Alkaline proteinase inhibitor of Pseudomonas aeruginosa - Interaction of native and N-terminally truncated inhibitor proteins with Pseudomonas metalloproteinases

The apr locus of Pseudomonas aeruginosa encodes alkaline proteinase (APR), a member of the metzincin metalloendopeptidase superfamily, and an 11.4-kDa alkaline proteinase inhibitor (APRin), We describe here the expression in Escherichia coli and characterization of full-length and N-terminally trunc ated APRin proteins. Fluorescence and circular dichroism spectra indicated that the recombinant proteins were folded into native-like structures. Anal ytical ultracentrifugation showed that APRin was monomeric and formed a 1:1 complex with APR. Binding of wild-type APRin to APR occurred with associat ion (k(on)) and dissociation (k(off)) rate constants of 0.29 +/- 0.06 x 10( 6) M-1 s(-1) and 1.15 +/- 0.08 x 10(-6) s(-1) to give an equilibrium dissoc iation constant (K-D) of similar to 4 x 10(-12) M (25 degrees C, pH 7.0, io nic strength 2.4 M). The association rate decreased by similar to 2-fold in 20% glycerol and increased by similar to 3-fold in 0.1 M NaCl. The glycero l effect suggests a diffusion-limited reaction, and the small salt effect i ndicates that electrostatic interactions contribute little to binding. Dele tion of residues 1-10, 1-6, or 6-10 abolished inhibition, and deletion of r esidues 1-2, 1-3, 1-4, and 1-5 resulted in a progressively decreased affini ty of APRin for APR (K-D = 0.12 mu M for the Delta(1-5) mutant). Substituti on of APRin residues 6-10 with a (Gly)(5) or (Pro)(5) linker restored inhib itory activity of the Delta(6-10) mutant but with a 100- and 50-fold reduct ion in K-D. Log k(on) for the full-length and truncated inhibitors correlat ed with the solvent-accessible surface area of their N-terminal regions, su ggesting that increased interactions and/or desolvation of these residues i n the transition state for binding contribute to the enhanced association r ate. Treatment of APRin with pseudolysin, also secreted by P. aeruginosa, r esulted in removal of residues 1-5. APRin was neither an inhibitor nor a su bstrate of other metzincins, including collagenase or gelatinases A or B.