Unfavorable Electrostatic and Steric Interactions in DNA Polymerase β E295K Mutant Interfere with the Enzyme’s Pathway

Mutations in DNA polymerase β (pol β) have
been associated
with approximately 30% of human tumors. The E295K mutation of pol
β has been linked to gastric carcinoma via interference with
base excision repair. To interpret the different behavior of E295K
as compared to wild-type pol β in atomic and energetic detail,
we resolve a binary crystal complex of E295K at 2.5 Å and apply
transition path sampling (TPS) to delineate the closing pathway of
the E295K pol β mutant. Conformational changes are important
components in the enzymatic pathway that lead to and ready the enzyme
for the chemical reaction. Our analyses show that the closing pathway
of E295K mutant differs from the wild-type pol β in terms of
the individual transition states along the pathway, associated energies,
and the active site conformation in the final closed form of the mutant.
In particular, the closed state of E295K has a more distorted active
site than the active site in the wild-type pol β. In addition,
the total energy barrier in the conformational closing pathway is
65 ± 11 kJ/mol, much higher than that estimated for both correct
(e.g., G:C) and incorrect (e.g., G:A) wild-type pol β systems
(42 ± 8 and 45 ± 7 kJ/mol, respectively). In particular,
the rotation of Arg258 is the rate-limiting step in the conformational
pathway of E295K due to unfavorable electrostatic and steric interactions.
The distorted active site in the closed relative to open state and
the high energy barrier in the conformational pathway may explain
in part why the E295K mutant is observed to be inactive. Interestingly,
however, following the closing of the thumb but prior to the rotation
of Arg258, the E295K mutant complex has a similar energy level as
compared to the wild-type pol β. This suggests that the E295K
mutant may associate with DNA with similar affinity, but it may be
hampered in continuing the process of chemistry. Supporting experimental
data come from the observation that the catalytic activity of wild-type
pol β is hampered when E295K is present: this may arise from
the competition between E295K and wild-type enzyme for the DNA. These
combined results suggest that the low insertion efficiency of E295K
mutant as compared to wild-type pol β may be related to a closed
form distorted by unfavorable electrostatic and steric interactions
between Arg258 and other key residues. The active site is thus less
competent for proceeding to the chemical reaction, which may also
involve a higher reaction barrier than the wild-type or may not be
possible in this mutant. Our analysis also suggests further experiments
for other mutants to test the above hypothesis and dissect the roles
of steric and electrostatic factors on enzyme behavior.