Identification of a pH sensor in the furin propeptide that regulates enzyme activation

The folding and activation of furin occur through two pH- and compartment-specific autoproteolytic steps. In the endoplasmic reticulum (ER), profurin folds under the guidance of its prodomain and undergoes an autoproteolytic excision at the consensus furin site Arg-Thr-Lys-Arg ¹⁰⁷ ↓ generating an enzymatically masked furin-propeptide complex competent for transport to late secretory compartments. In the mildly acidic environment of the trans-Golgi network/endosomal system, the bound propeptide is cleaved at the internal site ⁶⁹HRGVTKR⁷⁵ ↓, unmasking active furin capable of cleaving substrates in trans. Here, by using cellular, biochemical, and modeling studies, we demonstrate that the conserved His⁶⁹ is a pH sensor that regulates the compartment-specific cleavages of the propeptide. In the ER, unprotonated His⁶⁹ stabilizes a solvent-accessible hydrophobic pocket necessary for autoproteolytic excision at Arg¹⁰⁷. Profurin molecules unable to form the hydrophobic pocket, and hence, the furin-propeptide complex, are restricted to the ER by a PACS-2- and COPI-dependent mechanism. Once exposed to the acidic pH of the late secretory pathway, protonated His69 disrupts the hydrophobic pocket, resulting in exposure and cleavage of the internal cleavage site at Arg⁷⁵ to unmask the enzyme. Together, our data explain the pH-regulated activation of furin and how this His-dependent regulatory mechanism is amodel for other proteins.