Biophysical studies of the interaction between calmodulin and the R²⁸⁷-T³¹¹ region of human estrogen receptor ¦Á reveals an atypical binding process

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• 作者：Ludovic Carlier^a ; ^{ludovic.carlier@upmc.fr} ; Cillian Byrne^a ; ^b ; Emeric Miclet^a ; Sandrine Bourgoin-Voillard^c ; ^e ; ¹ ; Magali Nicaise^d ; Jean-Claude Tabet^c ; Michel Desmadril^d ; Guy Leclercq^b ; ^e ; Olivier Lequin^a ; Yves Jacquot^a ; ^b ; ^{yves.jacquot@upmc.fr}
• 关键词：Estrogen receptor ; Calmodulin ; Fluorescence spectroscopy ; NMR ; ITC ; Far-UV CD
• 刊名：Biochemical and Biophysical Research Communications
• 出版年：2012
• 出版时间：9 March, 2012
• 年：2012
• 卷：419
• 期：2
• 页码：356-361
• 全文大小：767 K

文摘

The transcriptional activity of human estrogen receptor ER¦Á is modulated by a number of coregulatory proteins among which calmodulin (CaM). Segment 295-311 in the hinge region of ER¦Á has previously been proposed to be the CaM binding site. In this work, we investigate the molecular mechanism of the interaction of CaM with peptides derived from the hinge region of ER¦Á, using a biophysical approach combining isothermal titration calorimetry, fluorescence, CD and NMR. The ER¦Á17p peptide, corresponding to the previously identified 295-311 region of ER¦Á, recruits mainly the C-terminal domain of Ca₄CaM, as shown by NMR spectroscopy. In contrast, a longer peptide, ER¦Á25p, extended on the N-terminal side (residues 287-311) interacts with both N- and C-terminal domains of Ca₄CaM. These results lead to a new delineation of the CaM binding site, encompassing residues 287-294. In particular, fluorescence spectroscopy reveals that the conserved W²⁹² residue is engaged within hydrophobic pockets on Ca₄CaM. ITC results show that ER¦Á25p binds Ca₄CaM with an atypical 2:1 stoichiometry and a dissociation constant in the micromolar range. Based on the NMR titration of Ca₄CaM by ER¦Á25p showing a biphasic behavior for several residues, we suggest that concerted conformational changes of CaM domains may be required to accommodate the binding of a second peptide. CD spectra indicate that ER¦Á25p partially folds into an ¦Á-helix upon binding to Ca₄CaM. Hence, ER¦Á25p is a new CaM-binding ligand that could be appropriate for the synthesis of derivatives able to control ER-dependent transcription, particularly in the context of hormone-dependent breast tumors.