Investigating the mechanism of the pH sensor domain of PC1 by NMR spectroscopy

PC1 is a protease belonging to the subtilisin-like family of proprotein convertases and is involved in the regulation of hormones, i.e. glucagon and insulin. Freshly expressed PC1 consists of an N-terminal prodomain and the catalytical domain and is inactive. The prodomain helps in the proper folding of the catalytic domain and keeps the catalytic domain inactive by a tight interaction. The activation occurs at slightly acidic pH, which typically occurs in the late Golgi. Then the PC1 prodomain is cleaved off and the protease is activated. The aim of the study is to investigate the role of histidines during the activation of the enzyme. 3D triple resonance experiments with 13C/15N-labeled protein samples were performed to obtain the backbone and the side chain NMR assignments. pH titration (pH range 7.4-6.0) with long range 1H-15N correlations were performed to analyze the protonation state of the histidines. The secondary structure prediction and the tertiary structure calculations were obtained at pH 7.4 using TALOS+ and CYANA. At neutral pH three of the four histidines are neutral (H72, H75, H85) one is protonated (H67). The pH titration revealed that the three neutral histidines, which are very close in the 3D structure, are protonated in the order: H75 - H85 - H72 and at pH 6.3 and below the prodomain starts to unfold. We conclude that the histidines act as a pH sensor of the protein, a cluster of three histidines get protonated at low pH, electrostatic repulsion leads to unfolding. Thus PC1 becomes sensible to pH and the cell environment, and it allows the activation of PC1 only in specific regions.