Abstract
We have determined the crystal structure of a 154-residue intein derived from the dnaB gene of Synechocystis sp. strain PCC6803 and refined it to a 2.0-A resolution. The x-ray structure suggests that this intein possesses two catalytic sites that appear to be separately responsible for splicing and cleavage of the N- and C-terminal scissile bonds. The conserved intein block F residues are the important components of a catalytic site for side chain cyclization of the last intein residue, Asn-154. The data suggest that the imidazole ring of His-143 is involved in the activation of the side chain Ndelta atom of Asn-154, leading to a nucleophilic attack on the carbonyl carbon of Asn-154. Substitution of His-143 with Ala or Gln resulted in the inhibition of C-terminal cleavage. His-153, Asp-136, and a water molecule appear to constitute an oxyanion binding site by contacting the carbonyl oxygen of Asn-154 to stabilize the transition state. The structure and mutagenesis data also support that the close contact between the hydroxyl groups of Thr-138 and Ser-155, whose side chain participates in an S --> O acyl shift, plays an important role in the nucleophile orientation. Our structural modeling suggests that this catalytic module is conserved in the C-terminal subdomains of inteins from diverse organisms.
Highlights
Protein splicing is a posttranslational processing event that involves the precise removal of an intervening sequence, an intein, from a protein precursor with concomitant ligation of the flanking protein sequences (N and C exteins) via a native peptide bond [1,2,3]
In vitro splicing of inteins in heterologous proteins suggests that inteins with the first C-extein residue contain sufficient information needed for the autocatalytic
The splicing domain of a dual function intein appears to be split by the endonuclease domain into two segments, the N- and C-terminal subdomains
Summary
The x-ray structures of the intein of the vacuolar ATPase subunit from Saccharomyces cerevisiae (Sce VMA intein) [21,22,23,24], the archaeal PI-PfuI intein from Pyrococcus furiosus (PIPfuI intein) [25], and the mini-intein of the GyrA protein from Mycobacterium xenopi (Mxe GyrA intein) [26] have revealed that inteins contain a horseshoe-like -strand scaffold termed the Hint (Hedgehog, intein) module [11]. Mutation of catalytic residues at the N- and C-terminal splice junctions of the Sce VMA inteins resulted in the trapping of the inactive precursor proteins for crystallization studies [22, 23] These investigations indicated that the conserved block B residues and the residues at the N-terminal splice site form an active center that facilitates cleavage of the N-terminal scissile peptide bond. The C-terminal catalytic site, formed by the conserved residues of blocks F and G, constitutes a charge relay system and an oxyanion binding site, which may play vital roles in catalyzing the cyclization of Asn-154 This intein structure reveals that the peptide bond between Asn-154 and the first C-extein residue, Ser-155, is in a distorted trans conformation. The implications of the existence of a catalytic site in each intein subdomain are discussed with respect to intein evolution
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