Structure of the branched intermediate in protein splicing

Zhihua Liu, Silvia Frutos, Matthew J. Bick, Miquel Vila-Perelló, Galia T. Debelouchina, Seth A. Darst, Tom W. Muir

Research output: Contribution to journalArticlepeer-review

22 Scopus citations


Inteins are autoprocessing domains that cut themselves out of host proteins in a traceless manner. This process, known as protein splicing, involves multiple chemical steps that must be coordinated to ensure fidelity in the process. The committed step in splicing involves attack of a conserved Asn side-chain amide on the adjacent backbone amide, leading to an intein-succinimide product and scission of that peptide bond. This cleavage reaction is stimulated by formation of a branched intermediate in the splicing process. The mechanism by which the Asn side-chain becomes activated as a nucleophile is not understood. Here we solve the crystal structure of an intein trapped in the branched intermediate step in protein splicing. Guided by this structure, we use protein-engineering approaches to show that intein-succinimide formation is critically dependent on a backbone-to-side-chain hydrogen-bond. We propose that this interaction serves to both position the side-chain amide for attack and to activate its nitrogen as a nucleophile. Collectively, these data provide an unprecedented view of an intein poised to carry out the rate-limiting step in protein splicing, shedding light on how a nominally nonnucleophilic group, a primary amide, can become activated in a protein active site.

Original languageEnglish (US)
Pages (from-to)8422-8427
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number23
StatePublished - Jun 10 2014

All Science Journal Classification (ASJC) codes

  • General


  • Expressed
  • Protein semisynthesis


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