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5 results on '"Tharun, Inga M"'

1. Estrogen receptor folding modulates cSrc kinase SH2 interaction via a helical binding mode

Author

Nieto, Lidia, Tharun, Inga M, Balk, Mark, Wienk, Hans, Boelens, Rolf, Ottmann, Christian, Milroy, Lech-Gustav, Brunsveld, Luc, Sub NMR Spectroscopy, NMR Spectroscopy, Chemical Biology, Sub NMR Spectroscopy, and NMR Spectroscopy

Subjects
SH2-Binding Motif, Protein Folding, Magnetic Resonance Spectroscopy, Molecular Sequence Data, Estrogen receptor, Plasma protein binding, Biology, Molecular Dynamics Simulation, Biochemistry, Models, Biological, Fluorescence, src Homology Domains, 03 medical and health sciences, 0302 clinical medicine, Taverne, Humans, Amino Acid Sequence, Binding site, Peptide sequence, 030304 developmental biology, 0303 health sciences, Binding Sites, General Medicine, src-Family Kinases, Receptors, Estrogen, 030220 oncology & carcinogenesis, Biophysics, Molecular Medicine, Phosphorylation, Protein folding, Female, Peptides, Proto-oncogene tyrosine-protein kinase Src, Protein Binding
Abstract

The estrogen receptors (ERs) feature, next to their transcriptional role, important nongenomic signaling actions, with emerging clinical relevance. The Src Homology 2 (SH2) domain mediated interaction between cSrc kinase and ER plays a key role in this; however the molecular determinants of this interaction have not been elucidated. Here, we used phosphorylated ER peptide and semisynthetic protein constructs in a combined biochemical and structural study to, for the first time, provide a quantitative and structural characterization of the cSrc SH2-ER interaction. Fluorescence polarization experiments delineated the SH2 binding motif in the ER sequence. Chemical shift perturbation analysis by nuclear magnetic resonance (NMR) together with molecular dynamics (MD) simulations allowed us to put forward a 3D model of the ER-SH2 interaction. The structural basis of this protein-protein interaction has been compared with that of the high affinity SH2 binding sequence GpYEEI. The ER features a different binding mode from that of the two-pronged plug two-hole socket model in the so-called specificity determining region. This alternative binding mode is modulated via the folding of ER helix 12, a structural element directly C-terminal of the key phosphorylated tyrosine. The present findings provide novel molecular entries for understanding nongenomic ER signaling and targeting the corresponding disease states.

Published
2015

2. Estrogen Receptor Folding Modulates cSrc Kinase SH2 Interaction via a Helical Binding Mode

Author

Sub NMR Spectroscopy, NMR Spectroscopy, Nieto, Lidia, Tharun, Inga M, Balk, Mark, Wienk, Hans, Boelens, Rolf, Ottmann, Christian, Milroy, Lech-Gustav, Brunsveld, Luc, Sub NMR Spectroscopy, NMR Spectroscopy, Nieto, Lidia, Tharun, Inga M, Balk, Mark, Wienk, Hans, Boelens, Rolf, Ottmann, Christian, Milroy, Lech-Gustav, and Brunsveld, Luc

Published
2015

4. Subtype-Specific Modulation of Estrogen Receptor–Coactivator Interaction by Phosphorylation

Author

Tharun, Inga M., Nieto, Lidia, Haase, Christian, Scheepstra, Marcel, Balk, Mark, Möcklinghoff, Sabine, Adriaens, Wencke, Dames, Sonja A., and Brunsveld, Luc

Abstract

The estrogen receptor (ER) is the number one target for the treatment of endocrine responsive breast cancer and remains a highly attractive target for new drug development. Despite considerable efforts to understand the role of ER post-translational modifications (PTMs), the complexity of these modifications and their impact, at the molecular level, are poorly understood. Using a chemical biology approach, fundamentally rooted in an efficient protein semisynthesis of tyrosine phosphorylated ER constructs, the complex role of the ER tyrosine phosphorylation is addressed here for the first time on a molecular level. The semisynthetic approach allows for the site-specific introduction of PTMs as well as biophysical probes. A combination of biophysical techniques, including NMR, with molecular dynamics studies reveals the role of the phosphorylation of the clinically relevant tyrosine 537 (Y537) in ERα and the analogous tyrosine (Y488) in ERβ. Phosphorylation has important effects on the dynamics of the ER Helix 12, which is centrally involved in receptor activity regulation, and on its interplay with ligand and cofactor binding, but with differential regulatory effects of the analogous PTMs on the two ER subtypes. Combined, the results bring forward a novel molecular model of a phosphorylation-induced subtype specific ER modulatory mechanism, alternative to the widely accepted ligand-induced activation mechanism.

Published
2015
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5. Estrogen Receptor Folding Modulates cSrc Kinase SH2 Interaction via a Helical Binding Mode.

Author

Nieto L, Tharun IM, Balk M, Wienk H, Boelens R, Ottmann C, Milroy LG, and Brunsveld L

Subjects
Amino Acid Sequence, Binding Sites, Female, Fluorescence, Humans, Magnetic Resonance Spectroscopy, Molecular Dynamics Simulation, Molecular Sequence Data, Peptides chemistry, Peptides genetics, Protein Binding, Protein Folding, Receptors, Estrogen chemistry, src Homology Domains, src-Family Kinases chemistry, Models, Biological, Receptors, Estrogen metabolism, src-Family Kinases metabolism
Abstract

The estrogen receptors (ERs) feature, next to their transcriptional role, important nongenomic signaling actions, with emerging clinical relevance. The Src Homology 2 (SH2) domain mediated interaction between cSrc kinase and ER plays a key role in this; however the molecular determinants of this interaction have not been elucidated. Here, we used phosphorylated ER peptide and semisynthetic protein constructs in a combined biochemical and structural study to, for the first time, provide a quantitative and structural characterization of the cSrc SH2-ER interaction. Fluorescence polarization experiments delineated the SH2 binding motif in the ER sequence. Chemical shift perturbation analysis by nuclear magnetic resonance (NMR) together with molecular dynamics (MD) simulations allowed us to put forward a 3D model of the ER-SH2 interaction. The structural basis of this protein-protein interaction has been compared with that of the high affinity SH2 binding sequence GpYEEI. The ER features a different binding mode from that of the "two-pronged plug two-hole socket" model in the so-called specificity determining region. This alternative binding mode is modulated via the folding of ER helix 12, a structural element directly C-terminal of the key phosphorylated tyrosine. The present findings provide novel molecular entries for understanding nongenomic ER signaling and targeting the corresponding disease states.

Published
2015
Full Text
View/download PDF

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