A β-hairpin epitope as novel structural requirement for protein arginine rhamnosylation

For canonical asparagine glycosylation, the primary amino acid sequence that directs glycosylation at specific asparagine residues is well-established. Here we reveal that a recently discovered bacterial enzyme EarP, that transfers rhamnose to a specific arginine residue in its acceptor protein EF-P, specifically recognizes a beta-hairpin loop. Notably, while the in vitro rhamnosyltransferase activity of EarP is abolished when presented with linear substrate peptide sequences derived from EF-P, the enzyme readily glycosylates the same sequence in a cyclized beta-hairpin mimic. Additional studies with other substrate-mimicking cyclic peptides revealed that EarP activity is sensitive to the method used to induce cyclization and in some cases is tolerant to amino acid sequence variation. Using detailed NMR approaches, we established that the active peptide substrates all share some degree of beta-hairpin formation, and therefore conclude that the beta-hairpin epitope is the major determinant of arginine-rhamnosylation by EarP. Our findings add a novel recognition motif to the existing knowledge on substrate specificity of protein glycosylation, and are expected to guide future identifications of rhamnosylation sites in other protein substrates

Synthesis of a tricyclic hexapeptide –via two consecutive ruthenium-catalyzed macrocyclization steps– with a constrained topology to mimic vancomycin's binding properties toward D-Ala-D-Ala dipeptide

A ring-closing metathesis (RCM) - peptide coupling - ruthenium-catalyzed azide alkyne cycloaddition (RuAAC) strategy was developed to synthesize a tricyclic hexapeptide in which the side chain to side chain connectivity pattern resulted in a mimic with a topology that effectively mimics the bioactivity of vancomycin as a potent binder of the bacterial cell wall D-Ala-D-Ala dipeptide sequence and more importantly being an effective inhibitor of bacterial growth

A peptide mimic of the chemotaxis inhibitory protein of Staphylococcus aureus: towards the development of novel anti-inflammatory compounds

Complement factor C5a is one of the most powerful pro-inflammatory agents involved in recruitment of leukocytes, activation of phagocytes and other inflammatory responses. C5a triggers inflammatory responses by binding to its G-protein-coupled C5a-receptor (C5aR). Excessive or erroneous activation of the C5aR has been implicated in numerous inflammatory diseases. The C5aR is therefore a key target in the development of specific anti-inflammatory compounds. A very potent natural inhibitor of the C5aR is the 121-residue chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS). Although CHIPS effectively blocks C5aR activation by binding tightly to its extra-cellular N terminus, it is not suitable as a potential anti-inflammatory drug due to its immunogenic properties. As a first step in the development of an improved CHIPS mimic, we designed and synthesized a substantially shorter 50-residue adapted peptide, designated CHOPS. This peptide included all residues important for receptor binding as based on the recent structure of CHIPS in complex with the C5aR N terminus. Using isothermal titration calorimetry we demonstrate that CHOPS has micromolar affinity for a model peptide comprising residues 7–28 of the C5aR N terminus including two O-sulfated tyrosine residues at positions 11 and 14. CD and NMR spectroscopy showed that CHOPS is unstructured free in solution. Upon addition of the doubly sulfated model peptide, however, the NMR and CD spectra reveal the formation of structural elements in CHOPS reminiscent of native CHIPS

A new perspective on fungal metabolites:Identification of bioactive compounds from fungi using zebrafish embryogenesis as read-out

There is a constant need for new therapeutic compounds. Fungi have proven to be an excellent, but underexplored source for biologically active compounds with therapeutic potential. Here, we combine mycology, embryology and chemistry by testing secondary metabolites from more than 10,000 species of fungi for biological activity using developing zebrafish (Danio rerio) embryos. Zebrafish development is an excellent model for high-throughput screening. Development is rapid, multiple cell types are assessed simultaneously and embryos are available in high numbers. We found that 1,526 fungal strains produced secondary metabolites with biological activity in the zebrafish bioassay. The active compounds from 39 selected fungi were purified by liquid-liquid extraction and preparative HPLC. 34 compounds were identified by a combination of chemical analyses, including LCMS, UV-Vis spectroscopy/ spectrophotometry, high resolution mass spectrometry and NMR. Our results demonstrate that fungi express a wide variety of biologically active compounds, consisting of both known therapeutic compounds as well as relatively unexplored compounds. Understanding their biological activity in zebrafish may provide insight into underlying biological processes as well as mode of action. Together, this information may provide the first step towards lead compound development for therapeutic drug development

