Mechanism of Action of Ribosomally Synthesized and Post-Translationally Modified Peptides

Abstract

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a natural product class that has undergone significant expansion due to the rapid growth in genome sequencing data and recognition that they are made by biosynthetic pathways that share many characteristic features. Their mode of actions cover a wide range of biological processes and include binding to membranes, receptors, enzymes, lipids, RNA, and metals as well as use as cofactors and signaling molecules. This review covers the currently known modes of action (MOA) of RiPPs. In turn, the mechanisms by which these molecules interact with their natural targets provide a rich set of molecular paradigms that can be used for the design or evolution of new or improved activities given the relative ease of engineering RiPPs. In this review, coverage is limited to RiPPs originating from bacteria.

Figure 1.

General biosynthetic scheme for RiPP maturation. The follower peptide is shown as dashes because it is not ubiquitous across known RiPP classes.

Figure 2.

Structures of thioether crosslinks and dehydrated amino acids commonly found in lanthipeptides. Shorthand notations are presented below the corresponding structure. Structures containing non-canonical LL-(methyl)lanthionine and D-allo-L-methyllanthionine stereochemistry are indicated with either a single or double asterisk in the shorthand notation, respectively.

Figure 3.

(A) Chemical structure of nisin Z (1) and lipid II (2). The A and B rings of nisin interact with the pyrophosphate moiety of lipid II (green) and the C-E rings of nisin (orange) are involved in pore formation. A hinge region (purple) is critical for pore formation. (B) NMR characterization of nisin (space filling) binding to a lipid II analog (sticks). (C) Zoom-in view showing the interactions between the pyrophosphate of lipid II (green and red) with the amide backbone of the nisin A ring (carbon atoms in grey). (D-E) Proposed model for lipid II mediated pore formation wherein nisin first interacts with lipid II through the above-described interaction, followed by pore formation through a complex of eight nisin molecules and four lipid II molecules. The molecular details of the arrangement of nisin and lipid II in the pore remain unknown.

Figure 4.

Shorthand notations for the structures of microbisporicin (3), mutacin 1140 (4), epidermin (5), epilancin 15X (6), geobacillin I (7), and subtilin (8). The nisin-like lipid II binding domains are indicated in blue, nisin-like pore forming domains are indicated in orange, and the hinge region, present in (8), is indicated in purple. The stereochemistry of the dihydroxyproline in (3) is not known. Throughout this review, in the shorthand notations the N-terminus is indicated by H- and the C-terminus by –OH.

Figure 5.

Chemical structures of SapT (9) and pinensin A (10) and B (11). The stereochemistry of the MeLan rings in SapT (9) was determined to be D-allo-L-methyllanthionine, the first lanthipeptide with such stereochemistry.

Figure 6.

Structures of the class II lanthipeptides mersacidin (12), lacticin 481 (13), bovicin HJ50 (14), nukacin ISK-1 (15), plantaricin C (16), and an alternative representation of plantaricin C (17). The 6-amino acid ring containing an Asp/Glu (orange) that is proposed to be the lipid II binding motif is shown in blue. The report on the structure of plantaricin C 16 deduced by NMR spectroscopy acknowledges that the distances between the β-carbons of the lanthionines in the proposed A and D-rings are ~8 Å, whereas these corresponding distances for the B and C rings are ~4 Å. We suggest an alternative possible ring pattern (17) based on the structures of mersacidin, lacticin 481, and nukacin ISK-1.^,

Figure 7.

Two different conformations of nukacin ISK-1 that interconvert on the timescale of seconds. Both structures were solved by NMR spectroscopy (PDB IDs: 5Z5Q and 5Z5R).

Figure 8.

Structures of select two-component lanthipeptides. Lacticin 3147 composed of Ltnα (18, also called LtnA1) and Ltnβ (19, also called LtnA2), haloduracin composed of Halα (20) and Halβ (21), and staphylococcin C55 consisting of SacAα (22) and SacAβ (23). The proposed lipid II binding motifs are colored blue with the Glu that is critical for antibacterial activity in orange.

Figure 9.

