Studying the Conformation of a Receptor Tyrosine Kinase in Solution by Inhibitor-Based Spin Labeling

Angewandte Chemie - International Edition

5 years ago

Studying the Conformation of a Receptor Tyrosine Kinase in Solution by Inhibitor-Based Spin Labeling

Michael Famulok, Gregor Hagelueken, Anton Schmitz, Dongsheng M. Yin, Jeffrey S. Hannam, Olav Schiemann

The synthesis of a spin label based on PD168393, a covalent inhibitor of a major anticancer drug target, the epidermal growth factor receptor (EGFR), is reported. The label facilitates the analysis of the EGFR structure in solution by pulsed electron paramagnetic resonance (EPR) spectroscopy. For various EGFR constructs, including near-full-length EGFR, we determined defined distance distributions between the two spin labels bound to the ATP binding sites of the EGFR dimer. The distances are in excellent agreement with an asymmetric dimer of the EGFR. Based on crystal structures, this dimer had previously been proposed to reflect the active conformation of the receptor but structural data demonstrating its existence in solution have been lacking. More generally, our study provides proof-of-concept that inhibitor-based spin labeling enables the convenient introduction of site-specific spin labels into kinases for which covalent or tight-binding small-molecule modulators are available. Synthesizing and employing a spin-labeled covalent inhibitor led to a versatile and convenient method to introduce paramagnetic residues at a defined site into a protein for EPR studies. The probe allows the determination of the conformation in solution of the near-full-length epidermal growth factor receptor (EGFR) bound to an inhibitor. This study is thus a significant step forward in EPR analysis of complex proteins.

Publisher URL: http://onlinelibrary.wiley.com/resolve/doi

DOI: 10.1002/anie.201703154