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

作者：Lin Ying; Fan Hong; Frederiksen Mathias; Zhao Kehao; Jiang Lei; Wang Zhaofu; Zhou Shaolian; Guo Weihui; Gao Jun; Li Shu; Harrington Edmund; Meier Peter; Scheufler Clemens; Xu Yao Chang; Atadja Peter; Lu Chris; Li En; Gu X Justin^*

来源：Analytical Biochemistry, 2012, 423(1): 171-177.

DOI：10.1016/j.ab.2012.01.019

• HMT
• Uncompetitive inhibitor
• G9a
• BIX-01294
• SAM
• Sinefungin
• Conformational change
• SET domain
• CJP702

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.

• 出版日期2012-4-1