(A) Shorthand structures of cytolysin composed of CylLL” (24) and CylLS” (25). Sites with non-canonical LL-stereochemistry are annotated with asterisks (see Figure 2). (B) Two conformations of CylLL” in the bent and extended positions elucidated by solution NMR 28 spectroscopy (PDB ID: 6VGT; BMRB 30710). (C) NMR solution structure of CylLS” (PDB ID: 6VGT; BMRB 30702).

Figure 10.

Chemical structure of cinnamycin (26), and its shorthand rendition (27). Structurally related molecules are shown with lysinoalanine crosslink shown in orange, (methyl)lanthionine rings shown in blue and hydroxyaspartate residues indicated in purple: duramycin (28), ancovenin (29), divamide A (30), and divamide B (31). Asp-OH, (3R)-hydroxy-aspartate.

Figure 11.

NMR structure of cinnamycin (26) bound to C12-lysophosphatidylethanolamine (labeled as PE). The carbons of hydroxyaspartate 15 (HyAsp15) are indicated in yellow and the carbon atoms of PE in pink (PDB ID: 2DDE). For clarity, only one carbon is shown of the C12 acyl chain of PE.

Figure 12.

Structures of labyrinthopeptin A1 (32) and labyrinthopeptin A2 (33). For the structure of labionin, see Figure 2.

Figure 13.

The structure of NAI-112 (34). The sugar stereochemistry has not yet been determined.

Figure 14.

Class III lanthipeptides. (A) Structures of SapB (35), AmfS (36), and catenulipeptin (37). (B) Sequence alignment of the core peptides of catenulipeptin and SapB illustrating the similar constellation of Cys and Ser residues, but different outcome of the cyclization process, resulting in labionins for the former and lanthionines for the latter.

Figure 15.

Chemical structures of ammosamides A (38), B (39) and C (40), lymphostin (41), and 3-thiaglutamate (42).

Figure 16.

(A) Chemical structure of ammosamide 272 (43). (B) Crystal structure showing the interaction between ammosamide 272 (carbons labeled in green) and the myosin 2 heavy chain (PDB ID: 4AE3). Side chains of myosin residues interacting with ammosamide 272 are shown in yellow.

Figure 17.

Crystal structure of ammosamide B (carbon atoms in cyan) bound to QR2 (purple and green; PDB 3UXE). Asn161 is located on monomer A (purple), while Thr71 is located on monomer B (green). The FAD carbons are shown in orange.

Figure 18.

Class V lanthipeptides cacaoidin (44) and lexapeptide (45). The D-Ala that was demonstrated to be important for antibacterial activity of lexapeptide is highlighted in red.

Figure 19.

Structure of microvionin (46).

Figure 20.

Generic scheme for the biosynthesis of azol(in)e on a peptide substrate by a trifunctional heterocyclase. In LAP biosynthesis, the C-D complex is composed of an E1-like protein (yellow) and an ATP-dependent YcaO protein (green), which together perform cyclodehydration of Cys, Ser, and/or Thr residues. The B protein (orange) is an FMN-dependent dehydrogenase that generates the azole. Abbreviations for (methyl)azol(in)es heterocycles used in this review are shown.

Figure 21.

Amino acid sequences of the precursor peptides encoding SLS-like cytolysins. Shown are the predicted leader and core regions for SLS, clostridiolysin S (CLS), listeriolyin S (LLS), stapholysin S (StsA), and the group G Streptococcus (GGS) SLS-like cytolysin. Residues in the SLS sequence that when replaced with Ala abolished cytolytic activity are shown in blue. The exact structure of any of these RiPPs has yet to be determined.

Figure 22.

(A) Chemical structure of plantazolicin (47). The plantazolicin precursor peptide is shown above, and the core peptide is shown in bold. The numbering scheme for (47) is reflected in the labels above the core peptide. The sole methyloxazoline (blue) can be selectively acid hydrolyzed, which results in an 8-fold reduction in MIC against B. anthracis. The N^α-dimethylations (orange) are essential for activity. (B) Minimal synthetic plantazolicin fragment (48) (the C-terminal methyl ester was utilized to facilitate chemical synthesis and to better serve as a mimic of the amino acid that follows).

Figure 23.

Chemical structure of microcin B17 (MccB17) (49). The MccB17 precursor peptide is shown above with the core peptide shown in bold. The numbering scheme for (49) is reflected in the labels above the core peptide.

