4216 Nucleic Acids Research, 2005, Vol. 33, No. 13 A more detailed understanding of the atomic determinants of cooperativity associated with RD binding to CBFb and DNA was obtained via interaction energy partitioning analysis between different regions of the RD and both CBFb and DNA. This was facilitated by partitioning RD into a variety of seg- ments as speciﬁed in Table 2 and shown in Figure 1. Several regions of the protein have been previously indicated to con- tribute to either RD–DNA or RD–CBFb interactions (13). Residues adjacent to the respective binding partner but not included in a deﬁned segment are referred to as Other-DNA and Other-CBFb and all residues not previously deﬁned are referred to as non-interacting-CBFb, allowing for the quanti- ﬁcation of their contributions to binding. Of note are the Wing residues, which are primarily important for DNA binding, while residues in Wing1 are proximal to the Wing residues, contacting both CBFb and the Wing residues. For all these segments of RD, interaction energies were calculated between the respective monomers in the dimers as well as in the trimer. As discussed above, cooperativity exists between both DNA and CBFb with respect to their binding to RD. Table 3 includes the change in RD–DNA interaction energies in both the trimer and the dimer complexes along with the change in interaction energy upon going from the RD–DNA dimer to the trimer, with those contributions broken down into the segments pre- sented in Table 2. Upon trimer formation the total RD–DNA interaction energy becomes more favorable, with three of the segments contributing signiﬁcantly to the more favorable interaction energy in the trimer. These include Wing, bA loop and Other-DNA segments. The largest contribution is from the Wing segment, consistent with its location in the vicinity of both the DNA and the CBFb. This location allows for Table 2. Definitions of regions of the RD interacting with DNA or CBFb Colora Lime Pink Purple Orange White Blue Cyan Yellow Brown White 67.0 11.5 13.7 1.8 42.9 109.5 Segment name DNA binding regions Wing bA loop bG strand Tail Other-DNA CBFb binding regions Wing1 Area1/2 N terminus Other-CBFb Non-interacting-CBFb Residues 139–145 77–84 166–169 170–177 54–76, 85–138, 146–165, 178 104–116 156–161 66–69 149–151 All remaining RD residues not interacting with CBFb aColor as shown in Figure 1. Red indicates residues adjacent to those omitted from the MD simulations. Table 3. RD region contributions to the RD–DNA interaction energy Region Wing bA Loop Tail bG strand Other-DNA Total CBFb–RD–DNA Complex RD–DNA DIE 1158.1 ± 9.9 899.7 ± 2.5 206.9 ± 8.7 418.1 ± 0.8 59.9 ± 10.3 2622.9 ± 13.4 1091.1 ± 10.1 888.2 ± 5.3 220.6 ± 5.8 416.3 ± 4.8 102.8 ± 20.9 2513.4 ± 11.9 interactions of CBFb to be communicated to Wing via Wing1 (see below for additional details), as discussed previously (13). Interestingly, the Other-DNA region makes a signiﬁcant con- tribution to cooperativity by its unfavorable contribution in the RD–DNA dimer becoming less unfavorable in the trimer. Such a change indicates that the binding of CBFb leads to a conformational change in the core region of RD that favors interactions with the DNA and, due to the Other-DNA region not being in direct contact with the DNA, suggests that long- range interactions contribute to binding cooperativity. To better understand the role of the Wing segment in facil- itation of DNA binding upon trimer formation, the contribu- tion of this region was broken down into a per residue basis (Table 4). Overall, the majority of the favorable interactions are made by Arg139, Arg142 and Lys144, as expected due to their positive charge and the polyanionic nature of DNA. With all three residues their interaction energy contribution becomes more favorable upon trimer formation. In addition, more favorable interactions occur with Ser140, Gly143 and Ser145, with the contribution of Gly143 being relatively large. Thus, speciﬁc residues in the Wing contribute to more favor- able interactions with the DNA upon formation of the ternary complex. Details of the role of CBFb and the Wing1 segment to these contributions will be discussed below. Similar to RD–DNA binding, the RD to CBFb interaction energy is increased upon trimer formation. Table 5 presents the energy breakdown into the different RD segment contri- butions. The overall cooperativity gain of 60 kcal/mol is due to the Wing1 and N-terminal segments. The contribution of the Wing segment to CBFb binding becomes less favorable whereas the contribution of this segment becomes more favor- able to DNA binding upon trimerization (Table 3). Such a scenario suggests a subtle balance between interactions of the Wing and Wing1 segments that contribute to the cooper- ative binding. Interestingly, as with the Other-DNA segment discussed above for RD–DNA binding, the N-terminal seg- ment makes a favorable contribution to the cooperativity of Table 4. Interaction energy of RD Wing residues with DNA RD residues CBFb–RD–DNA Complex RD–DNA DIE R139 S140 G141 R142 G143 K144 S145 452.4 ± 3.1 2.2 ± 0.2 0.6 ± 0.4 432.4 ± 8.6 19.7 ± 0.3 248.8 ± 1.4 2.0 ± 1.1 Region Wing Wing1 Area2 N-terminus Other-CBFb Non-interacting CBFb Total 0.0 ± 2.2 114.4 ± 8.9 65.2 ± 1.4 77.5 ± 4.8 12.2 ± 0.4 29.5 ± 3.8 298.7 ± 0.0 Energies in kcal/mol as mean ± SE, DIE ¼ CBFb–RD–DNA RD–DNA. Table 5. RD region contributions to the RD–CBFb interaction energy CBFb–RD–DNA Complex CBFb–RD DIE 428.1 ± 6.3 0.8 ± 0.6 7.6 ± 1.8 404.7 ± 8.8 8.4 ± 1.7 242.6 ± 2.0 0.4 ± 1.1 12.1 ± 2.6 70.9 ± 7.8 66.6 ± 1.3 29.4 ± 1.7 13.3 ± 0.2 46.4 ± 7.6 238.7 ± 2.9 24.3 3.0 7.0 27.7 11.3 6.2 1.6 12.1 43.5 1.4 48.1 1.1 16.9 60.0 Energies in kcal/mol as mean ± SE, DIE ¼ CBFb–RD–DNA RD–DNA. Energies in kcal/mol as mean ± SE, DIE ¼ CBFb–RD–DNA CBFb–RD.