PDBsum entry 1efb

Blood clotting

PDB id

1efb

Contents

			Protein chain

					136 a.a.*

* C-alpha coords only

References listed in PDB file

Key reference

Title

Structural insights into the protein splicing mechanism of pi-Scei.

Authors

B.W.Poland, M.Q.Xu, F.A.Quiocho.

Ref.

J Biol Chem, 2000, 275, 16408-16413. [DOI no: 10.1074/jbc.275.22.16408]

PubMed id

10828056

Abstract

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-A resolution crystal structure of a PI-SceI 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 miniprecursor to be purified and crystallized. The structure reveals both the N- and C-terminal scissile peptide bonds to be in distorted trans conformations (tau approximately 100 degrees ). Modeling of the wild-type PI-SceI based on the VMA29 structure indicates a large conformational change (movement of >9 A) must occur to 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.



	Figure 1. Fig. 1. a, schematic diagram of the chemical mechanism for protein splicing. The four-step reaction couples the excision of the intein (red) from the precursor protein with the ligation of the two exteins (blue and blue-green) via a native peptide bond. b, diagram of the new motif structure of PI-SceI according to Pietrokovski (9). Blocks N1-N4 and C1 and C2 (blue) contain residues involved in protein splicing; blocks EN1-EN4 (black) contain residues associated with the endonuclease/linker domain. Nucleophilic residues are highlighted below the block diagram (yellow letters in purple box), and highly conserved residues are shown in red. c, sequence alignment of the wild-type PI-SceI (VMA) and mutant miniprecursor ( VMA29). The red dash and arrow illustrate the N- and C-terminal splicing sites. Red residues in the VMA29 sequence indicate mutations made to the wild-type sequence (blue). Cys1 and Asn454 were mutated to Ala in order to block in vivo protein splicing.		Figure 3. Fig. 3. Close-up view of the zinc coordination at the C-terminal splicing junction of molecule B of VMA29. Zinc atom is the purple sphere.

	Headers