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pH‐induced conformational transitions of a molten–globule–like state of the inhibitory prodomain of furin: Implications for zymogen activation
Author(s) -
Bhattacharjya Surajit,
Xu Ping,
Xiang Hui,
Chrétien Michel,
Seidah Nabil G.,
Ni Feng
Publication year - 2001
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.41301
Subject(s) - furin , chemistry , zymogen , cleavage (geology) , stereochemistry , proprotein convertase , amino acid , residue (chemistry) , crystallography , enzyme , biochemistry , biology , lipoprotein , paleontology , ldl receptor , cholesterol , fracture (geology)
The endoprotease furin, which belongs to the family of mammalian proprotein convertase (PC), is synthesized as a zymogen with an N‐terminal, 81‐residue inhibitory prodomain. It has been shown that the proenzyme form of furin undergoes a multistep ‘autocatalytic’ removal of the prodomain at the C‐terminal side of the two consensus sites, R 78 ‐T‐K‐R 81 ∼ and R 44 ‐G‐V‐T‐K‐R 49 ∼. The furin‐mediated cleavage at R 44 ‐G‐V‐T‐K‐R 49 ∼, in particular, is significantly accelerated in an ‘acidic’ environment. Here, we show that under neutral pH conditions, the inhibitory prodomain of furin is partially folded and undergoes conformational exchanges as indicated by extensive broadening of the NMR spectra. Presence of many ring‐current shifted methyl resonances suggests that the partially folded state of the prodomain may still possess a ‘semirigid’ protein core with specific packing interactions among amino acid side chains. Measurements of the hydrodynamic radii and compaction factors indicate that this partially folded state is significantly more compact than a random chain. The conformational stability of the prodomain appears to be pH sensitive, in that the prodomain undergoes an unfolding transition towards acidic conditions. Our NMR analyses establish that the acid‐induced unfolding is mainly experienced by the residues from the C‐terminal half of the prodomain (residues R 44 –R 81 ) that contains the two furin cleavage sites. A 38‐residue peptide fragment derived from the entire pH‐sensitive C‐terminal region (residues R 44 –R 81 ) does not exhibit any exchange‐induced line broadening and adopts flexible conformations. We propose that at neutral pH, the cleavage site R 44 ‐G‐V‐T‐K‐R 49 ∼ is buried within the protein core that is formed in part by residues from the N‐terminal region, and that the cleavage site becomes exposed under acidic conditions, leading to a facile cleavage by the furin enzyme.