Structure of the branched intermediate in protein splicing.
Publication Type:Journal Article
Source:Proc Natl Acad Sci U S A, Volume 111, Issue 23, p.8422-7 (2014)
Keywords:Amides, Amino Acid Sequence, Asparagine, Catalytic Domain, DNA Gyrase, Electrophoresis, Polyacrylamide Gel, Exteins, Hydrogen Bonding, Inteins, Kinetics, Models, Molecular, Molecular Sequence Data, Molecular Structure, Mutation, Protein Splicing, Protein Structure, Secondary, Protein Structure, Tertiary, Proteins, Spectrometry, Mass, Electrospray Ionization
<p>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. </p>