Figure 24.

Chemical structure of klebsazolicin (50). Ser1 to Ala14 comprises the minimal bioactive core of klebsazolicin (blue). The klebsazolicin precursor peptide is shown above with the core peptide shown in bold. The numbering scheme for (50) is reflected in the labels above the core peptide.

Figure 25.

(A) Positioning of klebsazolicin (Kleb, dark purple) bound to the complex of the ribosome with tRNA (PDB ID: 5W4K). tRNA is bound in the A-site by A-tRNA (A: aminoacyl); P-site tRNA by P-tRNA (P: peptidyl); and E-site by E-tRNA (E: exit). (B) Overview of klebsazolicin blocking the exit tunnel. (C) Interactions of the 23S rRNA (carbon atoms in yellow) with klebsazolicin (carbon atoms in purple). Stacking interactions are shown as blue regions between regions of interest.

Figure 26.

Chemical structure of phazolicin (51). Residues in blue are involved in binding to nucleotides in the exit tunnel. The phazolicin precursor peptide is shown above with the core peptide shown in bold. The numbering scheme for (51) is reflected in the labels above the core peptide.

Figure 27.

Close-up of interactions between E. coli 23S rRNA (carbon atoms in pink) and phazolicin (carbon atoms in blue) (PDB ID: 6U48). Interactions and residues are shown. Stacking interactions are shown as yellow regions between residues of interest. The view in panel B has been rotated by 90°.

Figure 28.

Position of phazolicin (blue carbon residues) in relation to E. coli 50S ribosome uL4 (light green), uL22 (gold) loops (PDB ID: 6U48), and E. coli 23S rRNA (carbon atoms in pink) Key residues on both proteinaceous loops are highlighted.

Figure 29.

Chemical structure of goadsporin (52). Dehydroamino acids are shown in blue; azoles that can be substituted without loss of activity are shown in orange; Gly10 is shown in green. The goadsporin precursor peptide is shown above with the core peptide shown in bold. The numbering scheme for (52) is reflected in the labels above the core peptide.

Figure 30.

(A) Classification of thiopeptides based on the oxidation state of the central heterocyclic ring. (B) A generalized scheme of prokaryotic translation. Thiopeptides known to inhibit any steps in the process are noted. E, exit site; P, peptidyl site; A, aminoacyl site; EF-G, elongation factor G.

Figure 31.

Structures of thiostrepton (53) and micrococcin P1 (54) and P2 (55). Residues that project into the cavity between uL11 and the 23S rRNA are shown in blue. The C-terminal dehydroalanine tail of thiostrepton that is required for activity is highlighted in red. Conjugating chemical species to the penultimate Dha16 (magenta), as opposed to Dha17 results in an analog devoid of activity.

Figure 32.

(A) Isolated view of interactions of thiostrepton with uL11 (green) and helices 43 and 44 of 23S rRNA (pink) from the structure of thiostrepton bound to the 50S ribosomal subunit from D. radiodurans (PDB ID: 3CF5). Residues in the primary macrocycle of thiostrepton that bind between uL11 and the 23S rRNA are shown in cyan. CTD, C-terminal domain; NTD, N-terminal domain. (B) Close-up of the interactions between thiostrepton, uL11 and the 23S rRNA. Key residues on uL11 and the 23S rRNA are shown in yellow. Thz6 is stacked between Pro21 and Pro25 of uL11. The quinaldic acid moiety (QA, carbon atoms in olive) engages in stacking interactions with A1078 of 23S rRNA and Thz1 is stacked against A1106. These positions correspond to A1067 and A1095 respectively in E. coli 23S rRNA.

Figure 33.

Chemical structure of GE2270A (56), thiomuracin A (57), and amythiamycin (58).

Figure 34.

