Structure of the unliganded form of the proprotein convertase furin suggests activation by a substrate-induced mechanism

Abstract

Proprotein convertases (PCs) are highly specific proteases required for the proteolytic modification of many secreted proteins. An unbalanced activity of these enzymes is connected to pathologies like cancer, atherosclerosis, hypercholesterolaemia, and infectious diseases. Novel protein crystallographic structures of the prototypical PC family member furin in different functional states were determined to 1.8-2.0 Å. These, together with biochemical data and modeling by molecular dynamics calculations, suggest essential elements underlying its unusually high substrate specificity. Furin shows a complex activation mechanism and exists in at least four defined states: (i) the "off state," incompatible with substrate binding as seen in the unliganded enzyme; (ii) the active "on state" seen in inhibitor-bound furin; and the respective (iii) calcium-free and (iv) calcium-bound forms. The transition from the off to the on state is triggered by ligand binding at subsites S1 to S4 and appears to underlie the preferential recognition of the four-residue sequence motif of furin. The molecular dynamics simulations of the four structural states reflect the experimental observations in general and provide approximations of the respective stabilities. Ligation by calcium at the PC-specific binding site II influences the active-site geometry and determines the rotamer state of the oxyanion holeforming Asn295, and thus adds a second level of the activity modulation of furin. The described crystal forms and the observations of different defined functional states may foster the development of new tools and strategies for pharmacological intervention targeting furin.serine-protease | activation | specificity | conformational transition

Optimization of Substrate‐Analogue Furin Inhibitors

“…Notably, this is the only example among all of our substrate‐analogue inhibitors from previous and recent work in which decreased affinity was determined for a P5 4‐GMe‐Phac inhibitor relative to the unsubstituted Phac analogue. This can be only explained by differences in the overall binding mode of at least one of these two compounds, which must deviate from the experimentally determined crystal structures found for several 4‐Amba inhibitors . Surprisingly, exchanging the P1 m ‐substituted aniline structure in inhibitor 8 by the p ‐isomer also leads to a 3000‐fold higher IC 50 value of compound 6 , although both residues possess nearly identical p K a values.…”

“…Recently, we have developed highly potent substrate‐analogue furin inhibitors containing a C‐terminal 4‐amidinobenzylamide (4‐Amba) as shown in Figure . The binding mode of several of these inhibitors in complex with furin was characterized by crystal structure determination . The 4‐Amba group binds in a deep, well‐defined S1 pocket and is involved in numerous contacts with furin, thereby contributing to the picomolar potency of these analogues.…”

Elongated and Shortened Peptidomimetic Inhibitors of the Proprotein Convertase Furin

“…Notably, a reverse binding orientation was postulated for related substrate‐analogue inhibitors in a previous work . In contrast, a perfect fit was obtained for compounds 4 – 7 with an N‐terminal phenylacetyl residue or for derivatives 16 and 17 exhibiting an N‐terminal N α ‐guanidino group suggesting a single binding mode as observed in crystal structures with these amidinobenzylamide‐derived inhibitors . The latter modification contributed to an improved affinity and probably stabilizes the peptides against degradation by aminopeptidases, although this was not tested so far.…”

“…We have previously developed various substrate‐analogue furin inhibitors based on the lead structure phenylacetyl‐Arg‐Val‐Arg‐4‐amidinobenzylamide (inhibitor 1 , Figure ) . Further optimization by incorporation of basic P5 groups in combination with tert ‐leucine (Tle) at the P3 position has provided furin inhibitors with strongly improved potency, such as compounds 2 and 3 (Figure ) . The analysis of their crystal structures in complex with human furin revealed numerous polar interactions contributing to their low picomolar affinity .…”

Proprotein convertase inhibition: Paralyzing the cell’s master switches

“…Non-peptide inhibitors may circumvent these drawbacks, while still exerting a strong inhibitory effect on PCs. The elucidation of the crystal structure of furin has facilitated the modeling of these non-peptide inhibitors [54, 55]. Guanidinilated derivatives of 2-dideoxystreptamine, modeled using docking experiments and the crystal structure of furin as template, proved to be efficient (K i in the nanomolar range) inhibitors of furin in vitro [56].…”