Gene amplifications cause high-level resistance against albicidin in gram-negative bacteria.

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.

Discovery of the Lanthipeptide Curvocidin†and Structural Insights into its Trifunctional Synthetase CuvL.

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.

Iodine-Mediated Tryptathionine Formation Facilitates the Synthesis of Amanitins.

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.

Mechanism of intersubunit ketosynthase-dehydratase interaction in polyketide synthases.

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.

The Kalimantacin Polyketide Antibiotics Inhibit Fatty Acid Biosynthesis in Staphylococcus aureus by Targeting the Enoyl-Acyl Carrier Protein Binding Site of FabI.

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.

A cation-π interaction in a transmembrane helix of vacuolar ATPase retains the proton-transporting arginine in a hydrophobic environment.

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 (Arg735) 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-π interaction in TM7 between Arg735 and two highly conserved nearby aromatic residues, Tyr733 and Trp737 We propose a mechanism by which the transient, membrane-embedded cation-π complex provides the necessary energy to keep the charged side chain of Arg735 within the hydrophobic membrane. Such cation-π interactions may define a general mechanism to retain charged amino acids in a hydrophobic membrane environment.

Protein-protein interactions in trans-AT polyketide synthases.

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.

In Vitro Biosynthetic Studies of Bottromycin Expand the Enzymatic Capabilities of the YcaO Superfamily.

The bottromycins belong to the ribosomally synthesized and posttranslationally modified peptide (RiPP) family of natural products. Bottromycins exhibit unique structural features, including a hallmark macrolactamidine ring and thiazole heterocycle for which divergent members of the YcaO superfamily have been biosynthetically implicated. Here we report the in vitro reconstitution of two YcaO proteins, BmbD and BmbE, responsible for the ATP-dependent cyclodehydration reactions that yield thiazoline- and macrolactamidine-functionalized products, respectively. We also establish the substrate tolerance for BmbD and BmbE and systematically dissect the role of the follower peptide, which we show serves a purpose similar to canonical leader peptides in directing the biosynthetic enzymes to the substrate. Lastly, we leverage the expanded capabilities of YcaO proteins to conduct an extensive bioinformatic survey to classify known YcaO chemistry. This analysis predicts new functions remain to be uncovered within the superfamily.

Characterization of Protein-Protein Interfaces in Large Complexes by Solid-State NMR Solvent Paramagnetic Relaxation Enhancements.

Solid-state NMR is becoming a viable alternative for obtaining information about structures and dynamics of large biomolecular complexes, including ones that are not accessible to other high-resolution biophysical techniques. In this context, methods for probing protein-protein interfaces at atomic resolution are highly desirable. Solvent paramagnetic relaxation enhancements (sPREs) proved to be a powerful method for probing protein-protein interfaces in large complexes in solution but have not been employed toward this goal in the solid state. We demonstrate that 1H and 15N relaxation-based sPREs provide a powerful tool for characterizing intermolecular interactions in large assemblies in the solid state. We present approaches for measuring sPREs in practically the entire range of magic angle spinning frequencies used for biomolecular studies and discuss their benefits and limitations. We validate the approach on crystalline GB1, with our experimental results in good agreement with theoretical predictions. Finally, we use sPREs to characterize protein-protein interfaces in the GB1 complex with immunoglobulin G (IgG). Our results suggest the potential existence of an additional binding site and provide new insights into GB1:IgG complex structure that amend and revise the current model available from studies with IgG fragments. We demonstrate sPREs as a practical, widely applicable, robust, and very sensitive technique for determining intermolecular interaction interfaces in large biomolecular complexes in the solid state.

pE-DB: a database of structural ensembles of intrinsically disordered and of unfolded proteins.

The goal of pE-DB (http://pedb.vib.be) is to serve as an openly accessible database for the deposition of structural ensembles of intrinsically disordered proteins (IDPs) and of denatured proteins based on nuclear magnetic resonance spectroscopy, small-angle X-ray scattering and other data measured in solution. Owing to the inherent flexibility of IDPs, solution techniques are particularly appropriate for characterizing their biophysical properties, and structural ensembles in agreement with these data provide a convenient tool for describing the underlying conformational sampling. Database entries consist of (i) primary experimental data with descriptions of the acquisition methods and algorithms used for the ensemble calculations, and (ii) the structural ensembles consistent with these data, provided as a set of models in a Protein Data Bank format. PE-DB is open for submissions from the community, and is intended as a forum for disseminating the structural ensembles and the methodologies used to generate them. While the need to represent the IDP structures is clear, methods for determining and evaluating the structural ensembles are still evolving. The availability of the pE-DB database is expected to promote the development of new modeling methods and leads to a better understanding of how function arises from disordered states.

Synonymous constraint elements show a tendency to encode intrinsically disordered protein segments.

Synonymous constraint elements (SCEs) are protein-coding genomic regions with very low synonymous mutation rates believed to carry additional, overlapping functions. Thousands of such potentially multi-functional elements were recently discovered by analyzing the levels and patterns of evolutionary conservation in human coding exons. These elements provide a good opportunity to improve our understanding of how the redundant nature of the genetic code is exploited in the cell. Our premise is that the protein segments encoded by such elements might better comply with the increased functional demands if they are structurally less constrained (i.e. intrinsically disordered). To test this idea, we investigated the protein segments encoded by SCEs with computational tools to describe the underlying structural properties. In addition to SCEs, we examined the level of disorder, secondary structure, and sequence complexity of protein regions overlapping with experimentally validated splice regulatory sites. We show that multi-functional gene regions translate into protein segments that are significantly enriched in structural disorder and compositional bias, while they are depleted in secondary structure and domain annotations compared to reference segments of similar lengths. This tendency suggests that relaxed protein structural constraints provide an advantage when accommodating multiple overlapping functions in coding regions.

