A conserved threonine spring-loads precursor for intein splicing

Authors

  • Albert K. Dearden,

    1. Department of Physics, Applied Physics, and Astronomy, Rensselaer Polytechnic Institute, Troy, New York 12180
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  • Brian Callahan,

    1. Department of Biological Sciences and RNA Institute, University at Albany, Albany, New York 12222
    Current affiliation:
    1. State University of New York at Binghamton Department of Chemistry, 4400 Vestal Parkway East Binghamton, NY 13902
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  • Patrick Van Roey,

    1. Wadsworth Center, New York State Department of Health, Albany, New York 12201
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  • Zhong Li,

    1. Wadsworth Center, New York State Department of Health, Albany, New York 12201
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  • Utsav Kumar,

    1. Department of Physics, Applied Physics, and Astronomy, Rensselaer Polytechnic Institute, Troy, New York 12180
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  • Marlene Belfort,

    Corresponding author
    1. Department of Biological Sciences and RNA Institute, University at Albany, Albany, New York 12222
    • Biological Sciences, University at Albany, State University of New York, 1400 Washington Avenue, Albany, NY 12222
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  • Saroj K. Nayak

    Corresponding author
    1. Department of Physics, Applied Physics, and Astronomy, Rensselaer Polytechnic Institute, Troy, New York 12180
    2. School of Basic Sciences, Indian Institute of Technology, Bhubaneswar, India
    • Department of Physics, Applied Physics, & Astronomy, Rensselaer Polytechnic Institute, 110 Eighth Street, Troy, NY 12180-3590
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Abstract

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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