The bacterial protease inhibitor domains known as Streptomyces subtilisin inhibitors (SSI) are 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 “choanepins.” Among the proteins tested, only choanepin9 showed inhibitory activity against subtilisin as the target protease, accounting for 47% of the inhibitory activity of bacterial SSI. However, the binding affinity (expressed as the dissociation constant [K_d]) of choanepin9 measured via microscale thermophoresis was 21 nM, whereas that for bacterial SSI is 34 nM. The trend of binding and inhibitory activity suggests that the two inhibitors exhibit different inhibitory mechanisms for subtilisin protease. Interestingly, choanepin9 was identified as a monomer in studies in vitro, whereas bacterial SSI is a homodimer. Based on these observations, we constructed a monomeric bacterial SSI protein with decreased binding affinity to abrogate its inhibitory activity. By altering the reactive sites of choanepin9 deduced from the P1 and P4 sites of bacterial SSI, we reestablished that these residues in choanepins are also crucial for modulating inhibitory activity. These findings suggest that the fungal SSI 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 provides not only a clue to fungal pathogenesis via protease inhibition but also a template for the design of novel serine protease inhibitors.

中文翻译：

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从植物病原真菌葫芦属（Choanephora cucurbitarum）中分离出新型的单体真菌枯草杆菌蛋白酶抑制剂并进行分子表征。

在真菌中很少发现被称为链霉菌枯草杆菌蛋白酶抑制剂（SSI）的细菌蛋白酶抑制剂域。真菌病原体葫芦丝孢菌（Choanephora cucurbitarum） KUS-F28377的基因组分析揭示了11个SSI样结构域，它们在进化过程中被水平转移并相继发散。我们调查了命名为“ choanepins”的葫芦丝菌的真菌SSI样结构域的分子功能。在测试的蛋白质中，只有choanepin9表现出对枯草杆菌蛋白酶作为目标蛋白酶的抑制活性，占细菌SSI抑制活性的47％。但是，结合亲和力（表示为解离常数[ K _d]）通过微尺度热泳测量的choanepin9为21 nM，而细菌SSI为34 nM。结合和抑制活性的趋势表明，两种抑制剂对枯草杆菌蛋白酶的抑制机制不同。有趣的是，choanepin9在体外研究中被鉴定为单体，而细菌SSI是同型二聚体。基于这些观察，我们构建了具有降低的结合亲和力的单体细菌SSI蛋白，以消除其抑制活性。通过改变从细菌SSI的P1和P4位点推导的choanepin9的反应位点，我们重新建立了choanepins中的这些残基对于调节抑制活性也至关重要。这些发现表明，真菌SSI通过改变参与抑制反应性（反应位点）和结合亲和力（结构完整性）的残基进化为靶向特定的同源蛋白酶。在这项研究中鉴定的真菌SSI蛋白的功能不仅为通过蛋白酶抑制作用的真菌发病机理提供了线索，而且为设计新型丝氨酸蛋白酶抑制剂提供了模板。