Cortinarins have been described as cyclic peptides from mushrooms of the genus Cortinarius, and brought into context with mushroom intoxication, particularly causing kidney damage. Herein we report the first total synthesis of the bicyclic peptides cortinarin A and B using iodine-mediated Trp-Cys cyclization to establish their characteristic tryptathionine bridge. Peptide atropisomers, termed ansamers, were observed and characterized by NMR spectroscopy and molecular dynamics simulations. Conformational analysis showed that cortinarins adopt β-turn structures. Furthermore, cortinarins induce similar effects like the peptide hormone arginine vasopressin on the localization of aquaporin-2 in renal principal cells, which regulates water and salt balance in the body, but with a different mode of action. This work thus provides synthetic access to the otherwise elusive cortinarins for biological evaluation and as potential lead compounds for the treatment of renal disorders.

The peptide albicidin represents a highly promising lead structure which is a first-in-class antibiotic with remarkable potency against gram-negative bacteria. Past efforts in the synthesis of albicidin analogs focused on increasing hydrophilicity, broadening of the antibacterial profile and overcoming resistance. Herein, we present synthetic albicidin derivatives with variations in the N-terminal building block and characterize their antibacterial activity and DNA gyrase inhibition. Furthermore, we show that the N-terminus of albicidin greatly affects binding to the resistance factor AlbAL. This transcription regulator senses albicidin and triggers the biosynthesis of the binding protein AlbAS, thus reducing the free concentration of the antibiotic. Here we demonstrate uncoupling of the binding event from transcription activation for some derivatives, and even a few derivatives seemed insensitive to sequestration by AlbA. This approach could be a strategy to develop albicidin analogs escaping AlbA resistance.

In the fight against bacterial infections, particularly those caused by multi-resistant pathogens known as "superbugs", the need for new antibacterials is undoubted in scientific communities and is by now also widely perceived by the general population. However, the antibacterial research landscape has changed considerably over the past years. With few exceptions, the majority of big pharma companies has left the field and thus, the decline in R&D on antibacterials severely impacts the drug pipeline. In recent years, antibacterial research has increasingly relied on smaller companies or academic research institutions, which mostly have only limited financial resources, to carry a drug discovery and development process from the beginning and through to the beginning of clinical phases. This review formulates the requirements for an antibacterial in regard of targeted pathogens, resistance mechanisms and drug discovery. Strategies are shown for the discovery of new antibacterial structures originating from natural sources, by chemical synthesis and more recently from artificial intelligence approaches. This is complemented by principles for the computer-aided design of antibacterials and the refinement of a lead structure. The second part of the article comprises a compilation of antibacterial molecules classified according to bacterial target structures, e.g. cell wall synthesis, protein synthesis, as well as more recently emerging target classes, e.g. fatty acid synthesis, proteases and membrane proteins. Aspects of the origin, the antibacterial spectrum, resistance and the current development status of the presented drug molecules are highlighted.

Antibiotic resistance is a continuously increasing concern for public healthcare. Understanding resistance mechanisms and their emergence is crucial for the development of new antibiotics and their effective use. The peptide antibiotic albicidin is such a promising candidate that, as a gyrase poison, shows bactericidal activity against a wide range of gram-positive and gram-negative bacteria. Here, we report the discovery of a gene amplification-based mechanism that imparts an up to 1000-fold increase in resistance levels against albicidin. RNA sequencing and proteomics data show that this novel mechanism protects Salmonella Typhimurium and Escherichia coli by increasing the copy number of STM3175 (YgiV), a transcription regulator with a GyrI-like small molecule binding domain that traps albicidin with high affinity. X-ray crystallography and molecular docking reveal a new conserved motif in the binding groove of the GyrI-like domain that can interact with aromatic building blocks of albicidin. Phylogenetic studies suggest that this resistance mechanism is ubiquitous in gram-negative bacteria, and our experiments confirm that STM3175 homologs can confer resistance in pathogens such as Vibrio vulnificus and Pseudomonas aeruginosa.

Lanthipeptides are ribosomally-synthesized natural products from bacteria featuring stable thioether-crosslinks and various bioactivities. Herein, we report on a new clade of tricyclic class-IV lanthipeptides with curvocidin from Thermomonospora curvata as its first representative. We obtained crystal structures of the corresponding lanthipeptide synthetase CuvL that showed a circular arrangement of its kinase, lyase and cyclase domains, forming a central reaction chamber for the iterative substrate processing involving nine catalytic steps. The combination of experimental data and artificial intelligence-based structural models identified the N-terminal subdomain of the kinase domain as the primary site of substrate recruitment. The ribosomal precursor peptide of curvocidin employs an amphipathic α-helix in its leader region as an anchor to CuvL, while its substrate core shuttles within the central reaction chamber. Our study thus reveals general principles of domain organization and substrate recruitment of class-IV and class-III lanthipeptide synthetases.

