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

Abstract

Here we describe self-splicing proteins, called inteins, that function as redox-responsive switches in bacteria. Redox regulation was achieved by engineering a disulfide bond between the intein's catalytic cysteine and a cysteine in the flanking 'extein' sequence. This interaction was validated by an X-ray structure, which includes a transient splice junction. A natural analog of the designed system was identified in Pyrococcus abyssi, suggesting an unprecedented form of adaptive, post-translational regulation.

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Figure 1: Engineered redox-responsive intein precursor in vivo and in vitro.
Figure 2: Crystal structure of the CPGCDnaE intein.
Figure 3: Protein splicing by the P. abyssi MoaA intein is redox sensitive.

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Acknowledgements

We thank Z. Li for performing crystallization experiments and D. Smith for technical assistance; A.K. Dearden and S. Nayak (Rensselaer Polytechnic Institute) for sharing the results of their quantum mechanics–molecular mechanics (QM-MM) simulations; B. Pereira and G. Amitai for useful discussions; J. Dansereau for preparing figures and for useful comments; and M. Carl for manuscript preparation. We acknowledge the Wadsworth Center's Molecular Genetics Core for DNA sequencing and the Macromolecular Crystallography Core for equipment use. This work was supported by US National Institutes of Health grants GM39422 and GM44844 to M.B.

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Authors

Contributions

B.P.C. conceived the study; B.P.C., N.I.T., P.V.R. and M.B. designed research; B.P.C., N.I.T., M.J.S. and P.V.R. performed research; B.P.C., P.V.R., N.I.T. and M.B. analyzed data; and B.P.C., P.V.R., N.I.T. and M.B. wrote the paper.

Corresponding author

Correspondence to Marlene Belfort.

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Competing interests

The authors filed a provisional patent entitled “A redox trap to control intein activity” with the USPTO on 18 January 2011 (application no. 61/433,730).

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Supplementary Methods, Supplementary Figure 1 and Supplementary Table 1 (PDF 153 kb)

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Callahan, B., Topilina, N., Stanger, M. et al. Structure of catalytically competent intein caught in a redox trap with functional and evolutionary implications. Nat Struct Mol Biol 18, 630–633 (2011). https://doi.org/10.1038/nsmb.2041

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