Tertiary structure of porcine pancreatic elastase in complex with a potent inhibitor determined by neutron crystallography

Tamada, Taro; Kinoshita, Takayoshi*; Ohara, Takashi   ; Kurihara, Kazuo; Imai, Keisuke*; Kuroki, Ryota; Tada, Toshiji*

Porcine pancreatic elastase (PPE) is a serine protease classified in the chymotrypsin family. We determined two crystal structures of PPE in complex with peptidic inhibitor FR130180, which mimics the tetrahedral transition intermediate. One is the structure determined using 1.65 resolution neutron diffraction in the combination with 1.20 resolution X-ray diffraction data obtained using the same crystal. The other is the sub-angstrom X-ray structure determined to 0.94 resolution. The structural features obtained from neutron diffraction and X-ray diffraction were compared to understand the detailed scheme of interaction between PPE and its inhibitor. From the observation of hydrogen atom located between the active site His57 and Asp102 by neutron and high resolution X-ray diffraction experiment, it is revealed that the interaction is not a low barrier hydrogen bond, but a short ionic hydrogen bond. Moreover, using neutron diffraction data we show that the hydroxyl group of inhibitor FR13080 bound within the "oxy-anion hole" exhibits an oxy-anion-like tetrahedral intermediate.