Institute of Molecular Genetics of National Research Centre "Kurchatov Institute", Moscow, Russia; National Research Centre "Kurchatov Institute", Moscow, Russia; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia.
Institute of Molecular Genetics of National Research Centre "Kurchatov Institute", Moscow, Russia.
J Biol Chem. 2023 Apr;299(4):104585. doi: 10.1016/j.jbc.2023.104585. Epub 2023 Mar 6.
Emfourin (M4in) is a protein metalloprotease inhibitor recently discovered in the bacterium Serratia proteamaculans and the prototype of a new family of protein protease inhibitors with an unknown mechanism of action. Protealysin-like proteases (PLPs) of the thermolysin family are natural targets of emfourin-like inhibitors widespread in bacteria and known in archaea. The available data indicate the involvement of PLPs in interbacterial interaction as well as bacterial interaction with other organisms and likely in pathogenesis. Arguably, emfourin-like inhibitors participate in the regulation of bacterial pathogenesis by controlling PLP activity. Here, we determined the 3D structure of M4in using solution NMR spectroscopy. The obtained structure demonstrated no significant similarity to known protein structures. This structure was used to model the M4in-enzyme complex and the complex model was verified by small-angle X-ray scattering. Based on the model analysis, we propose a molecular mechanism for the inhibitor, which was confirmed by site-directed mutagenesis. We show that two spatially close flexible loop regions are critical for the inhibitor-protease interaction. One region includes aspartic acid forming a coordination bond with catalytic Zn of the enzyme and the second region carries hydrophobic amino acids interacting with protease substrate binding sites. Such an active site structure corresponds to the noncanonical inhibition mechanism. This is the first demonstration of such a mechanism for protein inhibitors of thermolysin family metalloproteases, which puts forward M4in as a new basis for the development of antibacterial agents relying on selective inhibition of prominent factors of bacterial pathogenesis belonging to this family.
Emfourin（M4in）是一种最近在细菌 Serratia proteamaculans 中发现的蛋白金属蛋白酶抑制剂，也是一个具有未知作用机制的新型蛋白蛋白酶抑制剂家族的原型。热稳定蛋白酶（thermolysin）家族的蛋白酶样蛋白酶（PLPs）是 emfourin 样抑制剂的天然靶标，这些抑制剂在细菌中广泛存在，在古菌中也有发现。现有数据表明，PLPs 参与了细菌之间以及细菌与其他生物体之间的相互作用，并可能与发病机制有关。可以说，emfourin 样抑制剂通过控制 PLP 活性参与了细菌发病机制的调节。在这里，我们使用溶液 NMR 光谱法测定了 M4in 的 3D 结构。得到的结构与已知的蛋白质结构没有明显的相似性。该结构被用于模拟 M4in-酶复合物，并用小角 X 射线散射对复合物模型进行了验证。基于模型分析，我们提出了抑制剂的分子机制，并通过定点突变得到了证实。我们表明，两个空间上接近的柔性环区对于抑制剂-蛋白酶相互作用至关重要。一个区域包含与酶的催化 Zn 形成配位键的天冬氨酸，另一个区域携带与蛋白酶底物结合位点相互作用的疏水性氨基酸。这种活性位点结构对应于非典型的抑制机制。这是首次证明热稳定蛋白酶家族金属蛋白酶的蛋白抑制剂具有这种机制，这为开发依赖于选择性抑制属于该家族的细菌发病机制中突出因素的抗菌剂提供了新的依据。