(A) Crystal structure of Ec EF-Tu (PDB ID: 1D8T) bound to GE2270A (orange carbon atoms) and GDP. Ec EF-Tu residues involved in hydrogen bonding are shown in yellow. Salt bridge that locks GE2270A into the binding pocket is shown in red dashed lines. The six amino acids that differ between P. rosea EF-Tu1 and Ec EF-Tu are shown in pink. (B) Overlay of the position of GE2270A (spheres) bound to Tt EF-Tu (PDB ID 2C77) superimposed onto the structure of T. aquaticus EF-Tu bound to GTP (PDB ID: 1EFT). GE2270A intrudes into the region between domains 1 and 2 of “on” state EF-Tu. (C) Overlay of the position of GE2270A (spheres) as bound to Tt EF-Tu superimposed onto the structure of T. aquaticus EF-Tu GNP (light green) bound to yeast Phe-tRNA (yellow, PDB ID: ITTT). GE2270A clashes with the 3’ end of the tRNA molecule (red arrow). Tt EF-Tu in panels (B) and (C) are otherwise hidden from view.

Figure 35.

EF-Tu in “on” (A) and “off” (B) states. EF-Tu adopts a compact state when bound to GTP or non-hydrolysable analogs such as GNP (PDB ID: 1EFT). EF-Tu adopts a relaxed state when bound to GDP (PDB ID: 1EFC).

Figure 36.

(A) Chemical structure of cyclothiazomycin (59), a thiopeptide that does not elicit the TipA response (i.e. lacks dehydroamino acids in the tail region), and (B) structures of those that do elicit the TipA response, thioxamycin (60), and thiotipin (61). The stereochemistry at Ala8 of thioxamycin is not defined.

Figure 37.

Promothiocin binding to TipA_S. (A) Chemical structure of promothiocin A (62). (B) Structure of apo TipA_S (PDB ID: 1N79). (C) Structure of TipA_S bound to promothiocin A (green, PDB ID: 2MBZ). Helices α6-α8 are disordered in the apo structure and become ordered upon thiopeptide binding.

Figure 38.

(A) Close up of TipA_S bound to promothiocin A (carbon atoms shown in green, PDB ID: 2MBZ). (B) Close up of TipA_S bound to nosiheptide (carbon atoms shown in teal, PDB ID: 2MC0). Key interacting residues on TipA_s are shown in yellow. π-π stacking interactions are shown as opaque yellow regions. “Pyr” denotes the pyridine ring in panels (A) and (B). Each thiopeptide and TipA_S interact mainly through hydrophobic packing. Limited hydrogen bonding contacts are made between the protein and the ligand. (C) Chemical structure of nosiheptide (63). (D) Generalized motif in various thiopeptides that is used for recognition by TipA_S.

Figure 39.

Structures of siomycin A (64), thiostrepton A hydrolyzed or methanolyzed at the ester in the B ring (65), and bisdehydroalanine fragment with anticancer activity (66).

Figure 40.

Chemical structures of thiopeptides that do not act as proteasome inhibitors. Thiocillin I (67), berninamycins A (68) and B (69), and YM-266183 (70).

Figure 41.

Chemical structure of bottromycin A2 (71), and its congeners B2 (72), C2 (73), and D (74). The methyl group on C_β of Phe6 (blue background) is critical for activity. The methyl ester (orange) is important because the corresponding carboxylic acid is inactive; however, replacement of the methyl ester with amides, ethyl/propyl esters, and propyl/isopropyl thioesters resulted in compounds with improved activity.

Figure 42.

Chemical structures of thioviridamide (75), pre-thioviridamide (76), thioalbamide (77), and thiostreptamide s4 (78).

Figure 43.

Chemical structures of the cyanobactins ulithiacyclamide (79), cycloxazoline (80), dendroamide A (81), agardhipeptin A (82), telomestatin (83), (R,R,R) QZ59 (84), (S,S,S) QZ59 (85), trunkamide (86), haliclonamide A (87), haliclonamide B (88), sphaerocyclamide (89), kawaguchipeptin A (90), and kawaguchipeptin B (91).

Figure 44.

Chemical structure of the patellamide A (92), and patellamides B-E (93–96).

Figure 45.

Chemical structures of unprocessed McC (97) and processed McC (98). Essential residues required for recognition by YejA are highlighted in blue.

Figure 46.

