Structural insights into the protein splicing mechanism of PI-SceI

PI-SceI is a member of a class of proteins (inteins) that excise themselves from a precursor protein and in the process ligate the flanking protein sequences (exteins). We report here the 2.1-Angstrom resolution crystal structure of a PI-SceZ miniprecursor (VMA29) containing 10 N-terminal extein residues and 4 C-terminal extein residues. Mutations at the N- and C-terminal splicing junctions, blocking in vivo protein splicing, allowed the mini-precursor to be purified and crystallized. The structure reveals both the N- and C-terminal scissile peptide bonds to be in distorted trans conformations (tau approximate to 100 degrees). Modeling of the wild-type PI-SceI based on the VMA29 structure indicates a large conformational change (movement of >9 Angstrom) must occur 60 allow transesterification to be completed. A zinc atom was discovered at the C-terminal splicing junction. Residues Cys(455), His(453) and Glu(80) along with a water molecule (Wat(53)) chelate the zinc atom. The crystal structure of VMA29 has captured the intein in its pre-spliced state.