Structural characterization of intrinsically disordered proteins by NMR spectroscopy.

Recent advances in NMR methodology and techniques allow the structural investigation of biomolecules of increasing size with atomic resolution. NMR spectroscopy is especially well-suited for the study of intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) which are in general highly flexible and do not have a well-defined secondary or tertiary structure under functional conditions. In the last decade, the important role of IDPs in many essential cellular processes has become more evident as the lack of a stable tertiary structure of many protagonists in signal transduction, transcription regulation and cell-cycle regulation has been discovered. The growing demand for structural data of IDPs required the development and adaption of methods such as 13C-direct detected experiments, paramagnetic relaxation enhancements (PREs) or residual dipolar couplings (RDCs) for the study of 'unstructured' molecules in vitro and in-cell. The information obtained by NMR can be processed with novel computational tools to generate conformational ensembles that visualize the conformations IDPs sample under functional conditions. Here, we address NMR experiments and strategies that enable the generation of detailed structural models of IDPs.

Transcription activation by the resistance protein AlbA as a tool to evaluate derivatives of the antibiotic albicidin.

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.

The neurotransmitter serotonin interrupts α-synuclein amyloid maturation.

Indolic derivatives can affect fibril growth of amyloid forming proteins. The neurotransmitter serotonin (5-HT) is of particular interest, as it is an endogenous molecule with a possible link to neuropsychiatric symptoms of Parkinson disease. A key pathomolecular mechanism of Parkinson disease is the misfolding and aggregation of the intrinsically unstructured protein α-synuclein. We performed a biophysical study to investigate an influence between these two molecules. In an isolated in vitro system, 5-HT interfered with α-synuclein amyloid fiber maturation, resulting in the formation of partially structured, SDS-resistant intermediate aggregates. The C-terminal region of α-synuclein was essential for this interaction, which was driven mainly by electrostatic forces. 5-HT did not bind directly to monomeric α-synuclein molecules and we propose a model where 5-HT interacts with early intermediates of α-synuclein amyloidogenesis, which disfavors their further conversion into amyloid fibrils.

The occurrence of ansamers in the synthesis of cyclic peptides.

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.

Solution structure and membrane binding of the toxin fst of the par addiction module.

The par toxin-antitoxin system is required for the stable inheritance of the plasmid pAD1 in its native host Enterococcus faecalis. It codes for the toxin Fst and a small antisense RNA which inhibits translation of toxin mRNA, and it is the only known antisense regulated toxin-antitoxin system in Gram-positive bacteria. This study presents the structure of the par toxin Fst, the first atomic resolution structure of a component of an antisense regulated toxin-antitoxin system. The mode of membrane binding was determined by relaxation enhancements in a paramagnetic environment and molecular dynamics simulation. Fst forms a membrane-binding alpha-helix in the N-terminal part and contains an intrinsically disordered region near the C-terminus. It binds in a transmembrane orientation with the C-terminus likely pointing toward the cytosol. Membrane-bound, alpha-helical peptides are frequently found in higher organisms as components of the innate immune system. Despite similarities to these antimicrobial peptides, Fst shows neither hemolytic nor antimicrobial activity when applied externally to a series of bacteria, fungal cells, and erythrocytes. Moreover, its charge distribution, orientation in the membrane, and structure distinguish it from antimicrobial peptides.

Dynamics and orientation of a cationic antimicrobial peptide in two membrane-mimetic systems.

In order to investigate the functional and structural properties of cationic alpha-helical peptides in two different membranes, we studied the 20-residue peptide maximin H6 in two membrane-mimetic systems by NMR spectroscopy using partially (15)N-labeled peptide and paramagnetic relaxation enhancements. Maximin H6, which is found in skin secretions of frogs of the Bombinae family, attacks gram-negative bacteria and acts haemolytically. While the peptide spontaneously folds into similar structures in both neutral dodecylphosphocholine (DPC) and negatively charged sodium dodecyl sulphate (SDS) micelles, its structure is more flexible in SDS as shown by (15)N relaxation measurements. In addition, it is bound closer to the surface of the micelle and rotated by approximately 70 degrees around its helix axis in the negatively charged membrane surrogate compared to the structure in DPC. This might form the basis for peptide-peptide interactions through a GxxxG motif, which could finally lead to membrane disruption and, thus, preferential attack of negatively charged microbial cell walls.

The peptide hormone ghrelin binds to membrane-mimetics via its octanoyl chain and an adjacent phenylalanine.

The peptide hormone ghrelin, which is the natural ligand of the membrane-bound growth hormone secretagogue receptor (GHS-R), regulates overall body and cell growth, energy homeostasis, carbohydrate, protein and lipid metabolism and water electrolyte balance. It contains an O-acyl linked octanoyl group on Ser3 and is the only peptide known to contain such a modification. Using solution state NMR spectroscopy and ultrafiltration we found that human ghrelin binds to membrane-mimetic environments via its octanoyl group as well as the aromatic moiety of Phe4. Relaxation enhancements in a paramagnetic environment reveal that both the octanoyl group on Ser3 and the aromatic group on Phe4 are inserted deep into the hydrophobic core of phosphocholine assemblies while the remaining peptide is freely mobile in solution. In contrast, no binding was observed for des-octanoyl ghrelin. Thus, the octanoyl chain, together with the Phe4 aromatic group of ghrelin, functions as a membrane anchor. Our results are in parallel with the previous finding that a bulky hydrophobic group on Ser3 and Phe4 of ghrelin are necessary for its function and thus indicate that membrane-binding is essential for ghrelin function.

Interaction between the scaffold proteins CBP by IQGAP1 provides an interface between gene expression and cytoskeletal activity.

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.