Schematic of McC processing. Unprocessed McC (97) is synthesized in the cytoplasm of the producer. The adenylation reaction converts the C-terminal Asn to an Asp amide. Met1-Ala6 are shown as blue circles, while the decorated Asp amide is shown as an orange circle with a star. After synthesis and export (left portion), unprocessed McC is imported into a susceptible cell via outer membrane transporters OmpC and OmpF, and the inner membrane transporter YejABEF (right portion). Unprocessed McC (97) is deformylated in the cytoplasm of the target cell, allowing for downstream proteolysis. Cellular proteases PepA, PepB, and PepN then remove the hexapeptide (blue circles) revealing the bioactive compound. Processed McC (98) inhibits the aminoacylation reaction carried out by AspRS.

Figure 47.

Peptide-cytidinylate moiety found on McC^Yps/Bam showing the cytidine and carboxymethyl moieties in blue (99). pro-McC^Yps (100) produced after TldD/E cleavage, the 11 amino acids that remain after cleavage are shown in orange. Neither the site of carboxymethylation nor the stereochemistry at phosphorus of these compounds have been established.

Figure 48.

Peptide sequences of characterized siderophore peptides. Residues colored in blue are predicted to be membrane spanning. The glycosylated trimer of N-(2,3 dihydroxybenzoyl)-L-serine (DHBS) is attached to the C-terminal serine carboxylate on MccE492m (101), and the bridging glucose is shown in orange. MccH47m and MccMm are thought to contain the same post-translational modification as MccE492m.

Figure 49.

Cartoon schematic of the MOAs for MccE492m and MccH47m. The FepA, Fiu, and Cir outer membrane receptors are responsible for recognizing the siderophore-peptide conjugate. The action of TonB is required to translocate the siderophore-peptide into the periplasm. MccE492(m) targets ManYZ in the occluded state to impede mannose import and utilizes the ManYZ as a scaffold for subsequent membrane insertion. Oligomerization of embedded MccE492 leads to pore formation, leakage of solutes (labeled M⁺) out of the cell, and eventual cell death. MccH47m targets ATP synthase and affects proton translocation.

Figure 50.

(A) Structure of pantocin A (102). (B) Reaction catalyzed by L-histidinol phosphate aminotransferase.

Figure 51.

Structure of and proposed model for methanobactin Cu(II) binding. (A) Chemical structure of methanobactin from Methylosinus trichosporium bound to Cu⁺ (103). (B) Crystal structure of methanobactin binding copper (PDB ID: 2XJH). (C) Proposed model for the process of copper binding to methanobactin in the structure shown in panel A.

Figure 52.

Schematic representation of the (A) agr and (B) fsr signaling pathways. The precursor peptide is synthesized with a leader region (orange). AgrB or FsrB is thought to install the thiolactone or lactone linkage (green) on the core peptide. The C-terminal portion of the AIP precursor peptide is removed and cyclized by AgrB. The AIP is secreted into the extracellular environment by an unknown mechanism. SpsB facilitates removal of the leader peptide. The GBAP leader peptide is thought to be removed by FsrB. Secreted proteases GelE (gelatinase, brown) and SprE (serine protease, blue) are factors associated with pathogenicity.

Figure 53.

Chemical structures of the AIPs: AIP-I (104), AIP-II (105), AIP-III (106), and AIP-IV (107). Critical hydrophobic residues, which are part of the “triangular knob” motif, are shown in blue (see Figure 54). Substitution of the residues shown in orange greatly perturbs the three-dimensional structure of the AIPs.

Figure 54.

NMR structures of native AIPs. (A) AIP-I (carbon atoms in purple), (B) AIP-II (carbon atoms in light green), (C) AIP-III (carbon atoms in cyan), and (D) AIP-IV (carbon atoms in orange). Hydrophobic residues inside the macrocycle are labeled. Orientations of the triangular knobs are shown in dark blue.

Figure 55.

NMR structures of (A) AIP-III, (B) AIP-III D4A, depicting the mutated residue in gold, (C) AIP-I, and (D) AIP-I D5A, depicting the mutated residue in gold. The triangular knob is lost in the AIP-III D4A variant and yields a more globular fold (orange line). The triangular knob is maintained in the AIP-I D5A variant, but the position of the exocyclic tail is different (green vs. blue line are pointing in opposite directions). These changes are likely to impact receptor binding.

Figure 56.

(A) Chemical structure of GBAP (108). Critical hydrophobic residues are shown in blue. (B) NMR structure of GBAP highlighting the triangular knob formed by Ile6-Phe7-Gly8.

