Detecting S-adenosyl-l-methionine-induced conformational change of a histone methyltransferase using a homogeneous time-resolved fluorescence-based binding assay

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• 作者：Ying Lin^a ; ¹ ; Hong Fan^a ; ¹ ; Mathias Frederiksen^b ; Kehao Zhao^a ; Lei Jiang^a ; Zhaofu Wang^a ; Shaolian Zhou^a ; Weihui Guo^a ; Jun Gao^a ; Shu Li^c ; Edmund Harrington^c ; Peter Meier^b ; Clemens Scheufler^b ; Yao-Chang Xu^a ; Peter Atadja^a ; Chris Lu^a ; En Li^a ; X. Justin Gu^a ; ^{justin.gu@novartis.com}
• 关键词：HMT ; Uncompetitive inhibitor ; G9a ; BIX-01294 ; SAM ; Sinefungin ; Conformational change ; SET domain ; CJP702
• 刊名：Analytical Biochemistry
• 出版年：2012
• 期刊代码：93_00032697
• 类别：imm
• 出版时间：1 April, 2012
• 卷：423
• 期：1
• 页码：171-177
• 文件大小：915 K

摘要

A homogeneous time-resolved fluorescence (HTRF)-based binding assay has been established to measure the binding of the histone methyltransferase (HMT) G9a to its inhibitor CJP702 (a biotin analog of the known peptide-pocket inhibitor, BIX-01294). This assay was used to characterize G9a inhibitors. As expected, the peptide-pocket inhibitors decreased the G9a-CJP702 binding signal in a concentration-dependent manner. In contrast, the S-adenosyl-l-methionine (SAM)-pocket compounds, SAM and sinefungin, significantly increased the G9a-CJP702 binding signal, whereas S-adenosyl-l-homocysteine (SAH) showed minimal effect. Enzyme kinetic studies showed that CJP702 is an uncompetitive inhibitor (vs. SAM) that has a strong preference for the E:SAM form of the enzyme. Other data presented suggest that the SAM/sinefungin-induced increase in the HTRF signal is secondary to an increased E:SAM or E:sinefungin concentration. Thus, the G9a-CJP702 binding assay not only can be used to characterize the peptide-pocket inhibitors but also can detect the subtle conformational differences induced by the binding of different SAM-pocket compounds. To our knowledge, this is the first demonstration of using an uncompetitive inhibitor as a probe to monitor the conformational change induced by compound binding with an HTRF assay.