A noncanonical mechanism of carboxypeptidase inhibition revealed by the crystal structure of the Tri-Kunitz SmCI in complex with human CPA4.

The crystal structure of SmCI (Sabellastarte magnifica carboxypeptidase inhibitor) has been determined in complex with human carboxypeptidase A4 (hCPA4). SmCI is composed by three BPTI/Kunitz domains, each one displaying high structural homology and functionality with serine protease inhibitors. Moreover, SmCI possesses a distinctive capability to inhibit metallo-carboxypeptidases, constituting a bifunctional metallocarboxy- and serine protease inhibitor. The structure of the 1:1 complex of SmCI with hCPA4 reveals a noncanonical mechanism of carboxypeptidase inhibition, which surprisingly occurs mainly via the N-terminal segment, which penetrates into the active site groove of the enzyme. Mutagenesis and biochemical analysis confirm the major role of the N-terminal segment in the inhibition of carboxypeptidases. SmCI represents a tri-Kunitz serine protease inhibitor adapted to inhibit metallo-carboxypeptidases and discloses an unusual mechanism of inhibition by the N-terminal segment, emulating the "classical" C-terminal substrate-like inhibition.