4OZ6

Structure of the Branched Intermediate in Protein Splicing

Summary for 4OZ6

• Entry DOI: 10.2210/pdb4oz6/pdb
• Descriptor: Mxe gyrA intein, ALA-MET-ARG-TYR, MAGNESIUM ION, ... (4 entities in total)
• Functional Keywords: intein, isomerase
• Biological source: Mycobacterium xenopi; More
• Total number of polymer chains: 2
• Total formula weight: 22286.44

Authors

Bick, M.J.,Liu, Z.,Frutos, S.,Vila-Perello, M.,Debelouchina, G.T.,Darst, S.A.,Muir, T.W. (deposition date: 2014-02-14, release date: 2014-05-14, Last modification date: 2024-10-30)

Primary citation

Liu, Z.,Frutos, S.,Bick, M.J.,Vila-Perello, M.,Debelouchina, G.T.,Darst, S.A.,Muir, T.W.
Structure of the branched intermediate in protein splicing.
Proc.Natl.Acad.Sci.USA, 111:8422-8427, 2014

PubMed Abstract: 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.

PubMed: 24778214
DOI: 10.1073/pnas.1402942111

Experimental method

X-RAY DIFFRACTION (2.786 Å)