The rising numbers of fatal infections with resistant pathogens emphasizes the urgent need for new antibiotics. Ideally, new antibiotics should be able to evade or overcome existing resistance mechanisms. The peptide antibiotic albicidin is a highly potent antibacterial compound with a broad activity spectrum but also with several known resistance mechanisms. In order to assess the effectiveness of novel albicidin derivatives in the presence of the binding protein and transcription regulator AlbA, a resistance mechanism against albicidin identified in Klebsiella oxytoca, we designed a transcription reporter assay. In addition, by screening shorter albicidin fragments, as well as various DNA-binders and gyrase poisons, we were able to gain insights into the AlbA target spectrum. We analysed the effect of mutations in the binding domain of AlbA on albicidin sequestration and transcription activation, and found that the signal transduction mechanism is complex but can be evaded. Further demonstrating AlbA's high level of specificity, we find clues for the logical design of molecules capable of avoiding the resistance mechanism.

a-amanitin and its derivatives are eight-membered, cyclic peptides that are additionally bridged with a tryptathionine cross-link. Their synthesis sometimes yields isomers, which haven't been classified unambiguously so far. Until now, it was not clear whether these isomers differing in the positioning of the trpytathionine bridge indeed exist and if yes, under which conditions they are formed and how they shall be named. In our work, we therefore investigated the occurrence of these isomers. We have identified the structure and the dynamics of the amanitin derivative 4a and its isomer 4b. Investigating the impact of the macrocyclisation site on the occurrence of this isomer, we published an optimised strategy for atroposelective synthesis (including precursors 3b and 3c). Here, we present the simulated structures of the ansamers 4a and 4b, which both are in very good agreement to crystal data, together with the precursors 3b and 3c. 4a and 4b are the amanitin derivatives, for whose classification we propose the term ansamers. For further instructions, please refer to 'README'. For the files required to reproduce the MD simulations, please refer to the Zenodo repository 'MD setup data for ansamer amanitin derivatives and precursors for atroposelective synthesis' available under DOI: 10.5281/zenodo.7125315.

α-Amanitin is a bicyclic octapeptide composed of a macrolactam with a tryptathionine cross-link forming a handle. Previously, the occurrence of isomers of amanitin, termed atropisomers has been postulated. Although the total synthesis of α-amanitin has been accomplished this aspect still remains unsolved. We perform the synthesis of amanitin analogs, accompanied by in-depth spectroscopic, crystallographic and molecular dynamics studies. The data unambiguously confirms the synthesis of two amatoxin-type isomers, for which we propose the term ansamers. The natural structure of the P -ansamer can be ansa-selectively synthesized using an optimized synthetic strategy. We believe that the here described terminology does also have implications for many other peptide structures, e.g. norbornapeptides, lasso peptides, tryptorubins and others, and helps to unambiguously describe conformational isomerism of cyclic peptides.

Amatoxins are strong inhibitors of RNA polymerase II, and cause cell death. Because of their cytotoxicity they are candidates for anti-cancer drugs, and understanding their structure-activity relationship is crucial. Amatoxins have a rigid bicyclic scaffold which consists of a cyclic octapeptide bridged by cysteine and tryptophan side chain forming a tryptathionine bridge. Here we show the influence of the N-methylation on the amatoxin scaffold by studying Gly5Sar-amanullin with MD simulations and NMR experiments. Since we have shown recently that the amatoxin scaffold allows for two isomeric forms (ansamers), we studied both isomers of Gly5Sar-amanullin. We found that both isomers of Gly5Sar-amanullin form two long-living conformations which is unusual for amatoxins, and that they are differently affected by the N-methylation. The natural Gly5Sar-amanullin forfeits the hydrogen bonds to Gly5 due to the N-methylation, which is expected from existing crystal structures for alpha-amanitin. Our results however indicate that this does not cause more flexibility due to a shift in the hydrogen bond pattern. In the unnatural isomer, we observe an interesting cis-trans-isomerisation of the backbone angles in Trp4 and Gly7, which is enabled by the N-methylation. We expect that our perspective on the effect of N-methylation in amatoxins could be a starting point for further SAR-studies which are urgently needed for the design of better anti-cancer agents.

