ch3616.qxd 11/24/1999 9:55 AM Page 672 672 Synergy and duality in peptide antibiotic mechanisms Dewey G McCafferty*, Predrag Cudic, Michael K Yu, Douglas C Behenna and Ryan Kruger The molecular mechanisms by which peptide antibiotics Synergistic peptide antibiotics disrupt bacterial DNA synthesis, protein biosynthesis, cell wall Synergism in targeting ribosomal protein biosynthesis biosynthesis, and membrane integrity are diverse, yet Thiopeptide antibiotics historically have been understood to follow a theme of one Thiostrepton and micrococcin are members of the class of antibiotic, one inhibitory mechanism. In the past year, thiazole-containing peptide antibiotics (or thiopeptide mechanistic and structural studies have shown a rich diversity antibiotics), which inhibit protein biosynthesis by binding in peptide antibiotic mechanism. Novel secondary targeting to the 23S rRNA subunit of the ribosome and preventing mechanisms for peptide antibiotics have recently been its proper function [1•–3•]. This family of antibiotics (see discovered, and the mechanisms of peptide antibiotics Figure 1) is characterized by highly modified peptide involved in synergistic relationships with antibiotics and backbones, in which thiazole, 4,2′-bisthiazole, didehy- proteins have been more clearly defined. In apparent response droalanine and didehydrobutyrine residues are formed to selective pressures, antibiotic-producing organisms have from oxidative cyclization and dehydration modifications elegantly integrated multiple functions and cooperative of cysteine, serine and threonine residues [4•]. interactions into peptide antibiotic design for the purpose of improving antimicrobial success. The 23S rRNA site where GTPase-driven elongation fac- tors EF-Tu and EF-G bind is one of the most important functional regions of the ribosome. This site is character- Addresses Department of Biochemistry and Biophysics and Johnson Research ized by two highly conserved regions of the 23S rRNA Foundation, The University of Pennsylvania School of Medicine, ribosomal subunit, the 17-nucleotide sarcin/ricin loop, and Philadelphia, PA 19104-6059, USA the 58-nucleotide domain associated with the ribosomal *e-mail: [email protected] protein L11. Binding of the L11 carboxyl terminus to the Current Opinion in Chemical Biology 1999, 3:672–680 58-nucleotide domain of the rRNA stabilizes its folded con- formation [5••,6••]. The amino-terminal domain of L11 is 1367-5931/99/$ — see front matter © 1999 Elsevier Science Ltd. highly conserved, proline-rich, and functions as a molecular All rights reserved. switch, mediating a conformational change that allows elon- Abbreviations gation factors to properly bind and propagate peptide EF elongation factor synthesis. Thiopeptide antibiotics bind to the 23S rRNA Lipid I undecaprenyl-pyrophosphoryl-MurNAc-pentapeptide and place a conformational constraint on protein L11, lock- Lipid II undecaprenyl-pyrophosphoryl-MurNAc-(GlcNAc)- ing it into a conformation that disfavors proper binding pentapeptide PBP penicillin-binding protein and/or function of EF-Tu and EF-G, effectively halting PE phosphatidylethanolamine protein synthesis [1•,2•]. Remarkably, thiostrepton-producing microorganisms have tailored this antibiotic not only to target a unique, function- Introduction ally-important conformational change in protein L11 but Peptide antibiotics are produced by bacterial, mam- also to manipulate this essential protein for the purpose of malian, insect, and plant organisms in defense against conferring cooperative, high-affinity binding and intimate invasive microbial pathogens. Evolution has crafted pep- specificity. Binding of thiostrepton and L11 protein togeth- tide antibiotics into functionally optimized compounds er to rRNA is synergistic and cooperative and essentially with defined specificity and higher order functionality. irreversible [7]; however, in the absence of rRNA, thiostrep- The number and composition of peptide antibiotics are ton and L11 do not associate and without L11 thiostrepton myriad, reflecting origins from ribosomal, post-transla- affinity for rRNA is comparatively weak [6••]. tional, or non-ribosomal biosynthetic means. Likewise their modes of action are equally diverse. As our under- The molecular basis for this unusual synergistic relation- standing of peptide antibiotic mechanisms steadily ship is rapidly becoming clearer. Resistant mutants and increases, two prevalent mechanistic themes are emerg- chemical footprinting experiments have been employed to ing: molecular synergy and functional duality. Molecular predict the target sites of thiostrepton and micrococcin. synergy is defined as the combined action of two or more High level resistance to thiostrepton is conferred both by antibiotics