A functional interplay between intein and extein sequences in protein splicing compensates for the essential block B histidine

Friedel, Kristina; Popp, Monika A.; Matern, Julian C. J.; Gazdag, E.M.; Thiel, Ilka V.; Volkmann, Gerrit; Blankenfeldt, Wulf; Mootz, Henning D.

doi:10.1039/c8sc01074a

Cited by 27 publications

(37 citation statements)

References 51 publications

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“…The highly conserved motif N3 : 10 histidine (His68) is in spatial hydrogen‐bond distance to the carbonyl group of the Glu1 residue. The key role of the histidine appears to be the distortion around the N‐scissile bond, consistent with other crystal structures of class 1 inteins and biochemical studies [5d,16] . Mutation of the residue in a H68A Int N precursor mutant ( 10 ) completely abolished protein trans ‐splicing, indicating its critical role (Figure 6B).…”

Section: Resultssupporting

confidence: 86%

“…A S65A mutant ( 12 ) was still active in protein trans ‐splicing but its 4‐fold impaired rate demonstrates the importance of Ser65 (Figure 6C). Interestingly, the twisted arrangement at the N‐scissile bond, with the distortion leading to the coordination of the −1 carbonyl oxygen with N3 : 10 histidine, has only been observed so far in two intein structures [5d,18] and the present case is the first example for a class 3 intein. This is noteworthy because as a class 3 intein the AceL NrdHF intein employs the motif C2 thiol instead of the immediately adjacent side chain of the motif N1 : 1 residue at position 1 of the intein for the nucleophilic attack.…”

Section: Resultsmentioning

confidence: 59%

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Biochemical and Structural Characterization of an Unusual and Naturally Split Class 3 Intein

et al. 2020

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Split inteins are indispensable tools for protein engineering because their ligation and cleavage reactions enable unique modifications of the polypeptide backbone. Three different classes of inteins have been identified according to the nature of the covalent intermediates resulting from the acyl rearrangements in the multistep protein-splicing pathway. Class 3 inteins employ a characteristic internal cysteine for a branched thioester intermediate. A bioinformatic database search of nonredundant protein sequences revealed the absence of split variants in 1701 class 3 inteins. We have discovered the first reported split class 3 intein in a metagenomics data set and report its biochemical, mechanistic and structural analysis. The AceL NrdHF intein exhibits low sequence conservation with other inteins and marked deviations in residues at conserved key positions, including a variation of the typical class-3 WCT triplet motif. Nevertheless, functional analysis confirmed the class 3 mechanism of the intein and revealed excellent splicing yields within a few minutes over a wide range of conditions and with barely detectable cleavage side reactions. A high-resolution crystal structure of the AceL NrdHF precursor and a mutagenesis study explained the importance and roles of several residues at the key positions. Tolerated substitutions in the flanking extein residues and a high affinity between the split intein fragments further underline the intein's future potential as a ligation tool.

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Section: Resultssupporting

confidence: 86%

Section: Resultsmentioning

confidence: 59%

Biochemical and Structural Characterization of an Unusual and Naturally Split Class 3 Intein

et al. 2020

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“…Meanwhile, it should be noted that low salt solution and high temperature may cause protein denaturation during ligation. Certainly, apart from sortase A-mediated ligation, other ligation methods may also be feasible, such as protein trans-splicing by split inteins (Friedel et al, 2019;Yao et al, 2020).…”

Section: Discussionmentioning

confidence: 99%

A Toolbox for Site-Specific Labeling of RecQ Helicase With a Single Fluorophore Used in the Single-Molecule Assay

Teng

Jiang

Huang

et al. 2020

Front. Mol. Biosci.

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Fluorescently labeled proteins can improve the detection sensitivity and have been widely used in a variety of biological measurements. In single-molecule assays, site-specific labeling of proteins enables the visualization of molecular interactions, conformational changes in proteins, and enzymatic activity. In this study, based on a flexible linker in the Escherichia coli RecQ helicase, we established a scheme involving a combination of fluorophore labeling and sortase A ligation to allow site-specific labeling of the HRDC domain of RecQ with a single Cy5 fluorophore, without inletting extra fluorescent domain or peptide fragment. Using single-molecule fluorescence resonance energy transfer, we visualized that Cy5-labeled HRDC could directly interact with RecA domains and could bind to both the 3 and 5 ends of the overhang DNA dynamically in vitro for the first time. The present work not only reveals the functional mechanism of the HRDC domain, but also provides a feasible method for site-specific labeling of a domain with a single fluorophore used in single-molecule assays.

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“…PTS takes place in four steps: (1) a nucleophilic attack by the first amino acid residue of either a cysteine or serine located in the N-terminal intein part on the carbonyl carbon of the preceding amino acid residue (−1 position) located in the flanking N-extein leads to an N-S or N-O acyl shift; (2) a linear (thio) ester intermediate being trans-esterificated by a nucleophilic attack of the first amino acid residue of the so-called C-extein (+1 position) forms a branched intermediate [95]; (3) the N-terminal intein part is now cleaved from its fusion protein and transferred to the N-extein. As a result, a succinimide ring is generated through cyclization of the conserved asparagine residue of the C-terminal intein part after the nucleophilic attack of the previously formed intein extein junction; (4) finally, the cleavage reaction of the C-terminal intein part followed by a spontaneous S-N or O-N acyl shift ligates the esterified C-and N-exteins by a native stable peptide bond [96,97]. The process of PTS is similar to native chemical ligation (NCL), which is well described elsewhere [98].…”

Section: Split Inteinsmentioning

confidence: 99%

Greatest Hits—Innovative Technologies for High Throughput Identification of Bispecific Antibodies

Hofmann

Krah

Sellmann

et al. 2020

IJMS

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Recent years have shown a tremendous increase and diversification in antibody-based therapeutics with advances in production techniques and formats. The plethora of currently investigated bi- to multi-specific antibody architectures can be harnessed to elicit a broad variety of specific modes of actions in oncology and immunology, spanning from enhanced selectivity to effector cell recruitment, all of which cannot be addressed by monospecific antibodies. Despite continuously growing efforts and methodologies, the identification of an optimal bispecific antibody as the best possible combination of two parental monospecific binders, however, remains challenging, due to tedious cloning and production, often resulting in undesired extended development times and increased expenses. Although automated high throughput screening approaches have matured for pharmaceutical small molecule development, it was only recently that protein bioconjugation technologies have been developed for the facile generation of bispecific antibodies in a ‘plug and play’ manner. In this review, we provide an overview of the most relevant methodologies for bispecific screening purposes—the DuoBody concept, paired light chain single cell production approaches, Sortase A and Transglutaminase, the SpyTag/SpyCatcher system, and inteins—and elaborate on the benefits as well as drawbacks of the different technologies.

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A functional interplay between intein and extein sequences in protein splicing compensates for the essential block B histidine

Abstract: Steric bulk can compensate for a catalytically critical histidine in an intein's active site and promote the N–S acyl shift.

Cited by 27 publications

References 51 publications

Biochemical and Structural Characterization of an Unusual and Naturally Split Class 3 Intein

Biochemical and Structural Characterization of an Unusual and Naturally Split Class 3 Intein

A Toolbox for Site-Specific Labeling of RecQ Helicase With a Single Fluorophore Used in the Single-Molecule Assay

Greatest Hits—Innovative Technologies for High Throughput Identification of Bispecific Antibodies

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