Molecular dynamics simulations and free energy calculations have been performed on the transcription factor Ndt80 either in complex with the native DNA sequence or with a mutant DNA with a switched central base pair, C5-G5′ to G5-C5′. This mutant has been shown to have a 100-fold decrease in binding affinity of Ndt80, and in this study we explain this both structurally and energetically. The major interactions between the protein and the DNA were maintained in the simulations, apart from around the mutation site. The crystal structure of the Ndt80-DNA complex revealed that R177 makes a base specific bidentate interaction with G5′ which is part of a conserved 5′-YpG-3′ step. In the simulation with the mutant DNA, the side chain of R177 changes conformation and makes three new stable hydrogen bonds to the DNA backbone. This in turn induces a conformational change in the DNA backbone of the T6′-G5′ step from the unusual BII state to the canonical BI state. The affinity difference for the protein–DNA complex with the native DNA compared with the mutant DNA is only about 3 kcal/mol. The free energy calculations of the base pair switch indicated a larger difference than what was found experimentally, about 7.7 kcal/mol, but this is explained in structural terms using the 10 ns simulations of the solvated complexes and the rearrangement of the R177 side chain. Proteins 2008. © 2008 Wiley-Liss, Inc.