3.2 Constructing the model of full-length AraC and DNA in the
presence of arabinose
In initial attempts to build a model of full-length AraC bound to DNA in
the presence of arabinose, we started with a straight helical structure
for I1 and I2 generated by a computer. However, we were
not able to achieve a satisfactory dock of straight I1 onto the
DBD. We then developed a DNA segment method (see Methods) to buildI1 and I2 out of experimentally-determined positions of
DNA atoms from the RCSB database. Upon docking, this process resulted in
an acceptable positioning of one of the HTHs of the DBD onto a
half-site. To dock both HTHs of each DBD onto a half-site, we had to
bend the DNA by adjusting the DNA roll values (Figures 2B, C).
Mutagenesis experiments have shown that there are points of contact
between N194 of the DBD and H126 and N139 of the dimerization domains
(DDs).9 H126 and N139 lie on the DD of different
subunits and are close to each other. Using this information and HDOCK
software, the DBD‑A/I1 complex was positioned onto the DDs with
N194 of DBD-A close to N139 of DD B and H126 of DD A (Figure 6). Due to
linker length constraints, the N-terminus of DBD-A had to be near the
C-terminus of DD A. (DD A and linker A are parts of subunit A.) The
distance between Cα of I167 of DD A and
Cα of M175 of DBD-A is 23 Å, which is consistent with
the length of linker A.
Biochemical measurements have shown that the two DBDs are bound toI1-I2 with a direct repeat orientation.6 To
build the model, the DBD-B/I2 complex had to be brought closer to
DD B, as shown in Figure 6. Large-scale bending of DNA was accomplished
in the 4-bp spacer region and first two bp of I2 . This process
enabled Cα of M175 of DBD-B to be within 25 Å of
Cα of I167 of DD B (Figure 7). Linkers A and B are
uncoiled relative to the conformation appearing in the crystal structure
1XJA. Table 1 shows the values used in adjusting the roll angles to
conduct large-scale bending of the DNA in the extended spacer region.
The full model of AraC/DNA is shown in Figure 7A. The two orange line
segments indicate a bend angle of 69°. Different lines can be
constructed tangent to the I1 contour resulting in a range of
possible DNA bend angles from 52° to 88°. Two straight line segments
(teal) indicate where the linkers are located. Linker B is clearly
visible in the middle of the figure; linker A is near the top and is
rotated to be almost hidden from view. Figure 7B and the schematic
diagram Figure 7D show the large-scale bend in the DNA and the domains
of AraC. The DDs are oriented such that DD B is on top of DD A. Figures
7C, E represent a rotated view of AraC/DNA in which the DNA appears
almost straight and both DDs are visible. The 5’ end of I1 is
near linker A and the N-terminus of DBD-A. Similarly the 5’ end ofI2 is near the N-terminus of DBD-B and linker B.
A Pymol session file (.pse) of the AraC/DNA complex is included in the
Supporting Information (Figure S3). Explicit hydrogens are not present
in the structure because HDOCK and Web 3DNA remove them. In the HDOCK
scoring function, the effects of hydrogens are incorporated in the
potentials associated with different heavy atom types and their
environments.26