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, James H.; Schwarz‐Linek, Ulrich

doi:10.1002/anie.201004340

Cited by 95 publications

(114 citation statements)

References 25 publications

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“…The latter also polarizes the C = O bond of the Asn or Asp side chain, resulting in a partial positive charge on Cγ (10,14). This is essentially a one-turnover autocatalytic reaction dependent on the polarity of the environment and the proximity of the reacting groups.…”

Section: Significancementioning

confidence: 99%

“…It includes not only Gram-positive pili but a number of other cell surface adhesins, known as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) (7). Examples of the latter include the collagen-binding A domain and repetitive B domains from the Staphylococcus aureus collagen-binding surface protein Cna (8,9), the fibronectin-binding protein FbaB from S. pyogenes (10), and the adhesin SspB from Streptococcus gordonii (11). In contrast to the Gram-positive pili, which are assembled from discrete protein subunits (pilins) by sortase enzymes (12), the MSCRAMMs are typically single polypeptides folded into many domains.…”

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confidence: 99%

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Autocatalytically generated Thr-Gln ester bond cross-links stabilize the repetitive Ig-domain shaft of a bacterial cell surface adhesin

Kwon

Squire

Young

et al. 2013

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

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Gram-positive bacteria are decorated by a variety of proteins that are anchored to the cell wall and project from it to mediate colonization, attachment to host cells, and pathogenesis. These proteins, and protein assemblies, such as pili, are typically long and thin yet must withstand high levels of mechanical stress and proteolytic attack. The recent discovery of intramolecular isopeptide bond cross-links, formed autocatalytically, in the pili from Streptococcus pyogenes has highlighted the role that such crosslinks can play in stabilizing such structures. We have investigated a putative cell-surface adhesin from Clostridium perfringens comprising an N-terminal adhesin domain followed by 11 repeat domains. The crystal structure of a two-domain fragment shows that each domain has an IgG-like fold and contains an unprecedented ester bond joining Thr and Gln side chains. MS confirms the presence of these bonds. We show that the bonds form through an autocatalytic intramolecular reaction catalyzed by an adjacent His residue in a serine protease-like mechanism. Two buried acidic residues assist in the reaction. By mutagenesis, we show that loss of the ester bond reduces the thermal stability drastically and increases susceptibility to proteolysis. As in pilin domains, the bonds are placed at a strategic position joining the first and last strands, even though the Ig fold type differs. Bioinformatic analysis suggests that similar domains and ester bond cross-links are widespread in Gram-positive bacterial adhesins. intramolecular ester bond | protein stability A striking feature of globular proteins is that despite the chemical diversity inherent in the side chains of their constituent amino acids, chemical reactions between these side chains are very rare. This may be explained by evolutionary selection, which minimizes reactions that could prejudice proper protein folding. Thus, the only common example of a covalent cross-link between protein side chains is the disulfide bond, which forms only in an appropriate redox environment when two Cys residues are brought together by protein folding. Nevertheless, some surprising examples of unexpected crosslinks have been brought to light by protein structure analysis or by the observation of unusual spectroscopic or biophysical properties. Examples include the Cys-Tyr bond in galactose oxidase (1), which provides a radical center; similar bonds in some catalases (2); the His-Tyr bond in cytochrome C oxidase (3); and the remarkable chromophore of GFP (4). These, and other examples, arise through intramolecular reactions facilitated by particular local environments.The recent discovery of isopeptide bonds joining Lys and Asn side chains in the proteins that make up pili on the Gram-positive bacterium Streptococcus pyogenes (5), as well as on other Gram-positive pathogens (6), has highlighted a class of proteins in which intramolecular cross-links seem to be remarkably prevalent. It includes not only Gram-positive pili but a number of other cell surface adhesins, known as mi...

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Section: Significancementioning

confidence: 99%

mentioning

confidence: 99%

Autocatalytically generated Thr-Gln ester bond cross-links stabilize the repetitive Ig-domain shaft of a bacterial cell surface adhesin

Kwon

Squire

Young

et al. 2013

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

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“…These intramolecular isopeptide bonds have now been characterized experimentally in a wide-range of pilus subunits from many Gram-positive pathogens (reviewed in refs. 7-9) and also in the MSCRAMM FbaB (10). Intramolecular isopeptide bonds are most commonly formed between the side chains of Lys and Asn residues ( Fig.…”

mentioning

confidence: 97%

“…Intramolecular isopeptide bonds are most commonly formed between the side chains of Lys and Asn residues ( Fig. 1) [although Lys-Asp bonds also exist (10,11)]. The residues comprising these bonds are strategically positioned to bridge the first and either penultimate or last β-strand of the Ig-like domains.…”

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confidence: 99%

Yet more intramolecular cross-links in Gram-positive surface proteins

Schwarz‐Linek

Banfield

2014

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

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“…Within CnaB2, there is a spontaneous reaction to form an isopeptide bond between Lys and Asp, catalyzed by an apposed Glu (Fig. 1A) (13,14). We previously showed that the CnaB2 domain could be split into two parts to enable protein-peptide ligation, via isopeptide bond formation between a peptide tag (i.e., SpyTag) and a protein domain (i.e., SpyCatcher) (15).…”

mentioning

confidence: 99%

SpyLigase peptide–peptide ligation polymerizes affibodies to enhance magnetic cancer cell capture

Fierer

Veggiani

Howarth

2014

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

156

162

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Individual proteins can now often be modified with atomic precision, but there are still major obstacles to connecting proteins into larger assemblies. To direct protein assembly, ideally, peptide tags would be used, providing the minimal perturbation to protein function. However, binding to peptides is generally weak, so assemblies are unstable over time and disassemble with force or harsh conditions. We have recently developed an irreversible protein-peptide interaction (SpyTag/SpyCatcher), based on a protein domain from Streptococcus pyogenes, that locks itself together via spontaneous isopeptide bond formation. Here we develop irreversible peptide-peptide interaction, through redesign of this domain and genetic dissection into three parts: a protein domain termed SpyLigase, which now ligates two peptide tags to each other. All components expressed efficiently in Escherichia coli and peptide tags were reactive at the N terminus, at the C terminus, or at internal sites. Peptide-peptide ligation enabled covalent and site-specific polymerization of affibodies or antibodies against the tumor markers epidermal growth factor receptor (EGFR) and HER2. Magnetic capture of circulating tumor cells (CTCs) is one of the most promising approaches to improve cancer prognosis and management, but CTC capture is limited by inefficient recovery of cells expressing low levels of tumor antigen. SpyLigase-assembled protein polymers made possible the isolation of cancerous cells expressing lower levels of tumor antigen and should have general application in enhancing molecular capture.bionanotechnology | synthetic biology | metastasis | antibody | nanobiotechnology

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

Cited by 95 publications

References 25 publications

Autocatalytically generated Thr-Gln ester bond cross-links stabilize the repetitive Ig-domain shaft of a bacterial cell surface adhesin

Autocatalytically generated Thr-Gln ester bond cross-links stabilize the repetitive Ig-domain shaft of a bacterial cell surface adhesin

Yet more intramolecular cross-links in Gram-positive surface proteins

SpyLigase peptide–peptide ligation polymerizes affibodies to enhance magnetic cancer cell capture

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