toward a single target molecule. Functional mutation of the conserved amino-terminal helix of L11 at duality is defined as one molecule having dual antibiotic Pro22, as well as by rRNA 2′-O-methylation at A1067 and activity. The purpose of this review is to survey recent mutation of nucleotides A1067 and A1095. Footprinting examples of the intriguing themes of synergy and duali- experiments confirmed protection of these nucleotide posi- ty in peptide antibiotic mechanism. tions with thiostrepton and micrococcin, suggesting that ch3616.qxd 11/24/1999 9:55 AM Page 673 Synergy and duality in peptide antibiotic mechanisms McCafferty et al. 673 Figure 1 Chemical structures of synergistic ribosome- targeting peptide antibiotics. N O O N OH N N H O HN R O O O 1 O O N O H O N O N O N O NH O O HO N Pristinamycin IIA Pristinamycin IA OH ONH2 ONH HN HN O O O S N N S H O N N S N H O S N H N N N S N N O H HO S HN O H N O N N O HO S HN HN O HO S HN O O O N HN HN OH H O S N N O S H N N O OH N HO S O Micrococcin Thiostrepton Current Opinion in Chemical Biology the two antibiotics bind to overlapping regions of rRNA. peptide-derived antibiotics that act synergistically to White and co-workers [6••] recently reported the crystal inhibit ribosomal peptidyl transfer during bacterial protein structure of the L11–rRNA complex, which confirmed the biosynthesis [8–10]. Streptogramin type A (pristi- hypothesis that A1067, A1089 of the rRNA and the amino- namycin IIA, virginiamycin M1) and type B terminal proline-rich helix of L11 are adjacent and suggests (pristinamycin IA, virginiamycin S1) antibiotics are macro- that the exposed cleft between the three juxtaposed cyclic lactone peptolides produced by Streptomyces strands is the candidate thiostrepton-binding site pristinaespiralis and Streptomyces virginiae. Type A strep- (Figure 2). From the structure of the L11–rRNA complex, togramins inactivate the donor (P) and acceptor (A) sites of synergistic binding could be explained by initial displace- the peptidyl transferase region of the 23S rRNA subunit by ment of a loosely-associated amino-terminal helix of L11 by blocking two of the peptide chain elongation steps: thiostrepton followed by back-binding of the displaced aminoacyl–tRNA binding to the A site; and peptide bond helix onto the rRNA–antibiotic complex, locking in the formation with peptidyl–tRNA at the P site. The type B tightly-associated ternary complex. Thiopeptides also con- streptogramins inhibit peptide bond formation by binding tain potentially reactive didehydroalanine residues, which to the 23S rRNA peptidyl transferase loop, forcing the could participate in covalent capture of either rRNA or L11. release of incomplete peptide chains [9,10]. This mode of Confirmation of structure of the ternary complex by X-ray action is similar to that of the 14-member macrolide fami- analysis is anxiously awaited and will, we hope, better our ly of antibiotics (which includes erythromycin) and also to understanding of the molecular basis of the observed syn- that of the lincosamides, and suggests that streptogramin B ergy and high affinity association. antibiotics share overlapping binding sites with these structurally unrelated antibiotics [9]. Streptogramin antibiotics The streptogramin antibiotics (also known as the syn- Separately, streptogramins A and B are bacteriostatic, yet ergimycins) consist of pairs of structurally unrelated when administered in combination in vivo the antibiotics ch3616.qxd 11/24/1999 9:55 AM Page 674 674 Biopolymers Figure 2 same RNA binding site (as shown by their overlapping footprints), suggesting that a unique heterodimeric antibi- otic complex forms upon contact with RNA that juxtaposes regions 2058–2062 with 2503–2506, simultaneously bridg- Thiostrepton binding site ing the two adjacent strands of the 23S rRNA. A1067 Synergism in membrane permeabilization A1095 As a defense mechanism against microbial pathogens, insects, mammals, amphibians, and plants produce antimi- crobial polypeptides such as the defensins, cecropins, magainins, and the type A lantibiotics [14•–16•,17,18•,19•]. Antimicrobial peptides in these classes are produced riboso- mally, and in some cases undergo further post-translational Pro22 modifications that are essential for export and optimal activ- ity. Typically cationic, peptides in these classes generally act by interacting with anionic membrane phospholipids caus- ing pore formation, which leads to disruption of the proton motive force, cell metabolite leakage, and cellular lysis. Within these classes (Table 1) a common theme of func- tional synergism between membrane targeting peptide antibiotics is rapidly emerging. Current Opinion in Chemical Biology Magainins Magainin 2 and PGLa are members of the magainin fami-