Figure 57.

Chemical structures of ComX168 (109) and ComX_RO-E-2 (110). The minimal structural features required for ComX_RO-E- activity are highlighted in blue. The inset shows a schematic of ComX pheromone production and activity. ComX peptides activate phosphorylation of ComA mediated by the ComP receptor. Phosphorylated ComA activates expression of surfactin biosynthesis, which is necessary for cellular differentiation in response to environmental changes and production of secreted proteases that are used for cells to escape biofilms.

Figure 58.

(A) Schematic structure and classification of lasso peptides. Important regions of a generic lasso peptide are shown. (B) Class I lasso peptides have two disulfide bonds (orange) that covalently join ring-to-loop and ring-to-tail. A large majority of lasso peptides belong to class II, which lack disulfide linkages; instead, the loop is sterically thought to be locked into place by the side chains of “plug” residues (orange circles) in the tail that are positioned below the ring and sometimes also above the ring. Class III lasso peptides are joined by ring-to-loop disulfides while class IV display a tail-to-tail disulfide linkage. The numbering of the residues in the schematic structure of class I shows that the lasso peptide is right-handed, which is the natural isomer for all currently known lasso peptides.

Figure 59.

Sequences and connectivity of lasso peptides referred to in this review. Green lines represent the isopeptide bond, while orange lines represent disulfide linkages.

Figure 60.

(A) Structure of endothelin type B receptor bound to the human hormone endothelin-1 peptide (PDB ID: 5GLH). The transmembrane (TM) domain is shown in green, while the intracellular domain is shown in orange. (B) Close up of endothelin-1 bound to the endothelin type B receptor. Portions of the linear tail of endothelin-1 (Ile19-Trp21) project into the cavity of the receptor. RES-701–1 and its congeners may bind in a similar way. Peptide sequences of the hormone endothelin 1 and the lasso peptide RES-701–1 are shown (the isopeptide linkage is shown in green and disulfide links in orange).

Figure 61.

Crystal structure of ClpC1 (PDB ID: 3WDB). The N-terminal portion of the protein (circled and labeled) is proposed to interact with lassomycin.

Figure 62.

(A) Surface model of E. coli RNAP β’ subunit bound to MccJ25 (PDB ID: 6N60). (B) Close up of bound MccJ25 (carbon atoms in green). MccJ25 interacts with the E. coli RNAP β’ subunit primarily through hydrophobic interactions. The activation loop (purple), F loop (red), and bridge helix (tan) and active site Mg²⁺ are also shown. (C) Location of resistance-conferring variants are shown as spheres in a wire model of E. coli RNAP β’ subunit. Spheres corresponding to residues in the activation loop (purple), F loop (red), or bridge helix (tan) are colored as in panel B. Sites of other variants are shown in blue.

Figure 63.

Co-crystal structures of FhuA-MccJ25 and FhuA-ferrichrome. (A) The co-crystal structure of MccJ25 (green spheres) bound to FhuA (light blue ribbons) demonstrates that the lasso peptide is bound to the exterior cavity of FhuA (PDB ID: 4CU4). Ferrichrome (brown spheres) bound to FhuA (PDB ID: 1BY5) occupies a similar region. (B) Close-up of the ferrichrome binding pocket. FhuA residues engaging in hydrogen-bonding interactions are shown in yellow (black numbering), while carbon atoms of ferrichrome are shown in brown. (C) Close-up of the MccJ25 binding pocket. Residues engaging in hydrogen-bonding interactions are shown in yellow (black numbering for FhuA, green borders for MccJ25; carbon atoms of MccJ25 shown in green).

Figure 64.

Capistruin binding to RNAP. Capistruin (blue carbon atoms) occupies a similar binding site as MccJ25 (green carbon atoms, see Figure 62) but is not positioned as close to the RNAP active site. The closest residue to the catalytically requisite Mg²⁺ ion is Arg15 (12.8 Å away), as compared to His5 in MccJ25 (5.9 Å).

Figure 65.

Chemical structure of tryptorubin A (111). The macrocycle formed by Trp2, Tyr3, and Trp5 is shown in blue.

Figure 66.

