RD residue Wing1 T104 V105 M106 A107 G108 N109 D110 E111 N112 Y113 S114 A115 E116 N-Terminus D66 S67 P68 N69 Area2 T154 N155 P156 P157 Q168 V169 A160 T161 Energies in kcal/mol as mean ± SE, DIE ¼ CBFb–RD–DNA CBFb–RD. RD–CBFb binding although it is spatially remote from the DNA in the ternary complex. This indicates that binding of DNA to form the trimer leads to global conformational changes in RD that favor RD–CBFb interactions, further indicating the contribution of long-range interactions to cooperativity. Residue breakdown of the interaction energy contributions of the Wing1 and N-terminal regions to CBFb–RD interac- tions is presented in Table 6. The energetic contribution of a number of the residues changes signiﬁcantly upon going from the CBFb–RD dimer to the trimer, with both favorable and unfavorable contributions occurring. The most signiﬁcant Table 6. Interaction energy of selected RD residues with CBFb CBFb–RD–DNA CBFb–RD DIE 1.2 ± 0.1 0.1 ± 0.1 6.9 ± 0.6 2.1 ± 0.3 0.8 ± 0.3 5.4 ± 0.3 3.2 ± 0.6 3.4 ± 1.3 5.2 ± 1.4 14.1 ± 0.9 14.8 ± 1.7 2.7 ± 0.9 66.5 ± 8.8 44.9 ± 4.4 9.9 ± 0.4 13.5 ± 0.3 9.3 ± 0.2 0.6 ± 0.4 4.6 ± 1.1 11.1 ± 0.4 12.1 ± 0.2 9.2 ± 0.5 16.3 ± 0.1 4.2 ± 0.1 8.3 ± 0.2 3.3 ± 0.8 0.7 ± 0.1 9.5 ± 0.2 1.2 ± 0.1 1.2 ± 0.1 4.7 ± 0.2 8.2 ± 1.0 1.2 ± 0.7 0.7 ± 0.6 27.6 ± 1.1 14.0 ± 0.3 1.5 ± 0.1 20.4 ± 7.8 1.0 ± 1.1 7.8 ± 0.5 12.8 ± 0.9 9.9 ± 0.3 0.1 ± 0.4 5.3 ± 0.4 10.1 ± 0.6 12.0 ± 0.1 10.4 ± 1.0 15.7 ± 0.1 4.2 ± 0.1 9.0 ± 0.1 2.1 0.6 2.6 0.9 0.4 0.7 5.0 4.6 4.5 13.5 0.8 1.2 46.1 45.9 2.1 0.7 0.6 0.7 0.7 1.0 0.1 1.2 0.6 0.0 0.7 Nucleic Acids Research, 2005, Vol. 33, No. 13 4217 contributions, both favorable, occur with Glu116 in Wing1 and Asp66 in the N-terminal segment. The importance of Glu116 for the binding of CBFb has not previously been described, although perturbed chemical shifts of Glu116 have been observed in NMR studies in the presence of either CBFb or DNA (13,17,46) and a signiﬁcant decrease in the order para- meter of Glu116 has been observed upon going from the RD–DNA dimer to the trimer (20). The interaction energies between RD Glu116 and CBFb in the binary and ternary complexes are 20 ± 8 and 67 ± 9 kcal/mol, respectively. In the present study, it was found that RD Glu116 interacts with CBFb residue Arg33. Experimentally, mutation of CBFb Arg33 to alanine increased the RD–CBFb dissociation con- stant in the ternary complex by 20-fold (18). This ﬁnding is consistent with the present result. Interestingly, the experi- mental change in KD was not accounted for in terms of any residue(s) in the RD. To understand this omission, distances between the two residues in the binary and ternary complexes as a function of time were obtained (Supplementary Figure S1). It is observed that in the ternary complex during the MD simulation, the sidechains of RD Glu116 and CBFb Arg33 move to within 2–3 s, in contrast to the OE2-NH2 distance being 9.4 s in the crystallographic structure of the ternary complex (13). In contrast, in the binary complex crystal struc- ture the OE2-NH2 distance is 12 s; this distance initially decreases to shorter values during the MD simulation, but upon further simulation time assumes a stable conformation with a distance of 12 s between the OE2-NH2 atoms. To better understand the signiﬁcant changes in the RD Glu116 and CBFb Arg33 distances for the different systems the experimental structure along with ﬁnal time frames from the 4.5 ns RD–CBFb binary and ternary simulations were obtained (Figure 3). In the RD–CBFb dimer, RD Glu116 and CBFb Arg33 are relatively far apart (Figure 3A). Upon going to the ternary complex in the MD simulation it is clear that a salt bridge forms between Glu116 and Arg33 (Figure 3B). The formation of this salt bridge is associated with a shift in the position of the Arg33 sidechain towards the RD, which is suggested to be associated with the electrostatic ﬁeld due to the presence of DNA in the ternary complex as evidenced by the favorable interactions between the CBFb Figure 3. RD–CBFb interactions involving RD residues Tyr113, Glu116, Arg118 and Arg139, and CBFb residues Arg33 and Arg35. Images from snapshots at 4.5 ns from the (A) RD–CBFb binary and (B) ternary MD simulations and (C) from the X-ray crystal structure. DNA is shown in red.