facilitating membrane anchoring. By semisynthesis, it has been suggested by a lattice contact seen in our crystals, wherein two demonstrated that the ramoplanin N-acyl chain is essential for dimers pack end-to-end, bridged by two CTAB molecules and antibiotic activity; as suggested earlier, we believe this reflects a two chloride ions. The two ions occupy a position that could be critical role for the fatty acyl chain in membrane insertion, which filled by the pyrophosphate group of Lipid II, and are shared is necessary if ramoplanin is to interact with the membrane- between the two facing Orn-10 side chains. Once two dimers anchored Lipid II (19, 20, 24). This fatty acyl modification is come together in such a manner, additional Lipid II molecules conserved between ramoplanins A1–A3, ramoplanose, and the could then bind at the uncomplexed ends of these dimers, recruit enduracidins A and B, underscoring its importance. additional dimers, and so on, ultimately forming a polymeric It is intriguing that the solution-phase ramoplanin monomer structure with a ramoplanin-Lipid II stoichiometry of 2:1. We do and the detergent-solublized dimer have almost identical not yet know if ramoplanin and Lipid II organize into supramo- backbone conformations. This suggests that interaction with lecular assemblies within the membrane, as is the case for the the membrane environment will not elicit a conformational pore-like complex formed by the nisin-Lipid II complex (40, 42). change in ramoplanin. The lack of a membrane-triggered However, it is intriguing to speculate that the ligand-induced conformational change implies that ramoplanin’s conforma- polymerization of ramoplanin that is observed with soluble Lipid tion is dictated principally by its sequence, as is typically found II analogues may reflect an intrinsic property of the antibiotic, BIOCHEMISTRY for larger polypeptides. The conformation of the antibiotic and that formation of such high order assemblies promotes the that recognizes Lipid II is therefore intrinsic and preformed, antibacterial activity. in contrast to the cases of nisin and mersacidin, which both undergo significant conformational rearrangements upon Methods Lipid II binding. This implies that membrane insertion does Ramoplanin Purification. Ramoplanin was a kind gift from Hoechst Celanese. not require Lipid II, and indeed we have observed by NMR Research grade preparations of ramoplanin contain a mixture of the A1–A3 methods that ramoplanin efficiently anchors to unilamellar forms, as well as additional impurities. Thus, before crystallization ramoplanin vesicles composed of physiologically relevant diacyl phosphati- factor A2 was purified to homogeneity by reversed-phase HPLC, using a dylethanolamine and phosphatidylglycerol phospholipids, but Jupiter 10 ␮m C18 column (300 Å, 250 ϫ 21.2 mm; Phenomenex) and a which contain no Lipid II. gradient from water to 70% acetonitrile, both containing 0.1% trifluoroacetic Although the membrane does not appear to elicit a confor- acid. The purified antibiotic exhibited the expected composition, as confirmed mational change in ramoplanin, it is nonetheless expected to by MALDI-TOF mass spectrometry, and displayed an absorption band at 272 perform two important structural roles, namely stabilizing the nm indicative of the cyclic lactone. No linear hydrolyzed ramoplanin was detected before crystallization, and significant amounts of hydrolysis were highly amphipathic ramoplanin dimer and positioning the dimer not observed during crystallogenesis. optimally for recognition of the Lipid II target. Similar effects have been observed with hydrophobic variants of glycopeptide Structure Determination. Large single crystals were grown by the hanging drop antibiotics (41). vapor diffusion method, mixing equal volumes of ramoplanin solution (20 mg/ml in water) and reservoir solution (0.15–0.3 M NaCl, 0.01–0.03 M MgCl2, A Model for Lipid II Recognition. The structure presented herein 0.01 M CTAB) and incubating at 295 K. Crystals could be obtained in similar suggests a clearer model for how ramoplanin may recognize the conditions using detergents other than CTAB, but no crystals were obtained Lipid II target in the context of the bacterial membrane. in the absence of detergent. Crystals used for phasing were soaked overnight Previous work had proposed a model by which the acyl chain in a solution containing 50% saturated ferrocene boronic acid in 0.5 M NaCl, linked to Asn-1 serves as a membrane anchor (19) and this is 0.05 M MgCl2, and 2 mM CTAB. Native and derivative data were measured at borne out in the crystal structure. The hydrophobic polyprenyl beamline X6A of the National Synchrotron Light Source and processed using XDS (43). A fluorescence scan was measured for the putative ferrocene deriv- tail of Lipid II will be inserted into the membrane interior, while ative, and data for this crystal were collected at a wavelength of 1.7375 Å, the polar disaccharide head group with its pendant peptide will corresponding to the peak at the iron K edge. Data collection statistics are be found in the aqueous region; the pyrophosphate group linking given in Table S3. Anomalous scatterer positions were identified using the head and tail will therefore be found at the membrane SHELXD, after which solvent flattening and phase extension in SHELXE were surface, where it will initially encounter Orn-10 and form a salt used to resolve the phase ambiguity and improve the map quality (44–46). The bridge. The Lipid II head group can then interact with the map produced by SHELXE showed clear and unambiguous density for each hydrophilic face of the ramoplanin dimer. The apex of the ramoplanin molecule within the dimer, as well as many solvent atoms and dimer’s hydrophilic face is largely occupied by the pair of detergent molecules. The structure was built using Coot (47) and refined using mannose disaccharides, leaving two possible regions for further phenix.refine (48). Stereochemical libraries were prepared using PRODRG (49) interaction between the Lipid II head group and the antibiotic and phenix.elbow. Statistics for the final refined model are given in Table S4. (Figs. 2, 3, and Fig. S5). One possibility is that the carbohydrate Final refined values for R and Rfree are 0.170 and 0.179, respectively, for all data between 25 and 1.4 Å. and pentapeptide of Lipid II pack against the ramoplanin dimer Once refinement was completed, we re-examined maps for the derivative interface, lying along the top of the dimer and interacting with and could find no evidence for the expected ferrocene-boronic acid derivati- both chains simultaneously. A second possible mechanism has zation reagent. Indeed, the mannose sugars, which were the expected attach- the GlcNAc-MurNAc disaccharide interacting with Hpg-6 and ment point for the boronic acid moiety, are disordered and could not be Hpg-7, whereas the pentapeptide wraps around the side of the resolved in the map. An anomalous difference map using calculated phases

Hamburger et al. PNAS Early Edition ͉ 5of6 Downloaded by guest on September 26, 2021 revealed that all of the anomalous scatters found correspond to chlorine (P.J.L.) and AI46611 (D.G.M.) from the National Institutes of Health (NIH). atoms, including the covalently bound chlorine atoms in Chp-17 and chloride J.B.H. is the recipient of a Crohn’s and Colitis Foundation postdoctoral fellow- ions contributed by the buffer. ship. Diffraction data for this project were measured at beam line X6A of the National Synchrotron Light Source (NSLS), funded by the National Institute of ACKNOWLEDGMENTS. We thank Dr. Nathan Nicely of the Duke Medical General Medical Sciences, NIH, under agreement GM-0080. The NSLS at Center Human Vaccine Institute X-ray Crystallography laboratory for assis- Brookhaven National Laboratory is supported by the U.S. Department of tance with these studies. This work was supported in part by grants GM079508 Energy under contract no. DE-AC02-98CH10886.

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