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.
New England Biolabs, Ipswich, Massachusetts 01938, United States. saleh@neb.com
Lana
L
Saleh
Maurice W
MW
Southworth
Nancy
N
Considine
Colleen
C
O'Neill
Jack
J
Benner
J Martin
JM
Bollinger
Francine B
FB
Perler
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000378:metabolism*; D001483:Base Sequence; D003545:Cysteine / Q000737:chemistry; D004229:Dithiothreitol / Q000737:chemistry; D047669:Exteins; D005456:Fluorescent Dyes / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D047668:Inteins; D007700:Kinetics; D017017:Methanococcus / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D019154:Protein Splicing; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000737:chemistry
D019843:Archaeal Proteins; D005456:Fluorescent Dyes; D010455:Peptides; D014443:Tyrosine; D003545:Cysteine; D004229:Dithiothreitol