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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.

No MeSH data available.


Proteolysis of factor XS195A by FurinWT or FurinT562R and its inhibition by Nb14.Factor XS195A was subjected to limited proteolysis by FurinWT or FurinT562R and analysed by SDS-PAGE. The bands of uncleaved factor XS195A (FX), cleaved factor XS195A (FX(cleaved)) and Nb14 are marked with arrowheads.
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f3: Proteolysis of factor XS195A by FurinWT or FurinT562R and its inhibition by Nb14.Factor XS195A was subjected to limited proteolysis by FurinWT or FurinT562R and analysed by SDS-PAGE. The bands of uncleaved factor XS195A (FX), cleaved factor XS195A (FX(cleaved)) and Nb14 are marked with arrowheads.

Mentions: Inhibition of furin by Nb14 can conveniently be monitored in limited proteolysis experiments with the furin substrate coagulation factor X31. Maturation of it requires cleavage by furin in between the catalytic domain and the EGF-2 domain. For cleavage assays we used the inactive Ser195Ala mutant (factor XS195A), showing largely reduced self-degradation compared to the wild type protease32. After incubation of both proteins a band shift from 42 to 38 kDa is observed (Fig. 3). FurinWT and FurinT562R essentially showed the same activity in the factor XS195A cleavage assay (Fig. 3). Apparently the introduced mutation affects neither the turnover of small peptidic substrates (see above) nor the proteolysis of protein substrates like factor XS195A. Cleavage of factor XS195A by FurinWT is largely reduced in the presence of 1 μM Nb14 indicating inhibition of furin by the antibody (Fig. 3). FurinT562R, however, performs very similarly with and without Nb14 in the reaction mixture (Fig. 3).


The structure of a furin-antibody complex explains non-competitive inhibition by steric exclusion of substrate conformers
Proteolysis of factor XS195A by FurinWT or FurinT562R and its inhibition by Nb14.Factor XS195A was subjected to limited proteolysis by FurinWT or FurinT562R and analysed by SDS-PAGE. The bands of uncleaved factor XS195A (FX), cleaved factor XS195A (FX(cleaved)) and Nb14 are marked with arrowheads.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037460&req=5

f3: Proteolysis of factor XS195A by FurinWT or FurinT562R and its inhibition by Nb14.Factor XS195A was subjected to limited proteolysis by FurinWT or FurinT562R and analysed by SDS-PAGE. The bands of uncleaved factor XS195A (FX), cleaved factor XS195A (FX(cleaved)) and Nb14 are marked with arrowheads.
Mentions: Inhibition of furin by Nb14 can conveniently be monitored in limited proteolysis experiments with the furin substrate coagulation factor X31. Maturation of it requires cleavage by furin in between the catalytic domain and the EGF-2 domain. For cleavage assays we used the inactive Ser195Ala mutant (factor XS195A), showing largely reduced self-degradation compared to the wild type protease32. After incubation of both proteins a band shift from 42 to 38 kDa is observed (Fig. 3). FurinWT and FurinT562R essentially showed the same activity in the factor XS195A cleavage assay (Fig. 3). Apparently the introduced mutation affects neither the turnover of small peptidic substrates (see above) nor the proteolysis of protein substrates like factor XS195A. Cleavage of factor XS195A by FurinWT is largely reduced in the presence of 1 μM Nb14 indicating inhibition of furin by the antibody (Fig. 3). FurinT562R, however, performs very similarly with and without Nb14 in the reaction mixture (Fig. 3).

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.