4218 Nucleic Acids Research, 2005, Vol. 33, No. 13 Arg33 and the DNA ( 252.2 ± 6.1 kcal/mol). In addition, energy partitioning analysis shows that Arg33 of CBFb overall disfavors RD–CBFb binding in the trimer with this effect signiﬁcantly increased in the RD–CBFb dimer by 40 kcal/mol [Arg33-RD(trimer) ¼ 11.0 ± 4.9 and Arg33- RD(dimer) ¼ 50.3 ± 3.3], further disfavoring the RD Glu116 to CBFb Arg33 interaction in the dimer. However, the question remains why is the RD Glu116 and CBFb Arg33 interaction not observed in the ternary complex crystal struc- ture (Figure 3C). Although speculative, it may be due to the presence of higher salt concentrations in the experimental study (200 mM NaCl) (13), which may partially screen the Arg33–DNA interactions. This screening combined with com- peting interactions involving other residues, such as the RD Tyr113 to CBFb Arg33 interaction that occurs in the crystal and RD–CBFb dimer MD structures, may lead to the subtle difference between the two systems. Supporting this possib- ility is the NMR observed decrease in the Glu116 order para- meter in the ternary complex (20), suggesting that in solution and at lower salt concentration the proposed salt bridge may be present. Other experimental results indicate a 5-fold increase in the dissociation constant upon formation of the ternary complex from the RD–DNA dimer (18), which may be related to Tyr113 properly orienting Arg33 for the actual binding event. Regardless, the RD Glu116 to CBFb Arg33 salt bridge along with the additional interactions leads to the more favor- able interaction energy between CBFb and RD in the ternary versus the binary complex (Table 3). Thus, it is predicted that Glu116 of RD has a signiﬁcant impact on the binding of CBFb to RD as well as on the cooperativity of RD binding to DNA and CBFb. Notably, binding of CBFb to the RD–DNA dimer has been shown via NMR to decrease the conformational variability of Glu116 (20), supporting the impact of RD inter- actions with its binding partners on this residue. The interaction energies for Met106, Ala107, Asn109, Asn112, Tyr113 and Ser114 (Table 6) are consistent with the experimental mutation data. Mutation of these residues decreases RD–CBFb binding in the experimental study (17,18), consistent with the favorable interaction energies of these residues with CBFb. However, mutation of Asn109 to alanine decreased the RD–CBFb binding by 60-fold while the calculated interaction energy is relatively small. This discrepancy may be due to mutation induced structural perturbations of the RD (i.e. partial unfolding), leading to the experimentally observed decrease in binding (18). Recently, it has been shown that polar residues at protein–protein inter- faces provide structural rigidity so as to maintain speciﬁcity and to reduce the entropic cost upon binding (47). Thus, Asn109 may play an important role in maintenance of the structural integrity of RD with a modest contribution to afﬁnity associated with direct interactions with CBFb. The N-terminal segment makes a signiﬁcant, favorable gain in interaction energy with CBFb upon going from the RD–CBFb binary to the ternary complex (Table 5). As shown in Table 6, the contribution was dominated by Asp66. However, experimental mutation of Asp66 to alanine only moderately affected the RD–CBFb binding strength (19). Visual inspection of the binary MD, ternary MD and crystal structures (Figure 4), along with energy partitioning analysis shows Asp66 to interact favorably, via its backbone carbonyl, with Lys11 of CBFb, with this interaction becoming a salt bridge involving the sidechains in the trimer. The difference between the RD–CBFb dimer MD and crystal structures and the trimer MD structure appears to be due to a competitive interaction between Asp66 with His163 in RD (Figure 4A and C, respectively). Consistent with the results discussed above, this suggests that the electrostatic ﬁeld imposed by the poly- anionic DNA in the ternary simulations disrupts the Asp66- His163 interaction seen in the dimer and helps in orienting Asp66 toward Lys11 (Figure 4C), with the presence of 200 mM NaCl in the experimental structure potentially damp- ing this interaction. Alternatively, the protonation state of His163 may inﬂuence the results. His163 was treated as neut- ral in the MD simulations; hypothetically, a different result might be obtained if His163 was treated as being protonated. Presumably this would create a stronger interaction between RD Asp66 and His163, such that this interaction would dominate in both the ternary and the binary MD simulations, with the Asp66 backbone carbonyl oxygen interacting with the Lys11 sidechain amino group in both cases. Such a scen- ario where the interaction of Asp66 with Lys11 involves the formers backbone atoms could explain why a signiﬁcant Figure 4. RD–CBFb interactions involving RD residues Asp66 and His163 and CBFb residue Lys11. 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.