Alignment of the microviridin core sequences. Residues that are involved in macrolactone (orange and purple background) and macrolactam (green background) formation are shown. The identity of residue at position 5 (blue background) largely dictates target protease specificity. A similar figure is reported in ref () but has errors at positions 12 and 13 in the last two entries.

Figure 67.

Structures of group I microviridins A (112), B (113), G (114), J (115), K (116), and L (117). Group I microviridins contain two macrolactones (shown in blue) and one macrolactam (shown in orange) which are installed by ATP grasp ligases. Residues involved in the microviridin L mutagenesis study are labeled.

Figure 68.

Structures of the group II microviridins C (118), D (119), E (120), and H (121). Group II microviridins contain two macrolactones (shown in blue) and one macrolactam (shown in orange) which are installed by ATP grasp ligases.

Figure 69.

Chemical structures of microviridin F (122), marinostatin (123), and plesiocin (124). The hairpin motifs in plesiocin are outlined in green.

Figure 70.

Co-crystal structure of bovine trypsin (teal) bound to microviridin J (PDB ID:4KTU). Residues Thr4, Arg5, and Leu6 of microviridin J (purple carbon atoms) mimic the substrate of trypsin. The Ser195 nucleophile is proximal to the scissile bond (black arrow), but peptide cleavage is prevented by the rigid microviridin J structure.

Figure 71.

Four peptidic protease inhibitors. (A) Marinostatin, (B) microviridin J, (C) SSI, and (D) OMTKY3. All four inhibitors adopt similar structural conformations of residues that mimic the P2-P2’ sites in substrates.

Figure 72.

(A) Crystal structure of dimeric AS-48 obtained in Tris-HCl pH 7.5 (left, PDB ID: 1O83). Hydrophobic helices are shown in orange, while hydrophilic helices are shown in blue. Crystal structure of dimeric AS-48 in the presence of the detergent decyl-β-D-maltoside (right, PDB ID: 1084). (B) Hydrophobic surface models of AS-48 in aqueous solution (left) and in the presence of detergent (right). Hydrophobic residues are shown in orange; hydrophilic residues are shown in blue. (C) Structure of carnocyclin A (PDB ID: 2KJF). Hydrophobic helices are shown in silver, while hydrophilic helices are shown in green. (D) NMR solution structure of acidocin B obtained in deuterated sodium dodecyl sulfate and D₂O (PDB ID: 2MWR). Amphipathic helices are shown in salmon, while the hydrophobic helix is shown in cyan.

Figure 73.

Cartoon representation of the toroidal pore arrangement of AS-48 (shown as blue cylinders) in the lipid bilayer. Three copies of AS-48 are hypothetically required for pore formation.

Figure 74.

Chemical structures of the pheganomycins (125–128) and deoxypheganomycin D (129). A substituent at position 4 of the aromatic ring appears important for bioactivity of these compounds (blue). The terminal aspartate (orange) of deoxypheganomycin D may contribute to its bioactivity. The proteinogenic portion of both compounds (boxed) is labeled with the original residues above the structures prior to post-translational modification.

Figure 75.

Chemical structure of crocagin A (130) and crocagin B (131). Crocagin B is dehydrogenated between Cα and Cβ of Tyr (C=C bond shown in blue).

Figure 76.

Glycocin subclasses and NMR structures. (A) Chemical structures of sublancin (132), glycocin F (133), and enterocin F4–9 (134). (B) NMR three-dimensional structures of sublancin (PDB ID: 2MIJ) and (C) glycocin F (PDB ID: 2KUY).

Figure 77.

(A) Schematic representation of the glucose PTS used to import glucose into B. subtilis. A relay mechanism transfers a phosphate from phosphoenolpyruvate (PEP) via several protein carriers to the hydroxyl at C6 of the incoming glucose. A separate phosphorylation mechanism is involved in catabolite repression. (B) Replacement of the glucose on sublancin with xylose (which lacks C6) did not compromise antimicrobial activity. R = sublancin aglycon.

Figure 78

Proposed model for glycocin F-receptor interactions. The O-linked GlcNAc in the loop between the helices is black while the C-terminal S-linked GlcNAc is white. (A) Both sugars bind the GlcNAc-specific PTS membrane spanning EIIC domain (purple) followed by (B) the loop GlcNAc binding its primary target (blue) that has yet to be identified. Disulfides are shown in orange.

