05 Feb 2021
05 Feb 2021
High Affinity Tamoxifen Analogues Retain Extensive Positional Disorder when Bound to Calmodulin
- 1Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, UK
- 2Department of Biological Sciences, School of Science, Birkbeck University of London, London WC1E 7HX, UK
- 3Medical Research Council Prion Unit, University College of London Institute of Neurology, Queen Square, London WCN1 3BG, UK
- 4Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK
- 5Manchester Institute of Biotechnology, University of Manchester, 131 Princess St, Manchester, M1 7DN, UK
- 1Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, UK
- 2Department of Biological Sciences, School of Science, Birkbeck University of London, London WC1E 7HX, UK
- 3Medical Research Council Prion Unit, University College of London Institute of Neurology, Queen Square, London WCN1 3BG, UK
- 4Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK
- 5Manchester Institute of Biotechnology, University of Manchester, 131 Princess St, Manchester, M1 7DN, UK
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 (Kd ~ 300 nM) saturates with a 2:1 idoxifene:CaM 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 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 2:1 idoxifene:CaM complex illustrates how high affinity binding occurs without the loss of extensive positional dynamics.
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Lilia Milanesi et al.
Status: open (until 17 Mar 2021)
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RC1: 'Comment on mr-2021-7', Walter Chazin, 23 Feb 2021
reply
This manuscript presents an investigation of the interaction of calmodulin with the small molecule Tamoxifen using solution NMR. Interestingly, Tamoxifen was found to bind with high affinity without occupying specific hydrophobic pockets. This was clearly evident from the inability to satisfy inter-molecular NOEs by a single structure of the complex. The experimental approach is sound and the experiments are well designed, including several well thought out controls. This includes an important titration carried out to control for chemical shift perturbations that arise from CD3OD. The data are properly interpreted. The manuscript is well written and clear. The manuscript is suitable for publication requiring only a few typographical/grammar adjustments.
- Line 184 – “were allowed”
- Line 266 – Remove “from:
- Line 395 – “have been”
- Line 420 – “was”
This review was performed primarily by postdoctoral fellow Randika (Randy) Perera, Ph.D.
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AC1: 'Reply on RC1', Jonathan Waltho, 23 Feb 2021
reply
Thank you both for reviewing our manuscript. We will make the changes that you noted.
Lilia Milanesi et al.
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Lilia Milanesi et al.
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