Splicing of the Mycobacteriophage Bethlehem DnaB Intein

Abstract

Inteins are single turnover enzymes that splice out of protein precursors during maturation of the host protein (extein). The Cys or Ser at the N terminus of most inteins initiates a four-step protein splicing reaction by forming a (thio)ester bond at the N-terminal splice junction. Several recently identified inteins cannot perform this acyl rearrangement because they do not begin with Cys, Thr, or Ser. This study analyzes one of these, the mycobacteriophage Bethlehem DnaB intein, which we describe here as the prototype for a new class of inteins based on sequence comparisons, reactivity, and mechanism. These Class 3 inteins are characterized by a non-nucleophilic N-terminal residue that co-varies with a non-contiguous Trp, Cys, Thr triplet (WCT) and a Thr or Ser as the first C-extein residue. Several mechanistic differences were observed when compared with standard inteins or previously studied atypical KlbA Ala1 inteins: (a) cleavage at the N-terminal splice junction in the absence of all standard N- and C-terminal splice junction nucleophiles, (b) activation of the N-terminal splice junction by a variant Block B motif that includes the WCT triplet Trp, (c) decay of the branched intermediate by thiols or Cys despite an ester linkage at the C-extein branch point, and (d) an absolute requirement for the WCT triplet Block F Cys. Based on biochemical data and confirmed by molecular modeling, we propose roles for these newly identified conserved residues, a novel protein splicing mechanism that includes a second branched intermediate, and an intein classification with three mechanistic categories.

Sequential formation of two branched intermediates during protein splicing of class three inteins

“…Inteins from extremophiles were key to defining all three currently known mechanisms of intein-mediated protein splicing (Brace et al 2010; Eryilmaz et al 2014; Mills et al 2014; Southworth et al 2000; Tori et al 2010; Volkmann and Mootz 2013; Xu et al 1994; Xu and Perler 1996). Over 500 intein genes have been identified in numerous archaea, eubacteria, single cell eukaryotes and viruses (Perler 2002), and the numbers are growing rapidly with the explosion of archaeal and bacterial genome sequences.…”

“…1) and conserved signature sequences (Tori et al 2010). All inteins have at least four conserved motifs (Blocks A, B, F and G) in the splicing domain (Perler 2002; Perler et al 1997; Pietrokovski 1994, 1998).…”

“…All inteins have at least four conserved motifs (Blocks A, B, F and G) in the splicing domain (Perler 2002; Perler et al 1997; Pietrokovski 1994, 1998). Amino acids (aa) within these conserved motifs are numbered using the block designation and the position within the block, separated by a colon (Perler 2002; Tori et al 2010). For example, the fourth amino acid in Block F is referred to as F:4.…”

“…For example, the fourth amino acid in Block F is referred to as F:4. Several amino acids in these conserved motifs are present at or near the intein active site where they directly act as nucleophiles and facilitating residues for catalysis or they assist in proper packing of the intein active site to align catalytic residues (Brace et al 2010; Eryilmaz et al 2014; Kawasaki et al 1997; Mills et al 2014; Romanelli et al 2004; Southworth et al 2000; Tori et al 2010; Volkmann and Mootz 2013; Xu et al 1994; Xu and Perler 1996). Catalytically important residues common to all classes of inteins include His B:10 (essential for N-terminal splice junction reactions), the intein penultimate His G:6 (assists in C-terminal splice junction reactions), the intein C-terminal Asn G:7 (responsible for C-terminal splice junction cleavage during resolution of the Block G branched intermediate, BI G ) and Ser +1 , Cys +1 or Thr +1 [the G:8 residue, responsible for BI G formation and conversion of a (thio)ester bond to an amide peptide bond between the ligated exteins].…”

Chemischer “Bypass” des Intein‐katalysierten N‐S‐Acyltransfers im Proteinspleißen

“…[1] Besonders faszinierend ist der N-S-(oder N-O) Acyltransfer an der stromaufwärts vom Intein gelegenen zu spaltenden Peptidbindung unter Bildung eines Thioesters (oder Oxoesters); siehe Schema 1 B. Diese Umlagerung ist der erste Schritt des Proteinspleißens in Standard-Inteinen [2] und wird vielfach für die Generierung von Proteinthioestern genutzt. [3] Obwohl die Umlagerung thermodynamisch nicht bevorzugt ist, sind dafür keine Cofaktoren oder Energieäquivalente erforderlich.…”