Structure and Mechanism of the Sphingopyxin I Lasso Peptide Isopeptidase

Fage, Christopher D.; Hegemann, Julian D.; Nebel, Annika J.; Steinbach, Roman M.; Zhu, Shaozhou; Linne, Uwe; Harms, Klaus; Bange, Gert; Marahiel, Mohamed A.

doi:10.1002/ange.201605232

Cited by 7 publications

(15 citation statements)

References 39 publications

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“…11−13 The current record holder for the largest lasso peptide is sphingopyxin I at 26 aa. 13,17 Leveraging our genome mining software, we have had a longstanding interest in finding the limits on size for lasso peptides.…”

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confidence: 99%

Pandonodin: A Proteobacterial Lasso Peptide with an Exceptionally Long C-Terminal Tail

2019

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Lasso peptides are a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) defined by their threaded-ring topology. The N-terminus of the peptide forms an isopeptide bond with an aspartate or glutamate side chain to create a 7−9 amino acid (aa) macrocyclic ring through which the rest of the peptide is threaded. The result is a highly constrained three-dimensional structure. Even though they share a threaded-ring feature, characterized lasso peptides vary greatly in sequence and size, ranging from 14 to 26 aa. Using genome mining, we identified a new lasso peptide gene cluster with a predicted lasso peptide that is 33 aa long. Here we report the heterologous expression of this new peptide, pandonodin, its NMR structure, and its unusual biophysical properties. Pandonodin has a long, proteolytically resistant 18residue tail of low sequence complexity, which limits its water solubility. Within this tail is a 6 aa disulfide-bonded macrocycle that serves as a steric lock to maintain the lasso structure. This disulfide bond is unusually stable, requiring both heat and high concentrations of reductants for cleavage. Finally, we also show that segments of the C-terminal tail of pandonodin can be replaced with arbitrary sequences, allowing for the construction of pandonodin−protein fusions.

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confidence: 99%

Pandonodin: A Proteobacterial Lasso Peptide with an Exceptionally Long C-Terminal Tail

2019

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“…This approach was taken to test the limits of the machinery. Additional Pro residues were incorporated at positions 4,6,14,16,[18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35]. In these variants, ring 1 or 2 would contain two or three Pro residues.…”

Section: Resultsmentioning

confidence: 99%

“…[1][2][3][4][5][7][8][9][10][11][12][13] A feature that is intrinsic to many RiPP systems is the high promiscuity of their biosynthetic machineries. 7,10,[14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29] With exception of residues involved in the posttranslational modifications, amino acid substitutions in the core region are generally tolerated well. Additionally, the corresponding processing enzymes allow access to complicated peptide topologies that are not readily accessible by synthetic means.…”

Section: Introductionmentioning

confidence: 99%

Assessing the Flexibility of the Prochlorosin 2.8 Scaffold for Bioengineering Applications

et al. 2019

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Cyclization is a common strategy to confer proteolytic resistance to peptide scaffolds. Thus, cyclic peptides have been the focus of extensive bioengineering efforts. Ribosomally synthesized and posttranslationally-modified peptides (RiPPs) are a superfamily of peptidic natural products that often contain macrocycles. In the RiPP family of lanthipeptides, macrocyclization is accomplished through formation of thioether crosslinks between cysteines and dehydrated serines/threonines. The recent production of lanthipeptide libraries and development of methods to display lanthipeptides on yeast or phage highlights their potential for bioengineering and synthetic biology. In this regard, the prochlorosins are especially promising as the corresponding class II lanthipeptide synthetase ProcM matures numerous precursor peptides with diverse core peptide sequences. To facilitate future bioengineering projects, one of its native substrates, ProcA2.8, was subjected in this study to in-depth mutational analysis to test the limitations of ProcM-mediated cyclization. Alanine scan mutagenesis was performed on all residues within the two rings, and multiple prolines were introduced at various positions. Moreover, mutation, deletion, and insertion of residues in the region linking the two lanthionine rings was tested. Additional residues were also introduced or deleted from either ring, and inversion of ring forming residues was attempted to generate diastereomers. The findings were used for epitope grafting of the RGD integrin binding epitope within prochlorosin 2.8, resulting in a low nanomolar affinity binder of the αvβ3 integrin that was more stable towards proteolysis and displayed higher affinity than the linear counterpart.

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“…Lasso peptide isopeptidases (IsoPs) from Sphingopyxis alaskensis belong to the ribosomally synthetized and post-translationally modified peptide (RiPP) family and to the S9C subfamily of prolyloligopeptidases. They function as antimicrobials, enzyme inhibitors, and receptor antagonists [42].…”

Section: Physiological Significancementioning

confidence: 99%

“…The formation of the dimer leads to covering the "naked" sticky edge and the catalytic pocket can be reached by a rather large and structured side entrance of the monomers, where the intact active site is located. A large side entrance is created by a propeller breaking insertion in blade 4, and its missing blade 5 (PDB: 5jrk) [42]. This is presumably important for the bulky lasso peptide substrate to approach the catalytic site.…”

Section: Providing Access To the Active Sitementioning

confidence: 99%

Achieving Functionality Through Modular Build-up: Structure and Size Selection of Serine Oligopeptidases

Kiss-Szemán

Harmat

Menyhárd

2019

CPPS

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Enzymes of the prolyl oligopeptidase family (S9 family) recognize their substrates not only by the specificity motif to be cleaved but also by size - they hydrolyze oligopeptides smaller than 30 amino acids. They belong to the serine-protease family, but differ from classical serine-proteases in size (80 kDa), structure (two domains) and regulation system (size selection of substrates). This group of enzymes is an important target for drug design as they are linked to amnesia, schizophrenia, type 2 diabetes, trypanosomiasis, periodontitis and cell growth. By comparing the structure of various members of the family we show that the most important features contributing to selectivity and efficiency are: (i) whether the interactions weaving the two domains together play a role in stabilizing the catalytic triad and thus their absence may provide for its deactivation: these oligopeptidases can screen their substrates by opening up, and (ii) whether the interaction-prone β-edge of the hydrolase domain is accessible and thus can guide a multimerization process that creates shielded entrance or intricate inner channels for the size-based selection of substrates. These cornerstones can be used to estimate the multimeric state and selection strategy of yet undetermined structures.

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Structure and Mechanism of the Sphingopyxin I Lasso Peptide Isopeptidase

Cited by 7 publications

References 39 publications

Pandonodin: A Proteobacterial Lasso Peptide with an Exceptionally Long C-Terminal Tail

Pandonodin: A Proteobacterial Lasso Peptide with an Exceptionally Long C-Terminal Tail

Assessing the Flexibility of the Prochlorosin 2.8 Scaffold for Bioengineering Applications

Achieving Functionality Through Modular Build-up: Structure and Size Selection of Serine Oligopeptidases

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