Specific and Selective Inhibitors of Proprotein Convertases Engineered by Transferring Serpin B8 Reactive-Site and Exosite Determinants of Reactivity to the Serpin α1PDX

Abstract

The molecular determinants of substrate specificity and selectivity in the proprotein convertase (PC) family of proteases are poorly understood. Here we demonstrate that the natural serpin family inhibitor, serpin B8, is a specific and selective inhibitor of furin relative to the other PCs of the constitutive protein secretion pathway, PC4, PC5, PACE4, and PC7 (PC4-PC7, respectively), and identify reactive-site (P6-P5' residues) and exosite elements of the serpin that contribute to this specificity and selectivity through studies of chimeras of serpin B8 and α1PDX, an engineered serpin inhibitor of furin. Kinetic studies revealed that the specificity and selectivity of the serpin chimeras for inhibiting PCs were determined by P6-P5 and P3-P2 residue-dependent recognition of the P4Arg-X-X-P1Arg PC consensus sequence and exosite-dependent recognition of the reactive loop P2' residue of the chimeras by the PCs. Both productive and nonproductive binding of the chimeras to PC4-PC7 but not to furin contributed to a decreased specificity for inhibiting PC4-PC7 and an increased selectivity for inhibiting furin. Molecular dynamics simulations suggested that nonproductive binding of the chimeras to the PCs was correlated with a greater conformational variability of the catalytic sites of PC4-PC7 relative to that of furin. Our findings suggest a new approach for designing selective inhibitors of PCs using α1PDX as a scaffold, as evidenced by our ability to engineer highly specific and selective inhibitors of furin and PC4-PC7.

Figure 1.

Amino acid residue sequence at the RCL of α1-antitrypsin derivatives and serpin B8. The amino acid residue sequences from the RCL of α1-antitrypsin variants and of serpin B8 were aligned based on the location of the protease cleavage site between the residues P1 and P1’. A natural mutant of α1-antitrypsin, the Pittsburgh variant, has an arginine substitution at the P1 residue that changes the serpin specificity to inhibit trypsin-type proteases (20). The α1-antitrypsin Portland variant (α1PDX) was engineered with arginine residues at the P4 and P1 positions to inhibit furin (15, 16). The serpin B8–5S5A variant is a stabilized form of serpin B8 in which cysteine residues were mutated, five to serine and five to alanine, including the one at the P1’ position (10). The α1PDX-serpin B8 Ρ6-Ρ5’ chimera is now designated the Chicago 1 derivative (α1ORD) due to its high furin selectivity. The α1PDX+YE exosites variant, is now designated the Chicago 2 derivative (α1MDW) due to its high selectivity for PC4, PC5, PACE4 and PC7 over furin.

Figure 2.

Reaction of the α1PDX-serpin B8 P6-P5’ chimera with PACE4. (A) Shown are plots of PACE4 residual activity at increasing concentrations of the α1PDX-serpin B8 P6-P5’ chimera. Progressive reactions of 2 nM PACE4 activity with 10 μΜ of the fluorogenic substrate pyr-Arg-Thr-Lys-Arg-amido-methylcoumarin under protease activity assay conditions were run as described in the Materials and Methods section. (B) Shown is the fitted plot of the dependence of residual PACE4 activity on increasing concentrations of the serpin chimera that were corrected for the fluorogenic substrate concentration [S]. The plot was fitted to the quadratic tight binding equation by Morrison (21). Similar results were obtained with PC4, PC5 and PC7, and K_d values are listed in Table 4. (C) Shown is a plot of residual PACE4 activity for the inhibition of 50 nM PACE4 by 1.4 μΜ serpin chimera. The initial drop in activity reflects the fast equilibrium binding for the formation of the Michaelis complex that precedes slow covalent complex formation.

Figure 3.

Reaction of the α1PDX-serpin B8 P6-P1 chimera with PACE4. (A) Shown are plots of PACE4 residual activity at increasing concentrations of the α1PDX-serpin B8 P6-P1 chimera. Progressive reactions of 5 nM PACE4 were run as described in legend to figure 2. (B) Shown is the fitted plot of the dependence of residual PACE4 activity on increasing concentrations of the serpin chimera as described in legend to figure 2. Similar results were obtained with PC4, PC5 and PC7, and K_d values are listed in Table 4. (C) Shown is a plot of residual PACE4 activity for the time course of inhibition of 50 nM PACE4 by 1.0 μΜ of the chimera. The large initial drop in activity reflects the fast equilibrium binding for the formation of the Michaelis complex that precedes slow covalent complex formation.

Figure 4.

Reaction of the α1PDX-serpin B8 P6-P5 chimera with PACE4. (A) Shown are plots of PACE4 residual activity at increasing concentrations of the α1PDX-serpin B8 P6-P5 chimera. Progressive reactions of 3 nM PACE4 were run as described in legend to figure 2. (B) Shown is the fitted plot of the dependence of residual PACE4 activity on increasing concentrations of the serpin chimera as described in legend to figure 2. Similar results were obtained with PC4, PC5 and PC7, and K_d values are listed in Table 4. (C) Shown is a plot of residual PACE4 activity for the time course of inhibition of 2 nM PACE4 by 50 nM of the chimera. The large initial drop in activity reflects the fast equilibrium binding for the formation of the Michaelis complex that precedes slow covalent complex formation.

Figure 5.

Molecular dynamic simulation analysis of the structure of PCs. The configuration of amino acid residues in the catalytic domain of furin, PC4, PC5, PACE4 and PC7 was simulated employing furin crystal structures and molecular models of the catalytic domain of PCs 4–7 based on the x-ray crystal structure of the ligand-free and ligand-bound furin, as described in the Materials and Methods section. Two different starting structure conditions were employed, i) ligand-free form, which is associated with inactive configurations, and ii) ligand-bound form, which is associated with active configurations. Shown are molecular dynamic trajectories from the simulated systems projected on the first two principal components (nm). These projections are presented as heat maps where blue represents highly populated states. The maps derived from (i) are labeled as APO, and the ones from (ii) as LIG. The arrows pointing down correspond to the starting frame, which is the furin crystal structure. The arrows pointing upward correspond to the last frame of the analysis. (A) Comparative MDS analysis of the PC catalytic domain. PCA performed over 313 Cα atoms of amino acid residues. (B) Comparative MDS analysis of the PC catalytic site. PCA performed over 5 Cα atoms of catalytic site amino acid residues (Aspl53, Hisl94, Gly255, Asn295 and Ser368).