Synthetic methods on the macrocyclization of peptides are of high interest since they facilitate the synthesis of various types of potentially bioactive compounds, e.g. addressing targets like protein-protein-interactions. Herein, we report on an efficient method to construct tryptathionine-cross-links in peptides between the amino acids Trp and Cys. This reaction not only is the basis for the total synthesis of the death cap toxin α-amanitin but also provides rapid access to various new amanitin analogues. This study for the first time presents a systematic compilation of structure-activity relations (SAR) of amatoxins with regard to RNA polymerase II inhibition and cytotoxicity with one amanitin derivative of superior RNAP II inhibition. The present approach paves the way for the synthesis of structurally diverse amatoxins as future payloads for antibody-toxin conjugates in cancer therapy.

Nonribosomal peptides are a structurally diverse and bioactive class of natural products constructed by multidomain enzymatic assembly lines known as nonribosomal peptide synthetases (NRPSs). While the core catalytic domains and even entire protein subunits of NRPSs have been structurally elucidated, little biophysical work has been reported on the docking domains that promote interactions—and thus transfer of biosynthetic intermediates—between subunits. In the present study, we closely examine the COM domains that mediate COMmunication between donor epimerization (E) and acceptor condensation (C) domains found at the termini of NRPS subunits. Through a combination of X-ray crystallography, circular dichroism spectroscopy, solution- and solid-state NMR spectroscopy, and molecular dynamics (MD) simulations, we provide direct evidence for an intrinsically disordered donor COM region that folds into a dynamic helical motif upon binding to a suitable acceptor. Furthermore, our NMR titration and carbene footprinting experiments illuminate the residues involved at the COM interaction interface, and our MD simulations demonstrate folding consistent with experimental data. Although our results lend credence to the previously proposed helix-hand mode of interaction, they also underscore the importance of viewing COM interfaces as dynamic ensembles rather than single rigid structures and suggest that engineering experiments should account for the interactions which transiently guide folding in addition to those which stabilize the final complex. Through activity assays and affinity measurements, we further substantiate the role of the donor COM region in binding the acceptor C domain and implicate this short motif as readily transposable for noncognate domain crosstalk. Finally, our bioinformatics analyses show that COM domains are widespread in natural product pathways and function at interfaces beyond the canonical type described above, setting a high priority for thorough characterization of these docking domains. Our findings lay the groundwork for future attempts to rationally engineer NRPS domain–domain interactions with the ultimate goal of generating bioactive molecules.

The enoyl-acyl carrier protein reductase enzyme FabI is essential for fatty acid biosynthesis in Staphylococcus aureus and represents a promising target for the development of novel, urgently needed anti-staphylococcal agents. Here, we elucidate the mode of action of the kalimantacin antibiotics, a novel class of FabI inhibitors with clinically-relevant activity against multidrug-resistant S. aureus. By combining X-ray crystallography with molecular dynamics simulations, in vitro kinetic studies and chemical derivatization experiments, we characterize the interaction between the antibiotics and their target, and we demonstrate that the kalimantacins bind in a unique conformation that differs significantly from the binding mode of other known FabI inhibitors. We also investigate mechanisms of acquired resistance in S. aureus and identify key residues in FabI that stabilize the binding of the antibiotics. Our findings provide intriguing insights into the mode of action of a novel class of FabI inhibitors that will inspire future anti-staphylococcal drug development.

Crosstalk between cellular pathways is often mediated through scaffold proteins that function as platforms for the assembly of signaling complexes. Based on yeast two-hybrid analysis, we report here the interaction between two complex scaffold proteins, CREB-binding protein (CBP) and the Ras GTPase-activating-like protein 1 (IQGAP1). Dissection of the interaction between the two proteins reveals that the central, thus far uncharacterized, region of IQGAP1 interacts with the HAT domain and the C-terminal intrinsically disordered region of CBP (termed ID5). Structural analysis of ID5 by solution NMR spectroscopy and SAXS reveals the presence of two regions with pronounced helical propensity. The ID5 region(s) involved in the interaction of nanomolar affinity were delineated by solution NMR titrations and pull-down assays. Moreover, we found that IQGAP1 acts as an inhibitor of the histone acetyltransferase (HAT) activity of CBP. In in vitro assays, the CBP-binding region of IQGAP1 positively and negatively regulates the function of HAT proteins of different families including CBP, KAT5 and PCAF. As many signaling pathways converge on CBP and IQGAP1, their interaction provides an interface between transcription regulation and the coordination of cytoskeleton. Disruption or alteration of the interaction between these scaffold proteins may lead to cancer development or metastatic processes, highlighting the importance of this interaction.

