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