Branched intermediate formation is the slowest step in the protein splicing reaction of the Ala1 KlbA intein from Methanococcus jannaschii.

We report the first detailed investigation of the kinetics of protein splicing by the Methanococcus jannaschii KlbA (Mja KlbA) intein. This intein has an N-terminal Ala in place of the nucleophilic Cys or Ser residue that normally initiates splicing but nevertheless splices efficiently in vivo [Southworth, M. W., Benner, J., and Perler, F. B. (2000) EMBO J.19, 5019-5026]. To date, the spontaneous nature of the cis splicing reaction has hindered its examination in vitro. For this reason, we constructed an Mja KlbA intein-mini-extein precursor using intein-mediated protein ligation and engineered a disulfide redox switch that permits initiation of the splicing reaction by the addition of a reducing agent such as dithiothreitol (DTT). A fluorescent tag at the C-terminus of the C-extein permits monitoring of the progress of the reaction. Kinetic analysis of the splicing reaction of the wild-type precursor (with no substitutions in known nucleophiles or assisting groups) at various DTT concentrations shows that formation of the branched intermediate from the precursor is reversible (forward rate constant of 1.5 × 10(-3) s(-1) and reverse rate constant of 1.7 × 10(-5) s(-1) at 42 °C), whereas the productive decay of this intermediate to form the ligated exteins is faster and occurs with a rate constant of 2.2 × 10(-3) s(-1). This finding conflicts with reports about standard inteins, for which Asn cyclization has been assigned as the rate-determining step of the splicing reaction. Despite being the slowest step of the reaction, branched intermediate formation in the Mja KlbA intein is efficient in comparison with those of other intein systems. Interestingly, it also appears that this intermediate is protected against thiolysis by DTT, in contrast to other inteins. Evidence is presented in support of a tight coupling between the N-terminal and C-terminal cleavage steps, despite the fact that the C-terminal single-cleavage reaction occurs in variant Mja KlbA inteins in the absence of N-terminal cleavage. We posit that the splicing events in the Mja KlbA system are tightly coordinated by a network of intra- and interdomain noncovalent interactions, rendering its function particularly sensitive to minor disruptions in the intein or extein environments.

Splicing of the mycobacteriophage Bethlehem DnaB intein: identification of a new mechanistic class of inteins that contain an obligate block F nucleophile.

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 Ala(1) 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.

NMR structure of a KlbA intein precursor from Methanococcus jannaschii.

Certain proteins of unicellular organisms are translated as precursor polypeptides containing inteins (intervening proteins), which are domains capable of performing protein splicing. These domains, in conjunction with a single residue following the intein, catalyze their own excision from the surrounding protein (extein) in a multistep reaction involving the cleavage of two intein-extein peptide bonds and the formation of a new peptide bond that ligates the two exteins to yield the mature protein. We report here the solution NMR structure of a 186-residue precursor of the KlbA intein from Methanococcus jannaschii, comprising the intein together with N- and C-extein segments of 7 and 11 residues, respectively. The intein is shown to adopt a single, well-defined globular domain, representing a HINT (Hedgehog/Intein)-type topology. Fourteen beta-strands are arranged in a complex fold that includes four beta-hairpins and an antiparallel beta-ribbon, and there is one alpha-helix, which is packed against the beta-ribbon, and one turn of 3(10)-helix in the loop between the beta-strands 8 and 9. The two extein segments show increased disorder, and form only minimal nonbonding contacts with the intein domain. Structure-based mutation experiments resulted in a proposal for functional roles of individual residues in the intein catalytic mechanism.

Inteins in Microbial Genomes: Distribution, Mechanism, and Function.

Inteins are self-splicing protein elements (134 to 608 amino acids). Over 125 inteins have been cataloged in InBase, the on-line intein database (http://www.neb.com/neb/inteins.html), which includes the Intein Registry[1]. Inteins naturally present in pathogenic microbes represent novel, yet unexploited drug targets. Understanding the chemistry of the splicing reaction has allowed the manipulation of inteins, which are now used in many protein engineering applications[2].

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

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.

NMR assignment of a KlbA intein precursor from Methanococcus jannaschii.

The backbone and side chain resonance assignments of a precursor of the KlbA intein from Methanococcus jannaschii have been determined, based on triple-resonance experiments with the uniformly [13C,15N]-labeled protein.

Protein splicing of the Deinococcus radiodurans strain R1 Snf2 intein.

Adjacent intein fragments fused to a Snf2/Rad54 helicase-related protein and Snf2/Rad54 helicase were reported for Deinococcus radiodurans R1, leading to the speculation that a frameshift was required for splicing or that trans splicing occurred. However, a type strain (ATCC 13939, RF18410) yielded a single protein that splices by the Ala1 protein splicing pathway, with splicing dependent on adjacent residues.

Bacteriophage-based genetic system for selection of nonsplicing inteins.

A genetic selection system that detects splicing and nonsplicing activities of inteins was developed based on the ability to rescue a T4 phage strain with a conditionally inactive DNA polymerase. This phage defect can be complemented by expression of plasmid-encoded phage RB69 DNA polymerase. Insertion of an intein gene into the active site of the RB69 DNA polymerase gene renders polymerase activity and phage viability dependent on protein splicing. The effectiveness of the system was tested by screening for thermosensitive splicing mutants. Development of genetic systems with the potential of identifying protein splicing inhibitors is a first step towards controlling proliferation of pathogenic microbes harboring inteins in essential proteins.