Topoisomer Differentiation of Molecular Knots by FTICR MS: Lessons from Class II Lasso Peptides

Zirah, Séverine; Afonso, Carlos; Linne, Uwe; Knappe, Thomas A.; Marahiel, Mohamed A.; Rebuffat, Sylvie; Tabet, Jean‐Claude

doi:10.1007/s13361-010-0028-1

Cited by 37 publications

(51 citation statements)

References 44 publications

(97 reference statements)

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“…The threading of the tail also contributes characteristic features to the MS 2 spectrum (35,36). Sterically linked b-and y-series fragments have previously been reported in MS 2 spectra of MccJ25 (9-11).…”

Section: Resultsmentioning

confidence: 74%

Precursor-centric genome-mining approach for lasso peptide discovery

Maksimov¹,

Pelczer²,

Link

2012

Proc. Natl. Acad. Sci. U.S.A.

134

232

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Lasso peptides are a class of ribosomally synthesized posttranslationally modified natural products found in bacteria. Currently known lasso peptides have a diverse set of pharmacologically relevant activities, including inhibition of bacterial growth, receptor antagonism, and enzyme inhibition. The biosynthesis of lasso peptides is specified by a cluster of three genes encoding a precursor protein and two enzymes. Here we develop a unique genome-mining algorithm to identify lasso peptide gene clusters in prokaryotes. Our approach involves pattern matching to a small number of conserved amino acids in precursor proteins, and thus allows for a more global survey of lasso peptide gene clusters than does homology-based genome mining. Of more than 3,000 currently sequenced prokaryotic genomes, we found 76 organisms that are putative lasso peptide producers. These organisms span nine bacterial phyla and an archaeal phylum. To provide validation of the genome-mining method, we focused on a single lasso peptide predicted to be produced by the freshwater bacterium Asticcacaulis excentricus. Heterologous expression of an engineered, minimal gene cluster in Escherichia coli led to the production of a unique lasso peptide, astexin-1. At 23 aa, astexin-1 is the largest lasso peptide isolated to date. It is also highly polar, in contrast to many lasso peptides that are primarily hydrophobic. Astexin-1 has modest antimicrobial activity against its phylogenetic relative Caulobacter crescentus. The solution structure of astexin-1 was determined revealing a unique topology that is stabilized by hydrogen bonding between segments of the peptide.bioinformatics | protein NMR

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Section: Resultsmentioning

confidence: 74%

Precursor-centric genome-mining approach for lasso peptide discovery

Maksimov¹,

Pelczer²,

Link

2012

Proc. Natl. Acad. Sci. U.S.A.

134

232

View full text Add to dashboard Cite

show abstract

“…Additionally, leptin is composed of the simplest knot [38] as opposite to the more complex topologies like the trefoil knots [39]. Proteins with a similar topology are the lasso peptides [40], where the C-terminal tail is threaded through and caught within an N-terminal macrolactam ring. While these proteins share the same topology, they are very small peptides (16–21 residues) with less then 5 residues threaded through the closed-loop.…”

Section: Resultsmentioning

confidence: 99%

The Unique Cysteine Knot Regulates the Pleotropic Hormone Leptin

Haglund

Sułkowska

et al. 2012

PLoS ONE

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Leptin plays a key role in regulating energy intake/expenditure, metabolism and hypertension. It folds into a four-helix bundle that binds to the extracellular receptor to initiate signaling. Our work on leptin revealed a hidden complexity in the formation of a previously un-described, cysteine-knotted topology in leptin. We hypothesized that this unique topology could offer new mechanisms in regulating the protein activity. A combination of in silico simulation and in vitro experiments was used to probe the role of the knotted topology introduced by the disulphide-bridge on leptin folding and function. Our results surprisingly show that the free energy landscape is conserved between knotted and unknotted protein, however the additional complexity added by the knot formation is structurally important. Native state analyses led to the discovery that the disulphide-bond plays an important role in receptor binding and thus mediate biological activity by local motions on distal receptor-binding sites, far removed from the disulphide-bridge. Thus, the disulphide-bridge appears to function as a point of tension that allows dissipation of stress at a distance in leptin.

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“…McjD variants for FRET were purified in a final buffer of 20 mM Tris pH 7.5, 150 mM NaCl, and 0.03% (w/w) n‐dodecyl β‐D‐maltoside (DDM). The substrate of McjD, the peptide MccJ25, was produced and purified as previously described (Zirah et al , 2011). …”

Section: Methodsmentioning

confidence: 99%

Conformational dynamics of the ABC transporter McjD seen by single‐molecule FRET

et al. 2018

Self Cite

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ABC transporters utilize ATP for export processes to provide cellular resistance against toxins, antibiotics, and harmful metabolites in eukaryotes and prokaryotes. Based on static structure snapshots, it is believed that they use an alternating access mechanism, which couples conformational changes to ATP binding (outward‐open conformation) and hydrolysis (inward‐open) for unidirectional transport driven by ATP. Here, we analyzed the conformational states and dynamics of the antibacterial peptide exporter McjD from Escherichia coli using single‐molecule Förster resonance energy transfer (smFRET). For the first time, we established smFRET for an ABC exporter in a native‐like lipid environment and directly monitor conformational dynamics in both the transmembrane‐ (TMD) and nucleotide‐binding domains (NBD). With this, we unravel the ligand dependences that drive conformational changes in both domains. Furthermore, we observe intrinsic conformational dynamics in the absence of ATP and ligand in the NBDs. ATP binding and hydrolysis on the other hand can be observed via NBD conformational dynamics. We believe that the progress made here in combination with future studies will facilitate full understanding of ABC transport cycles.

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Topoisomer Differentiation of Molecular Knots by FTICR MS: Lessons from Class II Lasso Peptides

Cited by 37 publications

References 44 publications

Precursor-centric genome-mining approach for lasso peptide discovery

Precursor-centric genome-mining approach for lasso peptide discovery

The Unique Cysteine Knot Regulates the Pleotropic Hormone Leptin

Conformational dynamics of the ABC transporter McjD seen by single‐molecule FRET

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