LAUSR

The bacterial protease inhibitor domain, Streptomyces subtilisin inhibitor (SSI), is rarely found in fungi. Genome analysis of a fungal pathogen, Choanephora cucurbitarum KUS-F28377, revealed 11 SSI-like domains that are horizontally transferred and sequentially diverged during evolution. We investigated the molecular function of fungal SSI-like domains of C. cucurbitarum, designated as 'Choanepins'. Among the tested proteins, only choanepin9 showed inhibitory activity against subtilisin as the target protease, and accounting for 47% of the inhibitory activity of bacterial SSI. However, the binding affinity (Kd) of choanepin9 measured via microscale thermophoresis was 21 nM compared with 34 nM of bacterial SSI. The trend of binding and inhibitory activity suggests that the two inhibitors exhibit different inhibitory mechanism for subtilisin protease. Interestingly, choanepin9 was identified as a monomer in studies in vitro, whereas bacterial SSI is a homodimer. Based on the observations, we constructed a monomeric bacterial SSI protein with decreased binding affinity to abrogate the inhibitory activity. By altering the reactive sites of choanepin9 deduced from the P1 and P4 sites of bacterial SSIs, we re-established that these residues in choanepins are also crucial for modulating the inhibitory activity. These findings suggest that the fungal SSIs were evolved to target specific cognate proteases by altering the residues involved in inhibitory reactivity (reactive sites) and binding affinity (structural integrity). The function of fungal SSI proteins identified in this study not only provides a clue to fungal pathogenesis via protease inhibition as well as a template for the design of novel serine protease inhibitor.Importance Until recently, Streptomyces subtilisin inhibitor (SSI) was reported and characterized only in bacteria. We found these SSI-like domains in a plant pathogenic fungus, Choanephora cucurbitarum KUS-F28377, which contains 11 sequentially diverged SSI-like domains. None of these fungal SSI-like domains were functionally characterized before. The active form of fungal SSI-like protein is a monomer compared with the homo-dimeric bacterial SSI. We constructed a synthetic monomer of bacterial SSI to demonstrate the modulation of its activity based on structural integrity and not reactive sites. Our results suggest the duplication and divergence of SSI-like domains of C. cucurbitarum within the genome to inhibit various cognate proteases during evolution by modulating both binding and reactivity. The molecular functional characterization of fungal SSI-like domains will be useful in understanding its biological role and future biotechnological applications.