The K48-linked polyubiquitin chains are important signals for proteasomal degradation and other biological processes. Their recognition of ubiquitin binding partners such as the UBA2 (ubiquitin associated domain 2) domain of hHR23a is via the canonical hydrophobic patch formed by L8, I44, and V70. In near physiological pH (pH 6.8), the K48-linked diubiquitin predominantly adopts the closed conformation in which the binding sites for ubiquitin-binding partners are buried in the inter-domain interface, and therefore are not available for binding. The K48-linked diubiquitin also can adopt an open conformation at acidic pH. However, the mechanism of the transition between the open and closed states is poorly understood. This study is aimed at elucidating the driving force for the exchange between the open and closed conformations of K48-linked diubiquitin. Using different mutations of H68 in diubiquitin and NMR methods, I found that the protonation state of the histidine side chain is crucial for controlling the equilibrium between open and closed conformations. I also found that H68 is essential for maintaining the integrity of the inter-domain interface. I concluded that there are at least four interactions involved in controlling the transitions between open and closed states. These are point-to-point repulsion (strongest), point-to-bulk repulsion (medium), bulk-to-bulk repulsion (weakest), and hydrophobic interaction. Based on these results, I also proposed a pre-open state model for K48-linked diubiquitin which assumes that the closed conformation of Ub2 opens by twisting instead of directly pulling two domains away from each other.