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

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.


Related in: MedlinePlus

Structure of the furin:RVKR-CMK:Nb14 complex.Human furin is shown as surface representation. The catalytic domain and the P-domain are coloured in yellow and brown, respectively. The dec-RVKR-CMK inhibitor is shown as a ball and stick model with magenta carbons. Nb14 is displayed as a cartoon representation (blue) with the central disulphide bond highlighted as spheres. The inset shows a part of the interaction interface of furin and Nb14. The surface of the P-domain is shown as a partially transparent surface. The peptide stretch Gly561-Thr562-Leu563 is shown as a stick model. The 2Fo−Fc electron density composite omit map (green mesh) is contoured at 1.0 σ.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Structure of the furin:RVKR-CMK:Nb14 complex.Human furin is shown as surface representation. The catalytic domain and the P-domain are coloured in yellow and brown, respectively. The dec-RVKR-CMK inhibitor is shown as a ball and stick model with magenta carbons. Nb14 is displayed as a cartoon representation (blue) with the central disulphide bond highlighted as spheres. The inset shows a part of the interaction interface of furin and Nb14. The surface of the P-domain is shown as a partially transparent surface. The peptide stretch Gly561-Thr562-Leu563 is shown as a stick model. The 2Fo−Fc electron density composite omit map (green mesh) is contoured at 1.0 σ.

Mentions: We have crystallised the ternary complex of human furin with decanoyl-RVKR-chloromethylketone (RVKR-CMK) and the inhibitory camelid antibody Nb14. Even optimised crystals grew as needle clusters. Using a whole needle-cluster, initial diffraction data were obtained to a limiting resolution of 2.9 Å at the synchrotron beamline BL14.1 of the Helmholtz-Zentrum Berlin (HZB) (Fig. S1a, Table S1). These data verified the presence of well diffracting mono-crystalline fragments within the mounted crystal bundles. The structure was solved by molecular replacement revealing the presence of two copies of the furin:RVKR-CMK:Nb14 complex in the asymmetric unit. To obtain better defined crystallographic data, rather small crystal fragments with approx. sizes of only 5 × 5 × 150 μm3 were separated from these needle clusters and were used for single crystal diffraction data collection at the micro-focus beamline P14 at the European Molecular Biology Laboratory (EMBL) in Hamburg (Fig. S1b, Table S1). The latter dataset was subsequently used for refinement of the furin:RVKR-CMK:Nb14 complex at 2.0 Å resolution at high quality with final R/Rfree factors of 16.3/19.7% (Fig. 1, Table S1).


The structure of a furin-antibody complex explains non-competitive inhibition by steric exclusion of substrate conformers
Structure of the furin:RVKR-CMK:Nb14 complex.Human furin is shown as surface representation. The catalytic domain and the P-domain are coloured in yellow and brown, respectively. The dec-RVKR-CMK inhibitor is shown as a ball and stick model with magenta carbons. Nb14 is displayed as a cartoon representation (blue) with the central disulphide bond highlighted as spheres. The inset shows a part of the interaction interface of furin and Nb14. The surface of the P-domain is shown as a partially transparent surface. The peptide stretch Gly561-Thr562-Leu563 is shown as a stick model. The 2Fo−Fc electron density composite omit map (green mesh) is contoured at 1.0 σ.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Structure of the furin:RVKR-CMK:Nb14 complex.Human furin is shown as surface representation. The catalytic domain and the P-domain are coloured in yellow and brown, respectively. The dec-RVKR-CMK inhibitor is shown as a ball and stick model with magenta carbons. Nb14 is displayed as a cartoon representation (blue) with the central disulphide bond highlighted as spheres. The inset shows a part of the interaction interface of furin and Nb14. The surface of the P-domain is shown as a partially transparent surface. The peptide stretch Gly561-Thr562-Leu563 is shown as a stick model. The 2Fo−Fc electron density composite omit map (green mesh) is contoured at 1.0 σ.
Mentions: We have crystallised the ternary complex of human furin with decanoyl-RVKR-chloromethylketone (RVKR-CMK) and the inhibitory camelid antibody Nb14. Even optimised crystals grew as needle clusters. Using a whole needle-cluster, initial diffraction data were obtained to a limiting resolution of 2.9 Å at the synchrotron beamline BL14.1 of the Helmholtz-Zentrum Berlin (HZB) (Fig. S1a, Table S1). These data verified the presence of well diffracting mono-crystalline fragments within the mounted crystal bundles. The structure was solved by molecular replacement revealing the presence of two copies of the furin:RVKR-CMK:Nb14 complex in the asymmetric unit. To obtain better defined crystallographic data, rather small crystal fragments with approx. sizes of only 5 × 5 × 150 μm3 were separated from these needle clusters and were used for single crystal diffraction data collection at the micro-focus beamline P14 at the European Molecular Biology Laboratory (EMBL) in Hamburg (Fig. S1b, Table S1). The latter dataset was subsequently used for refinement of the furin:RVKR-CMK:Nb14 complex at 2.0 Å resolution at high quality with final R/Rfree factors of 16.3/19.7% (Fig. 1, Table S1).

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.


Related in: MedlinePlus