IE/DR MHC class II molecules have an extensive H-bonding network under the bound peptide. In IEk, two α chain acidic amino acids in the core of this network were mutated to amides. At low pH, the mutant molecule exchanged peptide much more rapidly than the wild-type. The crystal structure of the mutant IEk revealed the loss of a single buried water molecule and a reorganization of the predicted H-bonding network. We suggest that these mutations enhance the transition of MHC class II to an open conformation at low pH allowing the bound peptide to escape. In wild-type IEk, the need to protonate these amino acids also may be a bottleneck in the return to a closed conformation after peptide binding.