The Deinococcus radiodurans Snf2 intein caught in the act: Detection of the Class 3 intein signature Block F branched intermediate

Abstract

Inteins are the protein equivalent of introns. They are remarkable and robust single turnover enzymes that splice out of precursor proteins during post-translational maturation of the host protein (extein). The Deinococcus radiodurans Snf2 intein is the second member of the recently discovered Class 3 subfamily of inteins to be characterized. Class 3 inteins have a unique sequence signature: (a) they start with residues other than the standard Class 1 Cys, Ser or Thr, (b) have a noncontiguous, centrally located Trp/Cys/Thr triplet, and (c) all but one have Ser or Thr at the start of the C-extein instead of the more common Cys. We previously proposed that Class 3 inteins splice by a variation in the standard intein-mediated protein splicing mechanism that includes a novel initiating step leading to the formation of a previously unrecognized branched intermediate. In this mechanism defined with the Class 3 prototypic Mycobacteriophage Bethlehem DnaB intein, the triplet Cys attacks the peptide bond at the N-terminal splice junction to form the class specific branched intermediate after which the N-extein is transferred to the side chain of the Ser, Thr, or Cys at the C-terminal splice junction to form the standard intein branched intermediate. Analysis of the Deinococcus radiodurans Snf2 intein confirms this splicing mechanism. Moreover, the Class 3 specific Block F branched intermediate was isolated, providing the first direct proof of its existence.

Expanding the Definition of Class 3 Inteins and Their Proposed Phage Origin

“…Splicing of the class 2 prototype (Methanococcus jannaschii KlbA intein) is initiated by a direct attack on the N-terminal splice junction peptide bond by Cys ϩ1 instead of the initial acyl shift that initiates splicing of class 1 inteins; the remaining reaction is identical to the class 1 mechanism (19). The splicing mechanism of class 3 inteins was defined using the mycobacteriophage Bethlehem DnaB intein (20) and the Deinococcus radiodurans Snf2 intein (2). Their splicing reaction is initiated by a nucleophilic attack on the N-terminal splice junction peptide bond by the WCT motif Cys F:4 , which results in formation of the class-specific branched intermediate (block F BI; VIII) (Fig.…”

“…3A). This result clearly indicates that the MP-Catera Gp206 intein is a class 3 intein, not a class 2 intein, because class 2 inteins require the ϩ1 amino acid for N-terminal cleavage (19) and class 3 inteins do not (2,20).…”

“…3). In class 1 and 2 inteins, mutations at F:4 inhibit splicing because this residue assists reactions at one or both splice junctions, depending on the individual intein (2,8,13,(19)(20)(21). In class 3 inteins, Ala substitution for this residue blocks all N-terminal cleavage reactions, including BI formation.…”

Canonical Protein Splicing of a Class 1 Intein That Has a Class 3 Noncanonical Sequence Motif

“…1) (2,14). Class 2 inteins can bypass the first step of splicing; splicing is initiated by attack of the downstream nucleophile on the N-terminal splice junction (13,14).…”

“…Class 3 inteins lack an N-terminal nucleophile and have a covariant Trp-Cys-Thr conserved triplet of noncontiguous residues. For the Mycobacterium phage Bethlehem DnaB and Deinococcus radiodurans Snf2 inteins, the Cys of the covariant triplet is responsible for initial attack on the N-terminal splice junction peptide bond, creating an internal branched thioester (2,14). The N extein is then transferred from the side chain of the internal Cys to the side chain of the Ser flanking the intein C terminus.…”