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Szatmári Tímea

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Szatmári Tímea
Effect of rational modification of disordered domains of the epidermal growth factor receptor on its biophysical characteristics

Aug 30 - szerda

15:30 – 17:00

II. Poszterszekció

P54

Effect of rational modification of disordered domains of the epidermal growth factor receptor on its biophysical characteristics

Tímea Szatmári¹, Péter Nagy¹

¹ Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen

The epidermal growth factor receptor (EGFR) belongs to the ErbB receptor tyrosine kinase family. The extracellular domain of EGFR consists of four subdomains (I-IV). Upon ligand binding, the extracellular (EC) domain of the receptor is rearranged, resulting in exposition of the dimerization arm of subunit II providing an opportunity to form homodimers or heterodimers. The 3D structure determined by the amino acid sequence affects protein function. Proteins in which the formation of an ordered structure is not complete are called intrinsically disordered proteins. Recently, an algorithm (FuzPred) was developed that predicts the tendency of proteins to form disordered regions and their structural changes upon interacting with other proteins. The amino acid sequence of the EGFR was analyzed by FuzPred, which determines the tendency of distinct sequence regions to constitute globular (assuming ordered conformations), disordered (intrinsically disordered adopting ordered conformation upon binding), or fuzzy regions (remaining disordered even in their bound state). The following mutations were designed based on the software prediction that alter the fuzziness and the molecular interactions of EGFR domain II. 1. Mutation T274G is predicted to result in increased dynamics and destabilization of the dimerization site. It still forms a dimer, but the interaction is weak. 2. Mutation Q276F is expected to decrease dynamics of the dimerization arm and create a rigid binding site. As a result, it is unable to form a dimer due to lack of flexibility. 3. Mutation K284Q is expected to decrease dynamics while strengthening the binding site. This mutation promotes dimerization. We successfully generated all three mutants (dark and EGFP tagged on the C terminal of the proteins) by site-directed mutagenesis and stably transfected CHO cells expressing the EGFP-tagged mutant EGFRs. We investigated the ligand binding and the cooperativity of the wild-type EGFR and its mutant variants. CHO cells transfected with the wild-type or the mutant EGFRs were labeled with a concentration series of fluorescently labeled EGF (TAMRA-EGF). Our results suggest that those mutants that were predicted to be less prone to dimerization bind EGF less cooperatively and with a slightly lower affinity. In our further experiment we would like to examine the dimerization process (FRET, N&B) itself and consequent transmembrane signaling.