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Structure of the branched intermediate in protein splicing

2014; National Academy of Sciences; Volume: 111; Issue: 23 Linguagem: Inglês

10.1073/pnas.1402942111

1091-6490

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

RNA modifications and cancer

Significance We report the crystal structure of an intein poised to carry out the rate-limiting step in protein splicing, namely the attack of a conserved Asn side-chain amide on the adjacent backbone amide, leading to resolution of the branched intermediate in the process. The structure reveals that the Asn assumes an unprecedented ready-to-attack conformational state. Guided by this structure, we used protein semisynthesis methods to show that a backbone-to-side-chain hydrogen-bond is critical to position the Asn side-chain for attack and activate it as a nucleophile. This mechanistic insight has general implications for the study of other enzymatic processes involving nucleophilic Asn and Gln residues. The study highlights the power of the combined structural and semisynthesis methods for dissecting protein catalysis.