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

The transcriptional activity of human estrogen receptor ERa is modulated by a number of coregulatory proteins among which calmodulin (CaM). Segment 295–311 in the hinge region of ERa 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 ERa, using a biophysical approach combining isothermal titration calorimetry, fluorescence, CD and NMR. The ERa17p peptide, corresponding to the previously identified 295–311 region of ERa, recruits mainly the C-terminal domain of Ca4CaM, as shown by NMR spectroscopy. In contrast, a longer peptide, ERa25p, 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 ERa25p binds Ca4CaM with an atypical 2:1 stoichiometry and a dissociation constant in the micromolar range. Based on the NMR titration of Ca4CaM by ERa25p 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 ERa25p partially folds into an a-helix upon binding to Ca4CaM. Hence, ERa25p 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.