4220 Nucleic Acids Research, 2005, Vol. 33, No. 13 Figure 6. RD Wing–Wing1 interaction in the MD simulation structures at 4.5 ns for (A) RD, (B) RD–CBFb, (C) RD–DNA and (D) the trimer. RD residues Arg139, Arg142, Lys144, Asp110 and Glu111 are shown. DNA is shown in red. discussed above (Table 1). Moreover, NMR experiments indicate all four of these residues to undergo changes in mobil- ity upon going from the RD–CBFb dimer to the trimer (20). Thus, these intramolecular interactions make a signiﬁcant contribution to the binding of RD to CBFb. In contrast to the RD–CBFb interaction, upon going from RD alone to the RD–DNA binary complex, the Wing–Wing1 total interaction energy becomes less favorable. This is consistent with the signiﬁcantly favorable interaction energy between RD and DNA in the binary complex, such that when the Wing residues interact with the DNA, the Wing–Wing1 interactions are diminished (Table 7). Similarly, the Wing– Wing1 interaction energy in the trimer is not signiﬁcantly different as that in RD alone. Visual analysis of the RD struc- ture in the monomer (Figure 6A), the RD–DNA complex (Figure 6C) and the ternary complex (Figure 6D), reveals the spatial relationships of residues Asp110, Glu111, Arg142 and Lys144 to be similar, consistent with the interaction energy contributions in these systems (Table 7). In the RD–DNA binary and ternary complexes, it is evident that the negatively charged DNA attracts Arg142 away from the Wing1 residues, contributing to the less favorable interactions. In the RD monomer, the lack of salt bridges between Arg142 and Asp110 or Glu111 may be related to repulsion between the two positively charged residues, Arg142 and Lys144, such that the system gains more stabilization by orienting Arg142 toward the solvent. The balance of attraction and repulsion taking place between closely situated positively and negat- ively charged residues suggests a subtle balance of inter- actions, such that their relative orientations are sensitive to the inﬂuence of solvent and the presence of DNA and CBFb. As stated above, formation of the ternary complex leads to a Wing–Wing1 interaction energy that is similar to that of the RD monomer. Energy partitioning analysis (Table 7) shows signiﬁcantly less favorable interaction energies involving Arg142, Asp110 and Glu111 as compared with the CBFb– RD binary complex. The less favorable interactions of Asp110 and Glu111 are consistent with the loss of salt bridges with Arg142, although more favorable interactions with Lys144 occur (Figure 6D). Thus, a subtle balance of intramolecular and intermolecular interactions appear to contribute to the observed cooperativity. Interaction of RD with DNA leads to favorable intermolecular interactions (Table 3) with a sim- ultaneous loss of Wing–Wing1 interactions (Table 7). How- ever, upon formation of the ternary complex, the binding of CBFb leads to the favorable Wing–Wing1 interactions being regained, which also enhances the Wing1–DNA interactions. Interestingly, the majority of residues predicted to make the largest contributions to cooperativity are charged (i.e. RD residues Asp66, Asp110, Glu111, Glu116, Arg139, Arg142 and Lys144). Consequently, the long-range character of electrostatic interactions may be an important factor in the observed cooperativity. The results suggest some interesting mutations that may impact cooperativity. For example, Asp to