NMR Spectroscopic and Theoretical Analysis of a Spontaneously Formed Lys–Asp Isopeptide Bond

Hagan, Robert M.; Björnsson, Ragnar; McMahon, S.A.; Schomburg, Benjamin; Braithwaite, Vickie; Bühl, Michæl; Naismith, J.H.; Schwarz‐Linek, Ulrich

doi:10.1002/ange.201004340

Cited by 13 publications

(14 citation statements)

References 24 publications

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“…These bonds have been shown to confer proteolytic, thermal and mechanical stability to the protein . Similar experiments with other intramolecular isopeptide bond containing domains have shown similar results . These properties likely allow the proteins to remain stable and endure the stresses associated with the harsh environments at the sites of infection.…”

Section: Discussionmentioning

confidence: 60%

Intramolecular isopeptide but not internal thioester bonds confer proteolytic and significant thermal stability to the S. pyogenes pilus adhesin Spy0125

et al. 2013

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Streptococcus pyogenes and other Gram-positive bacterial pathogens present long macromolecular filaments known as pili on their surface that mediate adhesion and colonization. These pili are covalent polymers, assembled by sortases. Typically, they comprise a putative adhesin at their tip, a backbone subunit present in multiple copies and a basal subunit that is covalently anchored to the peptidoglycan layer of the cell surface. The crystal structures of pilin subunits revealed the presence of unusual covalent linkages in these proteins, including intramolecular isopeptide and internal thioester bonds. The intramolecular isopeptide bonds in backbone pilins are important for protein stability. Here, using both the wild-type protein and a set of mutants, we assessed the proteolytic and thermal stability of the S. pyogenes pilus tip adhesin Spy0125, in the presence and absence of its intramolecular isopeptide and internal thioester bonds. We also determined a crystal structure of the internal thioester bond variant Spy0125Cys426Ala. We find that mutations in the intramolecular isopeptide bonds compromise the stability of Spy0125. Using limited proteolysis and thermal denaturation assays, we could separate the contribution of each intramolecular isopeptide bond to Spy0125 stability. In contrast, mutation in the internal thioester bond had a lesser effect on protein stability and the crystal structure is essentially identical to wild type. This work suggests that the internal thioester in Spy0125, although having a minor contributory role, is not required for protein stability and must have a different primary function, most likely mediating a covalent interaction with host cell ligands. Proteins 2014; 82:517–527. © 2013 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

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Section: Discussionmentioning

confidence: 60%

Intramolecular isopeptide but not internal thioester bonds confer proteolytic and significant thermal stability to the S. pyogenes pilus adhesin Spy0125

et al. 2013

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“…This may be due to the lacking element of the UR/Spacer region found in SfbI, or the presence of putative integrin binding sites as well as two CnaB domains, which are important structure‐defining elements. Interestingly, the CnaB2 domain of FbaB forms an isopeptide bond that renders the protein highly thermostable and acid resistant (Hagan et al ., 2010). These additional elements may either constitute or present EC‐specific binding sites for receptors that are not found on epithelial cells such HEp‐2.…”

Section: Discussionmentioning

confidence: 99%

The FbaB-type fibronectin-binding protein of Streptococcus pyogenes promotes specific invasion into endothelial cells

Amelung¹,

Nerlich²,

Rohde³

et al. 2011

Cellular Microbiology

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Summary Invasive serotype M3 Streptococcus pyogenes are among the most frequently isolated organisms from patients suffering from invasive streptococcal disease and have the potential to invade primary human endothelial cells (EC) via a rapid and efficient mechanism. FbaB protein, the fibronectin-binding protein expressed by M3 S. pyogenes, was herein identified as a potent invasin for EC. By combining heterologous gene expression with allelic replacement, we demonstrate that FbaB is essential and sufficient to trigger EC invasion via a Rac1-dependent phagocytosis-like uptake. FbaB-mediated uptake follows the classical endocytic pathway with lysosomal destination. FbaB is demonstrated to be a streptococcal invasin exhibiting EC tropism. FbaB thus initiates a process that may contribute to the deep tissue tropism and spread of invasive S. pyogenes isolates into the vascular EC lining.

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“…The plasmid containing the original SpyCatcher gene (Zakeri et al, 2012) was obtained from Addgene (Addgene plasmid #35044), EGFP was amplified from the Regan lab vector pPROEX HTa M EGFP-MEEVD (pPROEX HTa M is a modified version of the pPROEX HTa vector) (Cormack et al, 1996) (Invitrogen) and mCherry was amplified from pNAS1b (Addgene plasmid #61968) (Sawyer et al, 2014). SpyoIPD was generated by site-directed mutagenesis of FbaB-CnaB2-Asp556Ala (Hagan et al, 2010) to introduce the Ile552 to Ala mutation. The original SpyCatcher protein contains the point mutations Glu473Ile and Tyr508Met, but these are not included in the SpyoIPD designs.…”

Section: Molecular Biologymentioning

confidence: 99%

“…The original SpyCatcher protein contains the point mutations Glu473Ile and Tyr508Met, but these are not included in the SpyoIPD designs. The original SpyCatcher protein also has an additional 20 residues at the N-terminus that are not part of the isopeptide domain fold (Hagan et al, 2010), and two residues at the C-terminus (Arg-Ser), which are not present in FbaB. These 22 residues are not included in SpyoIPD.…”

Section: Molecular Biologymentioning

confidence: 99%

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A new method for post-translationally labeling proteins in live cells for fluorescence imaging and tracking

Hinrichsen

Lenz

Edwards

et al. 2017

Protein Engineering, Design and Selection

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We present a novel method to fluorescently label proteins, post-translationally, within live Saccharomycescerevisiae. The premise underlying this work is that fluorescent protein (FP) tags are less disruptive to normal processing and function when they are attached post-translationally, because target proteins are allowed to fold properly and reach their final subcellular location before being labeled. We accomplish this post-translational labeling by expressing the target protein fused to a short peptide tag (SpyTag), which is then covalently labeled in situ by controlled expression of an open isopeptide domain (SpyoIPD, a more stable derivative of the SpyCatcher protein) fused to an FP. The formation of a covalent bond between SpyTag and SpyoIPD attaches the FP to the target protein. We demonstrate the general applicability of this strategy by labeling several yeast proteins. Importantly, we show that labeling the membrane protein Pma1 in this manner avoids the mislocalization and growth impairment that occur when Pma1 is genetically fused to an FP. We also demonstrate that this strategy enables a novel approach to spatiotemporal tracking in single cells and we develop a Bayesian analysis to determine the protein's turnover time from such data.

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NMR Spectroscopic and Theoretical Analysis of a Spontaneously Formed Lys–Asp Isopeptide Bond

Cited by 13 publications

References 24 publications

Intramolecular isopeptide but not internal thioester bonds confer proteolytic and significant thermal stability to the S. pyogenes pilus adhesin Spy0125

Intramolecular isopeptide but not internal thioester bonds confer proteolytic and significant thermal stability to the S. pyogenes pilus adhesin Spy0125

The FbaB-type fibronectin-binding protein of Streptococcus pyogenes promotes specific invasion into endothelial cells

A new method for post-translationally labeling proteins in live cells for fluorescence imaging and tracking

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