How to deal with weak interactions in noncovalent complexes analyzed by electrospray mass spectrometry: Cyclopeptidic inhibitors of the nuclear receptor coactivator 1-STAT6
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Mass spectrometry, and especially electrospray ionization, is now an efficient tool to study noncovalent interactions between proteins and inhibitors. It is used here to study the interaction of some weak inhibitors with the NCoA-1/STAT6 protein with KD values in the µM range. High signal intensities corresponding to some nonspecific electrostatic interactions between NCoA-1 and the oppositely charged inhibitors were observed by nanoelectrospray mass spectrometry, due to the use of high ligand concentrations. Diverse strategies have already been developed to deal with nonspecific interactions, such as controlled dissociation in the gas phase, mathematical modeling, or the use of a reference protein to monitor the appearance of nonspecific complexes. We demonstrate here that this last methodology, validated only in the case of neutral sugar-protein interactions, i.e., where dipole-dipole interactions are crucial, is not relevant in the case of strong electrostatic interactions. Thus, we developed a novel strategy based on half-maximal inhibitory concentration (IC50) measurements in a competitive assay with readout by nanoelectrospray mass spectrometry. IC50 values determined by MS were finally converted into dissociation constants that showed very good agreement with values determined in the liquid phase using a fluorescence polarization assay.
- 3.Ganem, B.; Li, Y. T.; Henion, J. D. Observation of Noncovalent Enzyme-Substrate and Enzyme-Product Complexes by Ion-Spray Mass Spectrometry Observation of Noncovalent Enzyme-Substrate and Enzyme-Product Complexes by Ion-Spray Mass Spectrometry. J. Am. Chem. Soc. 1991, 113, 7818–7819.CrossRefGoogle Scholar
- 22.Seitz, M.; Maillard, L.; Obrecht, D.; Robinson, J. A. Molecular Characterization of the NCoA-1-STAT6 Interaction. Chem. Biochem. 2008, 9, 1318–1322.Google Scholar
- 23.Nikolovska-Coleska, Z.; Wang, R.; Fang, X.; Pan, H.; Tomita, Y.; Li, P.; Roller, P. P.; Krajewski, K.; Saito, N. G.; Stuckey, J. A.; Wang, S. Development and Optimization of a Binding Assay for the XIAP BIR3 Domain Using Fluorescence Polarization. Anal. Biochem. 2004, 332, 261–273.CrossRefGoogle Scholar
- 25.Razeto, A.; Ramakrishnan, V.; Litterst, C. M.; Giller, K.; Griesinger, C.; Carlomagno, T.; Lakomek, N.; Heimburg, T.; Lodrini, M.; Pfitzner, E.; Becker, S. Structure of the NCoA-1/SRC-1 PAS-B Domain Bound to the LXXLL Motif of the STAT6 Transactivation Domain. J. Mol. Biol. 2004, 336, 319–329.CrossRefGoogle Scholar
- 27.Mathur, S.; Badertscher, M.; Scott, M.; Zenobi, R. Critical Evaluation of Mass Spectrometric Measurement of Dissociation Constants: Accuracy and Cross-Validation Against Surface Plasmon Resonance and Circular Dichroism for the Calmodulin-Melittin system. Phys. Chem., Chem. Phys. 2007, 9, 6187–6198.CrossRefGoogle Scholar