锌金属蛋白酶仿生亲和配基设计及相互作用机制研究

Zinc metalloprotease has important theoretical significance and application value, but at present there still lacks an effective biomimetic affinity purification protocol for them, which limits their high-value applications. Efficient biomimetic affinity ligand is the critical factor for building the affinity purification technology. Our previous study showed that LupI, the specific inhibitor of zinc metalloprotease, possesses many excellent properties such as reversible inhibition and high temperature tolerance. However, the affinity between zinc metalloprotease and LupI is too strong to be eluded in the purification process. Meanwhile, the interaction mechanisms between the enzyme and ligand are still not clear, which limits its further research. This study, based on the structural information of co-crystallization of typical zinc metalloprotease and LupI, along with the computer-aided molecular design technology, the molecular modification of LupI will be carried out to obtain the efficient biomimetic affinity ligands. Then, an efficient biomimetic purification protocol will be built for zinc metalloprotease purification. Meanwhile, the interaction mechanism between the enzyme and affinity ligands will be illustrated by combining computational biology with molecular biology. This study will not only lay the theoretical basis for the high value application of zinc metalloprotease, but also provide references for affinity ligand screening and designagtion of other proteases.

锌金属蛋白酶具有重要的研究意义和应用价值。目前，锌金属蛋白酶缺乏有效的仿生亲和纯化方法，阻碍了其高值化应用。高效的仿生亲和配基是决定仿生亲和纯化技术成败的关键。前期研究中，我们发现锌金属蛋白酶特异性抑制剂LupI具有可逆抑制、性质稳定等优良特性。但是，LupI作为仿生亲和配基与锌金属蛋白酶结合过于牢固、不宜洗脱。两者之间的相互作用机制尚不明确，限制了对其进一步研究。本课题拟基于典型锌金属蛋白酶与抑制剂的共晶体结构以及计算机辅助分子设计对LupI进行分子改造，定向筛选仿生亲和配基，建立锌金属蛋白酶家族的特异性仿生亲和纯化方法；利用计算生物学与分子生物学技术相结合的方法，阐明锌金属蛋白酶家族与仿生亲和配基特异性识别的结构基础以及可逆性结合与解离的分子机制，揭示酶与配基之间的相互作用关系，为锌金属蛋白酶家族高值化应用提供理论依据，也为其他蛋白酶家族仿生亲和配基的筛选和设计提供借鉴。

锌金属蛋白酶应用广泛，高纯度制剂具有良好的药物开发价值。目前，锌金属蛋白酶缺乏有效的仿生亲和纯化方法，阻碍了其高值化应用。高效的仿生亲和配基是决定仿生亲和纯化技术成败的关键。本课题基于锌金属蛋白酶 MP 与抑制剂 LupI 的共结晶结构及计算生物学分析对 LupI 的三个重要功能域N端trunk结构、α-helix结构、C端V4-V5 Loop区域进行分子动力学模拟与分子改造，明确三个区域在抑制剂LupI与典型锌金属蛋白酶的相互作用关系。通过突变体活性检测筛选获得锌金属蛋白酶仿生亲和配基LupIPAQV/GGGG。制备仿生亲和纯化材料，仿生亲和材料对锌金属蛋白酶MP的解离常数（Kd）为 24.5 µg/mL, 最大结合能力（Qmax）为 25.1 mg/g。建立锌金属蛋白酶的高效仿生亲和纯化方法，将其应用于海洋微生物来源锌金属蛋白酶的纯化。在课题经费支持下发表多篇高水平学术论文，本研究不仅为微生物来源锌金属蛋白酶的高值化应用提供理论依据，也为其他家族蛋白酶仿生亲和配基的筛选和设计提供借鉴。