Insights into receptor structure and dynamics at the surface of living cells

Frederik Steiert, Peter Schultz, Siegfried Höfinger, Thomas D. Müller, Petra Schwille and Thomas Weidemann ()
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Frederik Steiert: Max Planck Institute of Biochemistry
Peter Schultz: Max Planck Institute of Biochemistry
Siegfried Höfinger: VSC Research Center, TU Wien
Thomas D. Müller: Lehrstuhl für Molekulare Pflanzenphysiologie und Biophysik - Botanik I
Petra Schwille: Max Planck Institute of Biochemistry
Thomas Weidemann: Max Planck Institute of Biochemistry

Nature Communications, 2023, vol. 14, issue 1, 1-15

Abstract: Abstract Evaluating protein structures in living cells remains a challenge. Here, we investigate Interleukin-4 receptor alpha (IL-4Rα) into which the non-canonical amino acid bicyclo[6.1.0]nonyne-lysine (BCNK) is incorporated by genetic code expansion. Bioorthogonal click labeling is performed with tetrazine-conjugated dyes. To quantify the reaction yield in situ, we develop brightness-calibrated ratiometric imaging, a protocol where fluorescent signals in confocal multi-color images are ascribed to local concentrations. Screening receptor mutants bearing BCNK in the extracellular domain uncovered site-specific variations of both click efficiency and Interleukin-4 binding affinity, indicating subtle well-defined structural perturbations. Molecular dynamics and continuum electrostatics calculations suggest solvent polarization to determine site-specific variations of BCNK reactivity. Strikingly, signatures of differential click efficiency, measured for IL-4Rα in ligand-bound and free form, mirror sub-angstrom deformations of the protein backbone at corresponding locations. Thus, click efficiency by itself represents a remarkably informative readout linked to protein structure and dynamics in the native plasma membrane.

Date: 2023
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DOI: 10.1038/s41467-023-37284-4

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