title: Biophysical studies of the interaction between calmodulin and the R²⁸⁷-T³¹¹ region of human estrogen receptor α reveals an atypical binding process.
abstract: The transcriptional activity of human estrogen receptor ERα is modulated by a number of coregulatory proteins among which calmodulin (CaM). Segment 295-311 in the hinge region of ERα 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 ERα, using a biophysical approach combining isothermal titration calorimetry, fluorescence, CD and NMR. The ERα17p peptide, corresponding to the previously identified 295-311 region of ERα, recruits mainly the C-terminal domain of Ca(4)CaM, as shown by NMR spectroscopy. In contrast, a longer peptide, ERα25p, extended on the N-terminal side (residues 287-311) interacts with both N- and C-terminal domains of Ca(4)CaM. These results lead to a new delineation of the CaM binding site, encompassing residues 287-294. In particular, fluorescence spectroscopy reveals that the conserved W(292) residue is engaged within hydrophobic pockets on Ca(4)CaM. ITC results show that ERα25p binds Ca(4)CaM with an atypical 2:1 stoichiometry and a dissociation constant in the micromolar range. Based on the NMR titration of Ca(4)CaM by ERα25p 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 ERα25p partially folds into an α-helix upon binding to Ca(4)CaM. Hence, ERα25p 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.
authors.affiliation: UPMC Univ Paris 06, UMR 7203 CNRS-UPMC-ENS, 4 Place Jussieu, 75005 Paris, France. ludovic.carlier@upmc.fr
authors.forename: Ludovic
authors.initials: L
authors.lastname: Carlier
authors.forename: Cillian
authors.initials: C
authors.lastname: Byrne
authors.forename: Emeric
authors.initials: E
authors.lastname: Miclet
authors.forename: Sandrine
authors.initials: S
authors.lastname: Bourgoin-Voillard
authors.forename: Magali
authors.initials: M
authors.lastname: Nicaise
authors.forename: Jean-Claude
authors.initials: JC
authors.lastname: Tabet
authors.forename: Michel
authors.initials: M
authors.lastname: Desmadril
authors.forename: Guy
authors.initials: G
authors.lastname: Leclercq
authors.forename: Olivier
authors.initials: O
authors.lastname: Lequin
authors.forename: Yves
authors.initials: Y
authors.lastname: Jacquot
mesh_terms: D055592:Biophysical Phenomena; D002147:Calmodulin / Q000737:chemistry*; D047628:Estrogen Receptor alpha / Q000737:chemistry*; D006801:Humans; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding; D025941:Protein Interaction Mapping
chemical_list: D002147:Calmodulin; D047628:Estrogen Receptor alpha; D010446:Peptide Fragments