Figure 79.

Schematic structure of pallidocin (135).

Figure 80.

Chemical structure of RaxX21 (136). Post-translational modiications are colored in blue. The C-terminus of the peptide contains residues that are important for recognition by XA21 (orange). Amino acid sequences of Arabidopsis PSY1, RaxX21 and Rax13 are shown (top), highlighting the position of the tyrosine residue in each (green). Additional chemical modifications are found on Arabidopsis PSY1 (Pro-4-hydroxylation and O-glycosylation).

Figure 81.

Structure of polytheonamide B (137, pTB). The N-terminal 5,5-dimethyl-2-oxohexanoate is indicated in pink. L-amino acids are shown in blue, while D-amino acids are shown in orange. SAR studies utilized derivatives of pTB (137a-i) that were modified at the N-terminal 5, 5-dimethyl-2-oxohexanoate group (R₁), residue 44 (R₂), β-substituents of residues 2, 22, 29, and 37 (X, and Y), and residue 47 (Z).

Figure 82.

(A) Solution NMR structure of polytheonamide B (PDB ID: 2RQO). The hydrogen-bonding network (dashed lines) occurring between D-Asn residues is presumed to stabilize a pore-forming conformation. (B) Polytheonamide B exhibits a relatively polar C-terminus (left) and a more nonpolar N-terminus (right). Hydrophobic residues are colored from orange (hydrophobic) to blue (hydrophilic).

Figure 83.

Bioactivity of polytheonamide B fragments. The C-terminal fragment D exhibited the highest membrane perturbation measured by carboxyfluorescein (CF) release from egg yolk phosphatidylcholine and cholesterol (10:1) liposomes but lacked ion transport ability measured by H⁺/Na⁺ exchange. Conversely, N-terminal fragments lacked membrane perturbation activity. Additions of N-terminal fragments to fragment D yielded a compound that lost membrane disruption activity but gained ion transport activity. Fragment IC₅₀ values were determined in vitro against murine p388 leukemia cells. Ion exchange and CF release values were estimated from the maximal percentage of each variable released over the time course of the experiment. Percentage exchange monitors loss of the starting material from inside the liposome, i.e. 5% exchange means 5% left the liposome interior. Figure adapted from reference.

Figure 84.

(A) Structure of darobactin (138). Strands β1 (blue) and β16 (orange) surround the lateral pore of BamA. (B) The open state of the lateral pore shown in ribbons. (C) The open state of the lateral pore highlights the hydrogen bonding contacts between β1 and β16. (D) The closed state of the lateral pore. (E) Location of resistance-conferring mutations in BamA, noting proximity to the lateral pore. (F) Close-up of β-strand mimicry interactions between darobactin (pink carbon atoms) and β1 of BamA (blue carbon atoms).

Figure 85.

(A) Chemical structure of subtilosin A (139). (B) Hydrophobicity surface map of subtilosin A (PDB ID: 1PXQ). Hydrophobic regions are shown in orange, while hydrophilic regions are shown in blue. The hydrophilic patch composed of Asn1, Lys2 and Trp34 is highlighted by the red curved line. The right panel shows the same surface rotated 180° about the vertical axis.

Figure 86.

The structures of the sactipeptides thuricin H (140), thuricin CD, composed of two components thuricin α and thuricin β (141, 142), and huazicin/thuricin Z (143). Thuricin Z/huazacin stereocenters marked with * do not have assigned stereochemistry.

Figure 87.

Chemical structures of sporulation killing factor (144), RumC (145), and streptosactin (146). NMR solution structure of RumC is shown in the inset (PDB ID: 6T33). Stereocenters marked with * do not have an assigned absolute stereochemistry.

Figure 88.

Structure of EpeX* (147). Val4 and Ile12 are epimerized to the D-configuration (blue).

Figure 89.

(A) Schematic of the lia cell stress response signaling pathway. (B) Schematic of the lia signaling pathway in response to EpeX*. EpeP is a transmembrane protease that processes and exports EpeX*. EpeAB is an ABC transporter involved in providing self-immunity against the antibacterial compound.

Figure 90.

Structure of pre-mycofactocin (148).