Limitations of computer modeling
To produce the large-scale bends in DNA, we adjusted the roll angles
using an approximation of 10 bp/turn. In experiments, biochemical
analyses have shown that straight DNA in solution has 10.4 ± 0.1
bp/turn, rather than 10 bp/turn as observed in the solid
state.34 In vivo , supercoiling of DNA leads to
a slight unwinding with the result of 11.1 bp/turn.35Because the corresponding parts of I1 and I2 half sites
are 21 bp apart, it suggests that in this region of DNA, the twist is
closer to 10.5 bp/turn to have the DBDs bind on the same side of the
DNA. For 10, 10.4, and 11.2 bp/turn, the total twist over the 38 bp
length of I1-spacer-I2 is three full helical turns and 288°,
235°, and 152° of the fourth helical turn, respectively. The incomplete
fourth turn would affect which side of the DNA the DBDs could bind to
and the relative rotation of the two DBDs. For producing tertiary and
quaternary models of protein/DNA complexes, the value of 10 bp/turn was
adequate for bending the DNA in a narrow step range (< 10
steps) but it introduces inaccuracies in the hydrogen bonding within
DNA, the plane of the bend, and the relative orientation of the two
DBDs.
To create the full model of the AraC/DNA complex, docking software was
used as one stage of the process to form protein-DNA and protein-protein
complexes. Our docking procedure however, cannot produce reliable
results for residue/base, base/base, and residue/residue interactions.
Some docking packages support the DNA sequence as
input.17 However, we found that when the DNA structure
is generated by a computer from a sequence, the DBD could not be
satisfactorily docked onto DNA. We had to divide the sequence into
segments and construct each segment from experimentally-determined
coordinates. While the DNA segment method initially positioned the DBD
on the DNA, it was still insufficient to achieve satisfactory docking in
that both HTHs of the DBD did not sit fully in the major groove of DNA.
Experiments have shown that protein and DNA both experience
conformational changes upon binding, and that when protein bends DNA,
there is an energy cost associated with distortion such that the DNA is
not in its lowest energy configuration.14 Docking
software should ideally allow for DNA flexibility and protein backbone
and side chain flexibility, which are difficult features for any
software package to possess.36, 37 We addressed this
issue partially by manually adjusting the bend in the DNA and submitting
the 3D structure to the docking software. This process was repeated
until satisfactory surface complementarity was achieved. However
detailed manipulations on the atomic scale were not conducted that would
result in the true energy of the DNA or the protein. We also observed
that upon docking, the two HTHs of the DBD can sit in the DNA major
groove in various ways. We had to make a judgment on which model was the
best; inaccuracies could have been introduced at this stage. Finally the
docking approach in this paper did not take into account effects of
solvation and salt.39 As a step towards determining
tertiary and quaternary structure of the AraC/DNA complex, docking was
useful; however conclusions about structure and interactions on an
atomic scale must be drawn carefully.