ja8b04794_si_001.pdf (1.5 MB)
Altered Coordination of Individual Catalytic Steps in Different and Evolved Inteins Reveals Kinetic Plasticity of the Protein Splicing Pathway
journal contribution
posted on 2018-08-15, 00:00 authored by Julian
C. J. Matern, Kristina Friedel, Jens Binschik, Kira-Sophie Becher, Zahide Yilmaz, Henning D. MootzProtein
splicing performed by inteins provides powerful opportunities
to manipulate protein structure and function, however, detailed mechanistic
knowledge of the multistep pathway to help engineering optimized inteins
remains scarce. A typical intein has to coordinate three steps to
maximize the product yield of ligated exteins. We have revealed a
new type of coordination in the Ssp DnaB intein,
in which the initial N–S acyl
shift appears rate-limiting and acts as an up-regulation switch to
dramatically accelerate the last step of succinimide formation, which
is thus coupled to the first step. The structure–activity relationship
at the N-terminal scissile bond was studied with atomic precision
using a semisynthetic split intein. We show that the removal of the
extein acyl group from the α-amino moiety of the intein’s
first residue is strictly required and sufficient for the up-regulation
switch. Even an acetyl group as the smallest possible extein moiety
completely blocked the switch. Furthermore, we investigated the M86
intein, a mutant with faster splicing kinetics previously obtained
by laboratory evolution of the Ssp DnaB intein, and
the individual impact of its eight mutations. The succinimide formation
was decoupled from the first step in the M86 intein, but the acquired
H143R mutation acts as a brake to prevent premature C-terminal cleavage
and thereby maximizes splicing yields. Together, these results revealed
a high degree of plasticity in the kinetic coordination of the splicing
pathway. Furthermore, our study led to the rational design of improved
M86 mutants with the highest yielding trans-splicing
and fastest trans-cleavage activities.