Conformational analyses of a partially‐folded bioactive prodomain of human furin

Abstract

The 81‐residue multifunctional prodomain of human furin adopts only a partially‐folded conformational state under near physiological conditions. By use of NMR spectroscopy, we demonstrate that the N‐terminal residues 1–46 of the prodomain in 50% trifluoroethanol (TFE) populates backbone conformations containing a short helix, a β‐strand and a helix‐loop‐helix super‐secondary structure with elements of tertiary interactions. 15N NMR relaxation measurements indicate that the helix‐loop‐helix region has similar motional characteristics in the fast picosecond to nanosecond timescales. On the other hand, the intervening segment (residues 47–65) is predominately unstructured with a long and highly flexible region surrounding the protease ‘activation loop’ followed by a partially helical segment in the C‐terminal end. Interestingly, the helix‐loop‐helix “fold” was found to be populated even when excised out of the full‐length prodomain, since a peptide fragment derived from residues Pro16‐Arg49 can also form the helix‐loop‐helix structure in aqueous solution in the absence of TFE. Structure analyses reveal that two helices orient in an antiparallel fashion directed by the sharing of hydrophobic residues involved in helix‐capping interactions. Very importantly, a positively‐charged Lys residue replacing His43 in the 16–49 fragment imparts stability to the super‐secondary structure at both acidic and neutral pH, while a hydrophobic residue Leu at position 43 appears to destabilize the helical conformation in the 31–44 region. As such, this study provides valuable insights into the structural properties of the furin prodomain in relation to its role in the folding of the furin zymogen and its inhibitory action toward furin. © 2007 Wiley Periodicals, Inc. Biopolymers 86: 329–344, 2007.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

NMR structure and localization of a large fragment of the SARS-CoV fusion protein: Implications in viral cell fusion

“…LFP was over-expressed in E. coli cells as a fusion protein containing 81 amino acid long human prodomain at the N-terminus [57][58][59]. A synthetic gene, with E. coli optimized codons, of the fusion protein was cloned (Shanghai ShineGene Molecular Biotech™) in a pET14b vector containing a six His-tag at the N-terminus for affinity purification of the fusion protein and a D-P sequence was introduced between the prodomain and LFP for formic acid mediated digestion of the fusion protein [57][58][59]. Note, in the synthetic gene of LFP, codon of residue Asp890 was changed to a codon of amino acid Ser to remove an internal D-P site in the LFP.…”

The Mechanism by Which a Propeptide-encoded pH Sensor Regulates Spatiotemporal Activation of Furin

“…This establishes a link between the increased thermodynamic stability of the H69L substitution and its ability to act as an inhibitor of MAT FUR , as indicated by the decrease in IC 50 . Taken together, the circular dichroism and fluorescence spectra, along with the analyses of thermodynamic stabilities, suggest that the nonprotonated mimic of the pH sensor subtly increases both secondary and tertiary structure, and enhances the overall thermodynamic stability and inhibitory function of H69L-PRO FUR (33) in the circular dichroism spectra suggests that glycerol induces the partially structured propeptide (34,35) to fold into a more stable state, and, thus, there are two distinct states in which the propeptide can exist, depending on its local environment (28). The second feature to note is the progressive stabilization of the secondary structure with increasing amounts of glycerol within isolated WT-PRO FUR and H69L-PRO FUR , measured using changes in ellipticity at 222 nm (Fig.…”

Selective and potent furin inhibitors protect cells from anthrax without significant toxicity

“…Furin is synthesized as a pre-proprotein which contains a signal peptide, a prodomain, a subtilisin-like catalytic domain, a middle P domain, a cysteine-rich region, a transmembrane anchor and a cytoplasmic tail. The N-terminal prodomain functions as a potent auto-inhibitor (Bhattacharjya et al, 2007, Fugere et al, 2002). To become proteolytically active, furin requires proteolytic removal of its inhibitory prodomain (Lazure, 2002, Thomas, 2002).…”