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Figure 2.
Figure 2. RuvB nucleotide recognition and an implied
strained ATP-bound conformation. (a) Details of the
nucleotide-binding site reveal that the phosphate groups are
coordinated by the Walker A motif (including Lys64 and Thr65)
with the ADP moiety contacted by residues of the sensor 2 motif
(Pro216 and Arg217). Sensor 1 (Thr158) and Walker B (Asp109 and
Glu110) motifs are located near the position of the g-phosphate
group. The isosurface of the simulated annealing omit difference
density is shown for ADP, contoured at 3s (green). (b)
Structure-based mutational analysis reveals the importance of
ATP hydrolysis in branch migration and the key roles played by
sensor 1, sensor 2, and arginine finger in RuvB. Biochemical
characterization of the DNA-dependent ATPase activity of RuvB
mutants[21] and branch migration of an in vitro reconstituted
RuvAB-Holliday junction complex.[51 and 52] Proteins scored as
inactive, "-", in branch migration activity are either wholly or
substantially compromised, as they showed less than 3 % of
wild-type activity after an incubation of 60 minutes. (c)
Overlay of the wild-type RuvB protein (blue) with structures of
the sensor 1 mutations Ala156Ser (yellow), Thr158Val (light
blue), and the Walker A mutation Lys64Arg (light brown). (d)
Overlay of the sensor 2 mutation Pro216Gly (yellow) with
wild-type RuvB, illustrating some of the structural
rearrangements required to accommodate the misregistered ATP
(Figure 2(c) in the nucleotide-binding site. (e) Details of ATP
binding from the Pro216Gly structure (red) and ADP binding from
the wild-type structure (blue) demonstrating the reorientation
of the both adenine and ribose moieties and the phosphate
misregistration, where the ATP g-phosphate group binds at the b
position and the ATP b-phosphate group binds at the a position.
This structure suggests that binding ATP in the appropriate
conformation channels binding energy into a strained RuvB
conformation. (f) Overlay of the arginine finger mutation
Arg170Ala (yellow) with wild-type RuvB, suggesting the dramatic
loss of ATPase and branch migration assay are due to loss of the
guanidium functionality, as structural perturbations are small.
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