Sequential formation of two branched intermediates during protein splicing of class three inteins

Tori, Kazuo; Perler, Francine B.

doi:10.1007/s00792-016-0876-0

Cited by 6 publications

(7 citation statements)

References 36 publications

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“…Conserved nucleophilic amino acids (AAs) within the structural context of the intein and residues of adjacent exteins are central to the chemistry of breaking and making peptide bonds during splicing. DnaBi1 utilizes a noncanonical mechanism known as class 3 splicing where an internal cysteine (C118), rather than a cysteine or serine at the first position of the intein, performs the initial nucleophilic attack ( 33 ). The formation of two sequential branched intermediates during DnaBi1 splicing requires, among other features, a serine as the first C-extein residue (+1S) ( 33 ).…”

Section: Introductionmentioning

confidence: 99%

“…DnaBi1 utilizes a noncanonical mechanism known as class 3 splicing where an internal cysteine (C118), rather than a cysteine or serine at the first position of the intein, performs the initial nucleophilic attack ( 33 ). The formation of two sequential branched intermediates during DnaBi1 splicing requires, among other features, a serine as the first C-extein residue (+1S) ( 33 ). Mutation of C118 blocks DnaBi1 excision, leading to accumulation of the unspliced and nonfunctional DnaB precursor ( 7 ).…”

Section: Introductionmentioning

confidence: 99%

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Conditional DnaB Protein Splicing Is Reversibly Inhibited by Zinc in Mycobacteria

Woods

Vangaveti

Egbanum

et al. 2020

mBio

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Inteins, as posttranslational regulatory elements, can tune protein function to environmental changes by conditional protein splicing (CPS). Translated as subdomains interrupting host proteins, inteins splice to scarlessly join flanking sequences (exteins). We used DnaB-intein1 (DnaBi1) from a replicative helicase of Mycobacterium smegmatis to build a kanamycin intein splicing reporter (KISR) that links splicing of DnaBi1 to kanamycin resistance. Using expression in heterologous Escherichia coli, we observed phenotypic classes of various levels of splicing-dependent resistance (SDR) and related these to the insertion position of DnaBi1 within the kanamycin resistance protein (KanR). The KanR-DnaBi1 construct demonstrating the most stringent SDR was used to probe for CPS of DnaB in the native host environment, M. smegmatis. We show here that zinc, important during mycobacterial pathogenesis, inhibits DnaB splicing in M. smegmatis. Using an in vitro reporter system, we demonstrated that zinc potently and reversibly inhibited DnaBi1 splicing, as well as splicing of a comparable intein from Mycobacterium leprae. Finally, in a 1.95 Å crystal structure, we show that zinc inhibits splicing through binding to the very cysteine that initiates the splicing reaction. Together, our results provide compelling support for a model whereby mycobacterial DnaB protein splicing, and thus DNA replication, is responsive to environmental zinc. IMPORTANCE Inteins are present in a large fraction of prokaryotes and localize within conserved proteins, including the mycobacterial replicative helicase DnaB. In addition to their extensive protein engineering applications, inteins have emerged as environmentally responsive posttranslational regulators of the genes that encode them. While several studies have shown compelling evidence of conditional protein splicing (CPS), examination of splicing in the native host of the intein has proven to be challenging. Here, we demonstrated through a number of measures, including the use of a splicing-dependent sensor capable of monitoring intein activity in the native host, that zinc is a potent and reversible inhibitor of mycobacterial DnaB splicing. This work also expands our knowledge of site selection for intein insertion within nonnative proteins, demonstrating that splicing-dependent host protein activation correlates with proximity to the active site. Additionally, we surmise that splicing regulation by zinc has mycobacteriocidal and CPS application potential.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Conditional DnaB Protein Splicing Is Reversibly Inhibited by Zinc in Mycobacteria

Woods

Vangaveti

Egbanum

et al. 2020

mBio

View full text Add to dashboard Cite

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“…Together, these findings confirmed a class 3 splicing mechanism for the AceL NrdHF intein. The equilibrium of the initial N−S acyl transfer on the side of the peptide bond and the tight coupling of asparagine cyclization to the previous steps explain the observed efficient suppression of off‐pathway reactions during protein splicing [4c] …”

Section: Resultsmentioning

confidence: 99%

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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“…Intein residues and flanking extein residues that assist these reactions are not shown, nor are tetrahedral intermediates. X represents the sulphur or oxygen atom in the side chain of Cys, Ser or Thr [ 104 ].…”

Section: Figurementioning

confidence: 99%

Inteins in Science: Evolution to Application

et al. 2020

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Inteins are mobile genetic elements that apply standard enzymatic strategies to excise themselves post-translationally from the precursor protein via protein splicing. Since their discovery in the 1990s, recent advances in intein technology allow for them to be implemented as a modern biotechnological contrivance. Radical improvement in the structure and catalytic framework of cis- and trans-splicing inteins devised the development of engineered inteins that contribute to various efficient downstream techniques. Previous literature indicates that implementation of intein-mediated splicing has been extended to in vivo systems. Besides, the homing endonuclease domain also acts as a versatile biotechnological tool involving genetic manipulation and control of monogenic diseases. This review orients the understanding of inteins by sequentially studying the distribution and evolution pattern of intein, thereby highlighting a role in genetic mobility. Further, we include an in-depth summary of specific applications branching from protein purification using self-cleaving tags to protein modification, post-translational processing and labelling, followed by the development of intein-based biosensors. These engineered inteins offer a disruptive approach towards research avenues like biomaterial construction, metabolic engineering and synthetic biology. Therefore, this linear perspective allows for a more comprehensive understanding of intein function and its diverse applications.

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Sequential formation of two branched intermediates during protein splicing of class three inteins

Cited by 6 publications

References 36 publications

Conditional DnaB Protein Splicing Is Reversibly Inhibited by Zinc in Mycobacteria

Conditional DnaB Protein Splicing Is Reversibly Inhibited by Zinc in Mycobacteria

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

Inteins in Science: Evolution to Application

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