Solid state NMR is a powerful method to obtain information on the structure and dynamics of protein complexes that, due to solubility and size limitations, cannot be achieved by other methods. Here, we present an approach that allows the quantification of microsecond conformational exchange in large protein complexes by using a paramagnetic agent to accelerate 15N R1ρ relaxation dispersion measurements and overcome sensitivity limitations. The method is validated on crystalline GB1 and then applied to a >300 kDa precipitated complex of GB1 with full length human immunoglobulin G (IgG). The addition of a paramagnetic agent increased the signal to noise ratio per time unit by a factor of 5, which allowed full relaxation dispersion curves to be recorded on a sample containing less than 50 μg of labelled material in 5 and 10 days on 850 and 700 MHz spectrometers, respectively. We discover a similar exchange process across the β-sheet in GB1 in crystals and in complex with IgG. However, the slow motion observed for a number of residues in the α-helix of crystalline GB1 is not detected in the complex.

Modular polyketide synthases and non-ribosomal peptide synthetases are molecular assembly lines that consist of several multienzyme subunits that undergo dynamic self-assembly to form a functional megacomplex. N- and C-terminal docking domains are usually responsible for mediating the interactions between subunits. Here we show that communication between two non-ribosomal peptide synthetase subunits responsible for chain release from the enacyloxin polyketide synthase, which assembles an antibiotic with promising activity against Acinetobacter baumannii, is mediated by an intrinsically disordered short linear motif and a β-hairpin docking domain. The structures, interactions and dynamics of these subunits were characterized using several complementary biophysical techniques to provide extensive insights into binding and catalysis. Bioinformatics analyses reveal that short linear motif/β-hairpin docking domain pairs mediate subunit interactions in numerous non-ribosomal peptide and hybrid polyketide-non-ribosomal peptide synthetases, including those responsible for assembling several important drugs. Short linear motifs and β-hairpin docking domains from heterologous systems are shown to interact productively, highlighting the potential of such interfaces as tools for biosynthetic engineering.

Vacuolar ATPases are multisubunit protein complexes that are indispensable for acidification and pH homeostasis in a variety of physiological processes in all eukaryotic cells. An arginine residue (Arg(735)) in transmembrane helix 7 (TM7) of subunit a of the yeast ATPase is known to be essential for proton translocation. However, the specific mechanism of its involvement in proton transport remains to be determined. Arginine residues are usually assumed to "snorkel" toward the protein surface when exposed to a hydrophobic environment. Here, using solution NMR spectroscopy, molecular dynamics simulations, and in vivo yeast assays, we obtained evidence for the formation of a transient, membrane-embedded cation-pi interaction in TM7 between Arg(735) and two highly conserved nearby aromatic residues, Tyr(733) and Trp(737). We propose a mechanism by which the transient, membrane-embedded cation-pi complex provides the necessary energy to keep the charged side chain of Arg(735) within the hydrophobic membrane. Such cation-pi interactions may define a general mechanism to retain charged amino acids in a hydrophobic membrane environment.

Modular polyketide synthases (PKSs) produce numerous structurally complex natural products that have diverse applications in medicine and agriculture. PKSs typically consist of several multienzyme subunits that utilize structurally defined docking domains (DDs) at their N and C termini to ensure correct assembly into functional multiprotein complexes. Here we report a fundamentally different mechanism for subunit assembly in trans-acyltransferase (trans-AT) modular PKSs at the junction between ketosynthase (KS) and dehydratase (DH) domains. This mechanism involves direct interaction of a largely unstructured docking domain (DD) at the C terminus of the KS with the surface of the downstream DH. Acyl transfer assays and mechanism-based crosslinking established that the DD is required for the KS to communicate with the acyl carrier protein appended to the DH. Two distinct regions for binding of the DD to the DH were identified using NMR spectroscopy, carbene footprinting, and mutagenesis, providing a foundation for future elucidation of the molecular basis for interaction specificity.

Covering: up to 2018 The construction of polyketide natural products by type I modular polyketide synthases (PKSs) requires the coordinated action of several protein subunits to ensure biosynthetic fidelity. This is particularly the case for trans-AT PKSs, which in contrast to most cis-AT PKSs, contain split modules and employ several trans-acting catalytic domains. This article summarises recent advances in understanding the protein-protein interactions underpinning subunit assembly and intra-subunit communication in such systems and highlights potential avenues and approaches for future research.