Skaggs School of Pharmacy and Pharmaceutical Sciences, Center for Discovery and Innovation in Parasitic Diseases, University of California San Diego, La Jolla, California (V.S., B.S., L.H.-A., A.D., L.M.P.) and Departamento de Física, Instituto de Biociências, Letras e Ciências Exatas, Universidade Estadual Paulista Julio de Mesquita Filho, São José do Rio Preto, São Paulo, Brazil (L.H.-A.).
Skaggs School of Pharmacy and Pharmaceutical Sciences, Center for Discovery and Innovation in Parasitic Diseases, University of California San Diego, La Jolla, California (V.S., B.S., L.H.-A., A.D., L.M.P.) and Departamento de Física, Instituto de Biociências, Letras e Ciências Exatas, Universidade Estadual Paulista Julio de Mesquita Filho, São José do Rio Preto, São Paulo, Brazil (L.H.-A.)
Mol Pharmacol. 2020 Dec;98(6):770-780. doi: 10.1124/molpharm.120.000092. Epub 2020 Oct 2.
Cytochromes P450 (P450, CYP) metabolize a wide variety of endogenous and exogenous lipophilic molecules, including most drugs. Sterol 14-demethylase (CYP51) is a target for antifungal drugs known as conazoles. Using X-ray crystallography, we have discovered a domain-swap homodimerization mode in CYP51 from a human pathogen, CYP51 (AcCYP51). Recombinant AcCYP51 with a truncated transmembrane helix was purified as a heterogeneous mixture corresponding to the dimer and monomer units. Spectral analyses of these two populations have shown that the CO-bound ferrous form of the dimeric protein absorbed at 448 nm (catalytically competent form), whereas the monomeric form absorbed at 420 nm (catalytically incompetent form). AcCYP51 dimerized head-to-head via N-termini swapping, resulting in formation of a nonplanar protein-protein interface exceeding 2000 Å with a total solvation energy gain of -35.4 kcal/mol. In the dimer, the protomers faced each other through the F and G -helices, thus blocking the substrate access channel. In the presence of the drugs clotrimazole and isavuconazole, the AcCYP51 drug complexes crystallized as monomers. Although clotrimazole-bound AcCYP51 adopted a typical CYP monomer structure, isavuconazole-bound AcCYP51 failed to refold 74 N-terminal residues. The failure of AcCYP51 to fully refold upon inhibitor binding in vivo would cause an irreversible loss of a structurally aberrant enzyme through proteolytic degradation. This assumption explains the superior potency of isavuconazole against The dimerization mode observed in this work is compatible with membrane association and may be relevant to other members of the CYP family of biologic, medical, and pharmacological importance. SIGNIFICANCE STATEMENT: We investigated the mechanism of action of antifungal drugs in the human pathogen . We discovered that the enzyme target [ sterol 14-demethylase (AcCYP51)] formed a dimer via an N-termini swap, whereas drug-bound AcCYP51 was monomeric. In the AcCYP51-isavuconazole complex, the protein target failed to refold 74 N-terminal residues, suggesting a fundamentally different mechanism of AcCYP51 inactivation than only blocking the active site. Proteolytic degradation of a structurally aberrant enzyme would explain the superior potency of isavuconazole against .
细胞色素 P450（P450，CYP）代谢广泛的内源性和外源性亲脂性分子，包括大多数药物。甾醇 14α-脱甲基酶（CYP51）是一种抗真菌药物的靶标，这些药物被称为唑类。利用 X 射线晶体学，我们在人类病原体 CYP51（AcCYP51）中发现了一种结构域交换同源二聚化模式。具有截断跨膜螺旋的重组 AcCYP51 被纯化作为二聚体和单体单元的异质混合物。对这两种群体的光谱分析表明，CO 结合的二价亚铁形式的二聚体蛋白在 448nm 处吸收（催化有效形式），而单体形式在 420nm 处吸收（催化无效形式）。AcCYP51 通过 N 端交换头对头二聚化，形成超过 2000Å 的非平面蛋白-蛋白界面，总溶剂化能增益为-35.4kcal/mol。在二聚体中，前体通过 F 和 G-螺旋彼此相对，从而阻断了底物进入通道。在药物克霉唑和伊曲康唑存在下，AcCYP51 药物复合物结晶为单体。尽管克霉唑结合的 AcCYP51 采用典型的 CYP 单体结构，但伊曲康唑结合的 AcCYP51 未能折叠 74 个 N 端残基。在体内结合抑制剂时，AcCYP51 不能完全折叠会导致结构异常的酶通过蛋白水解降解不可逆丢失。这种假设解释了伊曲康唑对人类病原体的优势。在这项工作中观察到的二聚化模式与膜结合兼容，并且可能与具有生物学、医学和药理学重要性的 CYP 家族的其他成员相关。意义声明：我们研究了抗真菌药物在人类病原体中的作用机制。我们发现，酶靶标[甾醇 14α-脱甲基酶（AcCYP51）]通过 N 端交换形成二聚体，而药物结合的 AcCYP51 是单体。在 AcCYP51-伊曲康唑复合物中，蛋白质靶标未能折叠 74 个 N 端残基，这表明 AcCYP51 失活的机制与仅阻断活性位点根本不同。结构异常的酶的蛋白水解降解可以解释伊曲康唑对人类病原体的优势。
