Multiple domains of endopin 2A for serpin cross-class inhibition of papain

The serpin endopin 2A inhibits the cysteine protease papain in cross-class inhibition. This study demonstrates the novel finding that both the non-RSL NH2-domain and the RSL domain with P1–P1′ residues participate in endopin 2A inhibition. Production of a chimeric mutant of endopin 2A with replacement of its NH2-domain with that of endopin 1 resulted in less effective inhibition of papain, indicated by its lower kass association rate constant compared to wild-type endopin 2A. This chimeric mutant formed complexes with papain, but at lower levels compared to that with wild-type endopin 2A. Papain degradation of a portion of the chimeric mutant suggested a role for the NH2-domain in regulating relative amounts of endopin 2A that enter the substrate pathway compared to the serpin inhibitory pathway. Furthermore, site-directed mutagenesis demonstrated that the RSL domain with intact P1–P1′ residues was necessary for inhibition. These findings indicate that the NH2-domain and the RSL region both participate in endopin 2A inhibition of papain.