pH-Induced conformational transitions of a molten-globule-like state of the inhibitory prodomain of furin: Implications for zymogen activation

Bhattacharjya, S Xu, P Xiang, H Chretien, M Seidah, NG Ni, F

S. Bhattacharjya et al., pH-Induced conformational transitions of a molten-globule-like state of the inhibitory prodomain of furin: Implications for zymogen activation, PROTEIN SCI, 10(5), 2001, pp. 934-942

58

INGLESE

Article

Biochemistry & Biophysics

PROTEIN SCIENCE

0961-8368 → ACNP

10

5

2001

934 - 942

ISI

0961-8368(200105)10:5<934:PCTOAM>2.0.ZU;2-L

The endoprotease furin, which belongs to the family of mammalian proprotein convertase (PC), is synthesized as a zymogen with an N-terminal, 81-residu e inhibitory prodomain. It has been shown that the proenzyme form of furin undergoes a multistep 'autocatalytic' removal of the prodomain at the C-ter minal side of the two consensus sites, R-78-T-K-R(81)similar to and R-44-G- V-T-K-R(49)similar to. The furin-mediated cleavage at R-44-G-V-T-K-R(49)sim ilar to, in particular, is significantly accelerated in an 'acidic' environ ment. Here, we show that under neutral pH conditions, the inhibitory prodom ain 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 specifi c packing interactions among amino acid side chains. Measurements of the hy drodynamic radii and compaction factors indicate that this partially folded state is significantly more compact than a random chain. The conformationa l stability of the prodomain appears to be pH sensitive, in that the prodom ain undergoes an unfolding transition towards acidic conditions. Our NMR an alyses establish that the acid-induced unfolding is mainly experienced by t he 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 d erived from the entire pH-sensitive C-terminal region (residues R-44-R-81) does not exhibit any exchange-induced line broadening and adopts flexible c onformations. We propose that at neutral pH, the cleavage site R-44-G-V-T-K -R(49)similar to is buried within the protein core that is formed in part b y 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.