Mechanistic insights into the C₅₅-P targeting lipopeptide antibiotics revealed by structure-activity studies and high-resolution crystal structures

AbstractThe continued rise of antibiotic resistance is a global concern that threatens to undermine many aspects of modern medical practice. Key to addressing this threat is the discovery and development of new antibiotics that operate by unexploited modes of action. The so-called calcium-dependent lipopeptide antibiotics (CDAs) are an important emerging class of natural products that provides a source of new antibiotic agents rich in structural and mechanistic diversity. Notable in this regard is the subset of CDAs comprising the laspartomycins and amphomycins/friulimicins that specifically target the bacterial cell wall precursor undecaprenyl phosphate (C55-P). In this study we describe the design and synthesis of new C55-P-targeting CDAs with structural features drawn from both the laspartomycin and amphomycin/friulimicin classes. Assessment of these lipopeptides revealed previously unknown and surprisingly subtle structural features that are required for antibacterial activity. High-resolution crystal structures further indicate that the amphomycin/friulimicin-like lipopeptides adopt a unique crystal packing that governs their interaction with C55-P and provides an explanation for their antibacterial effect. In addition, live-cell microscopy studies provide further insights into the biological activity of the C55-P targeting CDAs highlighting their unique mechanism of action relative to the clinically used CDA daptomycin.Microbial Biotechnolog

A combined solid- and solution-phase approach provides convenient access to analogues of the calcium-dependent lipopeptide antibiotics

The calcium-dependent lipopeptide antibiotics represent a promising new class of antimicrobials for use in combating drug-resistant bacteria. At present, daptomycin is the only such lipopeptide used clinically and displays potent antimicrobial activity against a number of pathogenic Gram-positive bacteria. Given the increasing need for new antibiotics, practical synthetic access to unnatural analogues of daptomycin and related antimicrobial lipopeptides is of value. We here report an efficient synthetic route combining solid- and solution-phase techniques that allows for the rapid preparation of daptomycin analogues. Using this approach, four such analogues, including two enantiomeric variants, were synthesized and their antimicrobial activities and hydrolytic stabilities evaluated.FWN – Publicaties zonder aanstelling Universiteit Leide

High-resolution NMR studies of structure and dynamics of human ERp27 indicate extensive interdomain flexibility

ERp27 (endoplasmic reticulum protein 27.7 kDa) is a homologue of PDI (protein disulfide-isomerase) localized to the endoplasmic reticulum. ERp27 is predicted to consist of two thioredoxinfold domains homologous with the non-catalytic b and b domains of PDI. The structure in solution of the N-terminal blike domain of ERp27 was solved using high-resolution NMR data. The structure confirms that it has the thioredoxin fold and that ERp27 is a member of the PDI family. 15N-NMR relaxation data were obtained and ModelFree analysis highlighted limited exchange contributions and slow internal motions, and indicated that the domain has an average order parameter S 2 of 0.79. Comparison of the single-domain structure determined in the present study with the equivalent domain within fulllength ERp27, determined independently by X-ray diffraction, indicated very close agreement. The domain interface inferred from NMR data in solution was much more extensive than that observed in the X-ray structure, suggesting that the domains flex independently and that crystallization selects one specific interdomain orientation. This led us to apply a new rapid method to simulate the flexibility of the full-length protein, establishing that the domains show considerable freedom to flex (tilt and twist) about the interdomain linker, consistent with the NMR data

Functional Relationship between Protein Disulfide Isomerase Family Members during the Oxidative Folding of Human Secretory Proteins

We systematically depleted PDI family members and show that whereas ERp72 and P5 contributed minimally to oxidative protein folding, PDI and ERp57 were the predominant catalysts. Depletion of PDI or ERp57 alone modestly delayed folding, but depletion of both led to generalized protein misfolding and degradation