Abstract
Using a combination of NMR and fluorescence measurements, we have investigated the structure and dynamics of the complexes formed between calcium-loaded calmodulin (CaM) and the potent breast cancer inhibitor idoxifene, a derivative of tamoxifen. High-affinity binding ( nM) saturates with a complex. The complex is an ensemble where each idoxifene molecule is predominantly in the vicinity of one of the two hydrophobic patches of CaM but, in contrast with the lower-affinity antagonists TFP, J-8, and W-7, does not substantially occupy the hydrophobic pocket. At least four idoxifene orientations per domain of CaM are necessary to satisfy the intermolecular nuclear Overhauser effect (NOE) restraints, and this requires that the idoxifene molecules switch rapidly between positions. The CaM molecule is predominantly in the form where the N and C-terminal domains are in close proximity, allowing for the idoxifene molecules to contact both domains simultaneously. Hence, the complex illustrates how high-affinity binding occurs without the loss of extensive positional dynamics.
Highlights
Calmodulin (CaM) is an important intracellular calcium receptor found in all eukaryotic cells
The anomalous behaviour of D64 and N137 may result from a sensitivity of these residues to the occupation of the other globular domain of CaM by idoxifene
The CaM molecule is predominantly in the form where the N- and C-terminal domains are in close proximity, meaning that the idoxifene molecules are able to contact both domains simultaneously
Summary
Calmodulin (CaM) is an important intracellular calcium receptor found in all eukaryotic cells. The four helices in each of the two globular domains undergo a large conformational change, where the domains become less compact and a hydrophobic pocket is opened (Finn et al, 1995; Kuboniwa et al, 1995; Zhang et al, 1995) These hydrophobic pockets play a central role in the binding of various CaM targets (Meador et al, 1992; Ikura et al, 1992; Craven et al, 1996; Osawa et al, 1998; Harmat et al, 2000; Kovesi et al, 2008).
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