Evaluation of the calmodulin-SOX9 interaction by "magnetic fishing" coupled to mass spectrometry.

Meghan J McFadden, Todd Hryciw, Arthur Brown, Murray S Junop, John D Brennan

Index: ChemBioChem. 15(16) , 2411-9, (2014)

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Abstract

Disruption of calmodulin (CaM)-based protein interactions has been touted as a potential means for modulating several disease pathways. Among these is SOX9, which is a DNA binding protein that is involved in chrondrocyte differentiation and regulation of the hormones that control sexual development. In this work, we employed a "magnetic fishing"/mass spectrometry assay in conjunction with intrinsic fluorescence to examine the interaction of CaM with the CaM-binding domain of SOX9 (SOX-CAL), and to assess the modulation of this interaction by known anti-CaM compounds. Our data show that there is a high affinity interaction between CaM and SOX-CAL (27±9 nM), and that SOX-CAL bound to the same location as the well-known CaM antagonist melittin; unexpectedly, we also found that addition of CaM-binding small molecules initially produced increased SOX-CAL binding, indicative of binding to both the well-known high-affinity CaM binding site and a second, lower-affinity binding site. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.