Structure of catalytically competent intein caught in a redox trap with functional and evolutionary implications

Callahan, Brian P.; Topilina, Natalya I.; Stanger, Matthew; Roey, P. Van; Belfort, Marlene

doi:10.1038/nsmb.2041

Cited by 75 publications

(111 citation statements)

References 31 publications

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“…The trap comprised an engineered disulfide bond between the catalytic Cys1 residue of the intein and a cysteine at the À3 position of the N-extein. 6 We noted that Gly-1, which corresponds to the N-extein junction residue in the precursor, adopts an unusual geometry, which is consistent with similar studies involving splicing reactions in which distortions are present. [7][8][9][10][11] The (u,W) angles of Gly-1 fall outside the typical range for this residue.…”

Section: Introductionsupporting

confidence: 74%

“…23 For our simulations, we analyzed a minimized DnaE intein based on the published precursor structure. 6 The model system consisted of an engineered disulfide loop that trapped the intermediate structure and all molecular fragments within an area extending 10 Å outward radially at the full QM level. To help simulate the natural constraints on the system that would otherwise not be present due to the removal of the surrounding intein, the outer most structures were held in place during all geometry optimizations.…”

Section: Methodsmentioning

confidence: 99%

“…A t1/2 of $40 min was determined from reaction progress curves, in reasonable accord with earlier studies. 6 The two mutants displayed slower kinetics. Less than 5% of the N-extein had been cleaved from the T69A variant after 18 h (lane 4) and after 48 h (not shown).…”

Section: Intein Splicing Activity Of Thr69 Mutantsmentioning

confidence: 99%

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A conserved threonine spring‐loads precursor for intein splicing

et al. 2013

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Protein splicing is an autocatalytic process where an ''intein'' self-cleaves from a precursor and ligates the flanking N-and C-"extein'' polypeptides. Inteins occur in all domains of life and have myriad uses in biotechnology. Although the reaction steps of protein splicing are known, mechanistic details remain incomplete, particularly the initial peptide rearrangement at the N-terminal extein/intein junction. Recently, we proposed that this transformation, an N-S acyl shift, is accelerated by a localized conformational strain, between the intein's catalytic cysteine (Cys1) and the neighboring glycine (Gly-1) in the N-extein. That proposal was based on the crystal structure of a catalytically competent trapped precursor. Here, we define the structural origins and mechanistic relevance of the conformational strain using a combination of quantum mechanical simulations, mutational analysis, and X-ray crystallography. Our results implicate a conserved, but largely unstudied, threonine residue of the Ssp DnaE intein (Thr69) as the mediator of conformational strain through hydrogen bonding. Further, the strain imposed by this residue is shown to position the splice junction in a manner that enhances the rate of the N-S acyl shift substantially. Taken together, our results not only provide fundamental understanding of the control of the first step of protein splicing but also have important implications in various biotechnological applications that require precursor manipulation.

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Section: Introductionsupporting

confidence: 74%

Section: Methodsmentioning

confidence: 99%

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A conserved threonine spring‐loads precursor for intein splicing

et al. 2013

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“…Our findings show that under conditions that include occasional growth of the population, the high fitness cost of the intein guarantees the survival of alleles with the empty target site, even in the absence of inteins without HEN activity. Although here we showed a fitness cost for the intein, several reports have indicated a potential positive role for intein presence, claiming a regulatory function under stress conditions (36)(37)(38)(39)(40). However, our finding that natural H. volcanii populations in the same sampling location tend to be mixed, having both inteinpositive and intein-negative strains sharing the same niche, does not support a strong selective pressure for intein retention in this case.…”

Section: Discussioncontrasting

confidence: 54%

Impact of a homing intein on recombination frequency and organismal fitness

Naor

Altman-Price

Soucy

et al. 2016

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

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Inteins are parasitic genetic elements that excise themselves at the protein level by self-splicing, allowing the formation of functional, nondisrupted proteins. Many inteins contain a homing endonuclease (HEN) domain and rely on its activity for horizontal propagation. However, successful invasion of an entire population will make this activity redundant, and the HEN domain is expected to degenerate quickly under these conditions. Several theories have been proposed for the continued existence of the both active HEN and noninvaded alleles within a population. However, to date, these models were not directly tested experimentally. Using the natural cell fusion ability of the halophilic archaeon Haloferax volcanii we were able to examine this question in vivo, by mating polB intein-positive [insertion site c in the gene encoding DNA polymerase B (polB-c)] and intein-negative cells and examining the dispersal efficiency of this intein in a natural, polyploid population. Through competition between otherwise isogenic intein-positive and intein-negative strains we determined a surprisingly high fitness cost of over 7% for the polB-c intein. Our laboratory culture experiments and samples taken from Israel's Mediterranean coastline show that the polB-c inteins do not efficiently take over an inteinless population through mating, even under ideal conditions. The presence of the HEN/intein promoted recombination when intein-positive and intein-negative cells were mated. Increased recombination due to HEN activity contributes not only to intein dissemination but also to variation at the population level because recombination tracts during repair extend substantially from the homing site. . HENs contribute to the horizontal transmission of these selfish elements into intronless or inteinless alleles, by cleaving the vacant allele to induce homologous recombination or reverse transcription, where the allele containing the intron or intein serves as template. Thus, the intein or intron effectively invades the vacant site and can later be passed on to daughter cells vertically.Paradoxically, if a HEN is highly successful in invading cells, it will saturate all target cells, and then its activity will no longer be under purifying selection. This may result in degeneration of the HEN domain due to accumulation of mutations and will prevent the future horizontal propagation of the intein in question. This phenomenon has been observed in group I introns of bacteriophages (7, 8), eukaryotes (9), and archaea (10). However, active HENs are often observed, and in most natural populations surveyed to date, there are in fact strains with inteins (or introns) containing degenerate HENs, whereas other isolates maintain a fully intact HEN (9). The homing cycle model (11, 12) resolves this paradox by hypothesizing that after the intein-/intron-containing allele has been fixed in the population and after the HEN has completely decayed due to the lack of selection for function of HEN activity, the splicing element (either intron or intein) ...

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“…Inteins are autocatalytic, thus bond breaking begins as soon as the intein folds into its native structure, and chemical intermediates generated will be processed before high-resolution structures can be trapped-or sometimes before the precursor is even purified! In lieu of antagonists or effective trapping strategies (8), the authors turned to expressed protein ligation (EPL). EPL is an intein-based method where engineered proteins with unnatural modifications can be prepared semisynthetically in a controlled stepwise manner (9).…”

mentioning

confidence: 99%