The transcriptional activity of human estrogen receptor ER alpha is modulated by a number of coregulatory proteins among which calmodulin (CaM). Segment 295-311 in the hinge region of ER alpha 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 alpha, using a biophysical approach combining isothermal titration calorimetry, fluorescence, CD and NMR. The ER alpha 17p peptide, corresponding to the previously identified 295-311 region of ER alpha, recruits mainly the C-terminal domain of Ca4CaM, as shown by NMR spectroscopy. In contrast, a longer peptide, ER alpha 25p, 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 W-292 residue is engaged within hydrophobic pockets on Ca4CaM. ITC results show that ER alpha 25p 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 alpha 25p 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 alpha 25p partially folds into an alpha-helix upon binding to Ca4CaM. Hence, ER alpha 25p 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. (C) 2012 Elsevier Inc. All rights reserved.