Molecular insights into antibiotic resistance - how a binding protein traps albicidin

The worldwide emergence of antibiotic resistance poses a serious threat to human health. A molecular understanding of resistance strategies employed by bacteria is obligatory to generate less-susceptible antibiotics. Albicidin is a highly potent antibacterial compound synthesized by the plant-pathogenic bacterium Xanthomonas albilineans. The drug-binding protein AlbA confers albicidin resistance to Klebsiella oxytoca. Here we show that AlbA binds albicidin with low nanomolar affinity resulting in full inhibition of its antibacterial activity. We report on the crystal structure of the drug-binding domain of AlbA (AlbAS) in complex with albicidin. Both α-helical repeat domains of AlbAS are required to cooperatively clamp albicidin, which is unusual for drug-binding proteins of the MerR family. Structure-guided NMR binding studies employing synthetic albicidin derivatives give valuable information about ligand promiscuity of AlbAS. Our findings thus expand the general understanding of antibiotic resistance mechanisms and support current drug-design efforts directed at more effective albicidin analogs

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.Deutsche Forschungsgemeinschaft http://dx.doi.org/10.13039/501100001659Research Training Group RTG 2473 "Bioactive Peptides"RTG 2473 "Bioactive Peptides"Peer Reviewe

Beyond the limit

Strukturelle Untersuchungen mittels Lösungs-NMR Spektroskopie sind für supramolekulare Maschinen mit Molekulargewichten von mehr als 150 kDa nur beschränkt möglich. Die Festkörper-NMR mit Probenrotation im sogenannten magischen Winkel (MAS) stellt dagegen eine molekulargewichtsunabhängige Methode dar. Im Rahmen dieser Arbeit wurde eine neue Methode entwickelt, die die MAS NMR Spektroskopie an supramolekularen Komplexen in Lösung erlaubt. Proteinlösungen bilden demnach durch MAS und dessen Ultrazentrifugationseffekt homogene Proteinsedimente aus, in denen die rotatorische Diffusion großer Proteinkomplexe überwiegend aufgehoben ist. Auf diese Weise können klassische Festkörper-NMR Methoden angewandt werden, ohne dass Präzipitations- oder Kristallisationsverfahren erforderlich sind. In Kombination mit Proteindeuterierung, Protonendetektion sowie paramagnetischer Relaxationsverstärkung ermöglichte diese neuartige Methode die Zuordnung von Rückgrat-Amidresonanzen des 20S Proteasoms mit einem Molekulargewicht von 1,1 MDa. Weiterhin wurde diese Methode zur Untersuchung des kleinen Hitzeschockproteins alpha-B-Crystallin und dessen Cu(II)-Bindungseigenschaften genutzt. Das Chaperon (600 kDa) spielt eine wesentliche Rolle in der zellulären Proteinhomeostase. Verschiedenste NMR Techniken und andere biophysikalische Methoden zeigen, dass die konservierte alpha-Crystallin-Domäne ein Cu(II)-Ion nahe der Monomer-Monomer Interaktionsfläche mit pikomolarer Affinität bindet. Die Cu(II)-induzierte Freilegung von Substrat-Interaktionsflächen und Veränderungen in der dynamischen Quartärstruktur modulieren so die oligomere Architektur und die Chaperonaktivität von alpha-B-Crystallin. Die hier erstmals beschriebene MAS NMR Spektroskopie von sedimentierten Biomolekülen legt einen wichtigen Grundstein für zukünftige Struktur- und Dynamikuntersuchungen an großen molekularen Maschinen.Structural investigations of large biomolecules by solution-state NMR are challenging in case the molecular weight of the complex exceeds 150 kDa. Magic-angle-spinning (MAS) solid-state NMR is a powerful tool for the characterization of biomolecular systems irrespective of their molecular weight. In this work, an approach was developed, which enables the investigation of supramolecular modules by MAS NMR. Protein solutions can yield fairly homogeneous sediments due to the ultracentrifugal forces during MAS. Since rotational diffusion is impaired, typical solid-state NMR techniques can thus be applied without the need of precipitation or crystallization. This new approach in combination with protein deuteration, proton-detection and paramagnetic relaxation enhancement enabled the observation and the assignment of backbone amide resonances of a 20S proteasome assembly with a molecular weight of 1.1 MDa. Similarly, the approach was used to characterize the small heat-shock protein alpha-B-crystallin with respect to its Cu(II)-dependent chaperone activity. The chaperone (600 kDa) plays an essential role in cellular protein homeostasis. We show that the conserved alpha-crystallin core domain is the elementary Cu(II)-binding unit specifically coordinating one Cu(II) ion near to the dimer interface with picomolar binding affinity. We suggest that Cu(II)-binding unblocks potential client binding sites and alters quaternary dynamics of both the dimeric building block as well as the higher-order assemblies of alpha-B-crystallin. In summary, MAS NMR employed to biomolecules in solution is a very promising tool to explore structural and dynamic properties of large biological machines with no upper size limit

Harnessing fungal nonribosomal cyclodepsipeptide synthetases for mechanistic insights and tailored engineering

Nonribosomal peptide synthetases represent potential platforms for the design and engineering of structurally complex peptides. While previous focus has been centred mainly on bacterial systems, fungal synthetases assembling drugs like the antifungal echinocandins, the antibacterial cephalosporins or the anthelmintic cyclodepsipeptide (CDP) PF1022 await in-depth exploitation. As various mechanistic features of fungal CDP biosynthesis are only partly understood, effective engineering of NRPSs has been severely hampered. By combining protein truncation, in trans expression and combinatorial swapping, we assigned important functional segments of fungal CDP synthetases and assessed their in vivo biosynthetic capabilities. Hence, artificial assembly line components comprising of up to three different synthetases were generated. Using Aspergillus niger as a heterologous expression host, we obtained new-to-nature octa-enniatin (4 mg L-1) and octa-beauvericin (10.8 mg L-1), as well as high titers of the hybrid CDP hexa-bassianolide (1.3 g L-1) with an engineered ring size. The hybrid compounds showed up to 12-fold enhanced antiparasitic activity against Leishmania donovani and Trypanosoma cruzi compared to the reference drugs miltefosine and benznidazole, respectively. Our findings thus contribute to a rational engineering of iterative nonribosomal assembly lines

Deuterium-Labeled Precursor Feeding Reveals a New pABA-Containing Meroterpenoid from the Mango Pathogen Xanthomonas citri pv. mangiferaeindicae

WOS: 000378758200007PubMed ID: 27232656A new para-aminobenzoic-acid-containing natural product from the mango pathogenic organism Xanthomonas citri pv. mangiferaeindicae is described. By means of stable isotope precursor feeding combined with nontargeted LC-MS/MS, the generated spectra were clustered and visualized in a molecular network. This led to the identification of a new member of the meroterpenoids, termed xanthomonic acid, which is composed of an isoprenylated Para-amino benzoic acid. In vitro cytotoxicity assays demonstrated activity of xanthomonic acid against several human cancer cell lines by induction of autophagy.German Academic Exchange Service (Deutscher Akademischer Austauschdienst)Deutscher Akademischer Austausch Dienst (DAAD); Egyptian Ministry of Higher EducationScience and Technology Development Fund (STDF)The authors thank Dr. Sebastian Kemper (Technical University of Berlin) for the instrumentation of the NMR experiments and Dr. P. Ballar Kirmizibayrak (Ege University) for support and guidance on the autophagy study. H.S. thanks the collaborative funding and support from the German Academic Exchange Service (Deutscher Akademischer Austauschdienst) and the Egyptian Ministry of Higher Education