Simonov Alexandr N, Holien Jessica K, Yeung Joyee Chun In, Nguyen Ann D, Corbin C Jo, Zheng Jie, Kuznetsov Vladimir L, Auchus Richard J, Conley Alan J, Bond Alan M, Parker Michael W, Rodgers Raymond J, Martin Lisandra L
School of Chemistry, Monash University, Clayton, Victoria, Australia.
ACRF Rational Drug Discovery Centre, St. Vincent's Institute of Medical Research, Fitzroy, Victoria, Australia.
PLoS One. 2015 Nov 20;10(11):e0141252. doi: 10.1371/journal.pone.0141252. eCollection 2015.
Cytochrome P450c17 (P450 17A1, CYP17A1) is a critical enzyme in the synthesis of androgens and is now a target enzyme for the treatment of prostate cancer. Cytochrome P450c17 can exhibit either one or two physiological enzymatic activities differentially regulated by cytochrome b5. How this is achieved remains unknown. Here, comprehensive in silico, in vivo and in vitro analyses were undertaken. Fluorescence Resonance Energy Transfer analysis showed close interactions within living cells between cytochrome P450c17 and cytochrome b5. In silico modeling identified the sites of interaction and confirmed that E48 and E49 residues in cytochrome b5 are essential for activity. Quartz crystal microbalance studies identified specific protein-protein interactions in a lipid membrane. Voltammetric analysis revealed that the wild type cytochrome b5, but not a mutated, E48G/E49G cyt b5, altered the kinetics of electron transfer between the electrode and the P450c17. We conclude that cytochrome b5 can influence the electronic conductivity of cytochrome P450c17 via allosteric, protein-protein interactions.
细胞色素P450c17（P450 17A1，CYP17A1）是雄激素合成中的一种关键酶，目前是前列腺癌治疗的靶标酶。细胞色素P450c17可表现出受细胞色素b5差异调节的一种或两种生理酶活性。其实现方式尚不清楚。在此，我们进行了全面的计算机模拟、体内和体外分析。荧光共振能量转移分析表明，在活细胞中细胞色素P450c17与细胞色素b5之间存在紧密相互作用。计算机模拟建模确定了相互作用位点，并证实细胞色素b5中的E48和E49残基对活性至关重要。石英晶体微天平研究确定了脂质膜中的特异性蛋白质-蛋白质相互作用。伏安分析表明，野生型细胞色素b5而非突变型E48G/E49G细胞色素b5改变了电极与P450c17之间的电子转移动力学。我们得出结论，细胞色素b5可通过变构、蛋白质-蛋白质相互作用影响细胞色素P450c17的电子传导性。
