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The structure of a furin-antibody complex explains non-competitive inhibition by steric exclusion of substrate conformers

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ABSTRACT

Proprotein Convertases (PCs) represent highly selective serine proteases that activate their substrates upon proteolytic cleavage. Their inhibition is a promising strategy for the treatment of cancer and infectious diseases. Inhibitory camelid antibodies were developed, targeting the prototypical PC furin. Kinetic analyses of them revealed an enigmatic non-competitive mechanism, affecting the inhibition of large proprotein-like but not small peptidic substrates. Here we present the crystal structures of furin in complex with the antibody Nb14 and of free Nb14 at resolutions of 2.0 Å and 2.3 Å, respectively. Nb14 binds at a site distant to the substrate binding pocket to the P-domain of furin. Interestingly, no major conformational changes were observed upon complex formation, neither for the protease nor for the antibody. Inhibition of furin by Nb14 is instead explained by steric exclusion of specific substrate conformers, explaining why Nb14 inhibits the processing of bulky protein substrates but not of small peptide substrates. This mode of action was further supported by modelling studies with the ternary factor X-furin-antibody complex and a mutation that disrupted the interaction interface between furin and the antibody. The observed binding mode of Nb14 suggests a novel approach for the development of highly specific antibody-based proprotein convertase inhibitors.

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Interaction of Nb14 with FurinWT or FurinT562R.The UV280 absorption is shown as a function of the elution volume of the GPC column. Fractions under the peaks (black bars) were analysed by SDS-PAGE (insets). The premixed GPC sample was loaded as control to the SDS-PAGE (S). (a) GPC run of Nb14 and FurinWT, marked in the inset as open and filled arrowheads, respectively. (b) GPC run of Nb14 and FurinT562R, marked in the inset as open and filled arrowheads, respectively.
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f2: Interaction of Nb14 with FurinWT or FurinT562R.The UV280 absorption is shown as a function of the elution volume of the GPC column. Fractions under the peaks (black bars) were analysed by SDS-PAGE (insets). The premixed GPC sample was loaded as control to the SDS-PAGE (S). (a) GPC run of Nb14 and FurinWT, marked in the inset as open and filled arrowheads, respectively. (b) GPC run of Nb14 and FurinT562R, marked in the inset as open and filled arrowheads, respectively.

Mentions: Next, we analysed the binding properties of FurinWT and FurinT562R in analytical gel permeation chromatography (GPC) experiments. If an equimolar mixture of FurinWT and Nb14 is subjected to gel permeation chromatography, the proteins form the expected complex and co-elute in a single peak (Fig. 2a). Pre-mixed Nb14 and mutant FurinT562R, however, elute as two separated peaks from the GPC-column (Fig. 2b). In conclusion, the mutation Thr562Arg at the interaction interface of furin disrupts complex formation with Nb14.


The structure of a furin-antibody complex explains non-competitive inhibition by steric exclusion of substrate conformers
Interaction of Nb14 with FurinWT or FurinT562R.The UV280 absorption is shown as a function of the elution volume of the GPC column. Fractions under the peaks (black bars) were analysed by SDS-PAGE (insets). The premixed GPC sample was loaded as control to the SDS-PAGE (S). (a) GPC run of Nb14 and FurinWT, marked in the inset as open and filled arrowheads, respectively. (b) GPC run of Nb14 and FurinT562R, marked in the inset as open and filled arrowheads, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC5037460&req=5

f2: Interaction of Nb14 with FurinWT or FurinT562R.The UV280 absorption is shown as a function of the elution volume of the GPC column. Fractions under the peaks (black bars) were analysed by SDS-PAGE (insets). The premixed GPC sample was loaded as control to the SDS-PAGE (S). (a) GPC run of Nb14 and FurinWT, marked in the inset as open and filled arrowheads, respectively. (b) GPC run of Nb14 and FurinT562R, marked in the inset as open and filled arrowheads, respectively.
Mentions: Next, we analysed the binding properties of FurinWT and FurinT562R in analytical gel permeation chromatography (GPC) experiments. If an equimolar mixture of FurinWT and Nb14 is subjected to gel permeation chromatography, the proteins form the expected complex and co-elute in a single peak (Fig. 2a). Pre-mixed Nb14 and mutant FurinT562R, however, elute as two separated peaks from the GPC-column (Fig. 2b). In conclusion, the mutation Thr562Arg at the interaction interface of furin disrupts complex formation with Nb14.

View Article: PubMed Central - PubMed

ABSTRACT

Proprotein Convertases (PCs) represent highly selective serine proteases that activate their substrates upon proteolytic cleavage. Their inhibition is a promising strategy for the treatment of cancer and infectious diseases. Inhibitory camelid antibodies were developed, targeting the prototypical PC furin. Kinetic analyses of them revealed an enigmatic non-competitive mechanism, affecting the inhibition of large proprotein-like but not small peptidic substrates. Here we present the crystal structures of furin in complex with the antibody Nb14 and of free Nb14 at resolutions of 2.0 Å and 2.3 Å, respectively. Nb14 binds at a site distant to the substrate binding pocket to the P-domain of furin. Interestingly, no major conformational changes were observed upon complex formation, neither for the protease nor for the antibody. Inhibition of furin by Nb14 is instead explained by steric exclusion of specific substrate conformers, explaining why Nb14 inhibits the processing of bulky protein substrates but not of small peptide substrates. This mode of action was further supported by modelling studies with the ternary factor X-furin-antibody complex and a mutation that disrupted the interaction interface between furin and the antibody. The observed binding mode of Nb14 suggests a novel approach for the development of highly specific antibody-based proprotein convertase inhibitors.

No MeSH data available.