Nucleic Acids Research, 2005, Vol. 33, No. 13 4219 Figure 5. RD–CBFb interactions involving RD residue Thr161 and CBFb residue Asn104. 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. contribution of this residue to binding was not observed experimentally. Area2 residues make favorable contributions to the inter- action energy between RD and CBFb (Table 5). Although this region does not contribute to the cooperativity, as evidenced by the similar contribution in the binary and the ternary com- plexes, the large favorable interaction energy contribution makes it of interest to investigate the role of individual resi- dues in this region to binding (Table 6). The majority of residues in this region make favorable contribution to the interaction energy. Experimental studies found Thr161 to be a ‘hot spot’ residue (18). The mutation of Thr161 to alanine increased the RD–CBFb dissociation constant in the ternary complex by 40-fold (18). The importance of Thr161 is thought to be due to its interaction with Asn104 of CBFb. Indeed, visual inspection of the RD–CBFb binary MD, ternary MD and crystal structures shows two hydrogen bonds between these residues [Asn(NH2)-Thr(OG1) and Asn(OD1-Thr(HN)] (Figure 5). However, calculation of the residue–residue inter- action energy between RD Thr161 and CBFb Asn104 yields a value of 4.9 ± 0.4 kcal/mol, including the contribution from the Thr HN atom, which does not correlate well with the change in dissociation constant in the T161A mutation. Alternatively, calculation of the contribution of Thr161 to the strain energy of RD yields values of 21.0 ± 1.8 and 26.5 ± 0.2 kcal/mol in the RD monomer and RD–CBFb dimer, respectively. Interestingly, it has been reported that the mutation of Thr161 to alanine leads to decreased exchange broadening in RD alone in NMR experiments, indicating a decrease in the conformational ﬂexibility of residue 161 and surrounding residues (18). This observation combined with the present energetic results suggests that Thr161 may play a role in maintaining the conformation of the local region of RD, thereby impacting binding, rather than contributing directly to protein–protein interactions. Such a role of Thr161 is con- sistent with the idea that conserved polar residues at protein– protein binding sites provide structural rigidity (47). The role of such conserved residues is primarily structural rather than functional. As discussed previously (13) and as emphasized above, the region of RD that includes the segments Wing and Wing1 appears to be of central importance for binding cooperativity. Presented in Table 7 are the interaction energies between Table 7. Interaction energies between individual Wing and Wing1 residues in RD alone and in the three complexes Residue RD RD–CBFb RD–DNA CBFb–RD–DNA Wing residues to Wing1 R139 74.4 ± 1.0 S140 1.5 ± 0.5 G141 2.9 ± 0.5 R142 68.9 ± 2.3 G143 3.5 ± 0.2 K144 213.6 ± 5.0 S145 1.9 ± 0.4 Wing1 residues to Wing T104 0.4 ± 0.0 V105 0.3 ± 0.0 M106 0.4 ± 0.1 A107 0.8 ± 0.0 G108 0.6 ± 0.1 N109 3.1 ± 0.7 D110 153.2 ± 0.9 E111 142.8 ± 6.6 N112 2.3 ± 0.7 Y113 0.2 ± 0.2 S114 0.9 ± 0.0 A115 1.9 ± 0.1 E116 61.8 ± 0.8 Totala 359.9 81.0 ± 1.8 1.2 ± 0.6 7.6 ± 1.0 118.6 ± 11.1 6.0 ± 0.2 185.2 ± 2.5 0.9 ± 0.5 0.4 ± 0.0 0.2 ± 0.0 0.5 ± 0.1 0.9 ± 0.0 0.8 ± 0.1 6.1 ± 0.5 158.9 ± 1.4 168.9 ± 7.4 5.6 ± 0.7 0.1 ± 0.1 1.0 ± 0.1 1.2 ± 0.1 62.2 ± 0.7 398.7 75.7 ± 1.0 1.3 ± 0.0 2.3 ± 1.0 61.1 ± 2.0 0.5 ± 0.0 206.2 ± 5.0 2.0 ± 0.0 0.4 ± 0.0 0.2 ± 0.0 0.4 ± 0.1 0.7 ± 0.1 0.5 ± 0.1 0.8 ± 1.7 149.4 ± 0.5 126.0 ± 5.6 2.3 ± 0.7 0.5 ± 0.2 1.1 ± 0.1 2.0 ± 0.1 66.5 ± 1.8 342.5 75.5 ± 0.8 2.2 ± 0.2 8.1 ± 0.4 65.9 ± 1.3 0.6 ± 0.1 213.4 ± 3.9 0.7 ± 0.3 0.4 ± 0.0 0.1 ± 0.0 0.2 ± 0.0 0.8 ± 0.0 0.6 ± 0.0 6.7 ± 0.2 141.4 ± 1.8 149.9 ± 3.4 5.3 ± 0.2 0.5 ± 0.3 0.9 ± 0.1 1.0 ± 0.1 63.3 ± 1.2 363.8 Energies in kcal/mol. aTotal interaction energy between Wing and Wing1 residues. Wing and Wing1, including a breakdown of the interaction of Wing residues with all Wing1 residues and vice versa. Upon going from RD to the RD–CBFb dimer, the Wing–Wing1 total interaction energy becomes signiﬁcantly more favorable, even though favorable intermolecular interactions occur between Wing1 and CBFb in the RD–CBFb binary complex (Table 5). This suggests that the binding of CBFb to RD, while forming a favorable interaction of CBFb with Wing1 also induces a conformational change in Wing1 that favors the Wing–Wing1 interaction. Indeed, Figure 6 shows that two salt bridges, Arg142-Glu111 and Lys144-Asp110, are present in the RD–CBFb dimer (Figure 6B); this represents a gain in interaction energy over the hydrogen bonds between Lys144 and Asp110 and Glu111 in RD alone (Figure 6A). Indeed, these favorable interactions contribute to the gain in favorable strain energy upon formation of the RD–CBFb dimer