Conformational changes in G-CSF/Receptor complex as investigated by isotope-edited FTIR spectroscopy

Conformations of G-CSF and the extracellular domain of its receptor as well as their complex have been investigated by employing isotope-edited FTIR spectroscopy. To determine unambiguously the protein conformations of G-CSF and the receptor in the complex, we have prepared uniformly 13C/15N isotope labeled G-CSF to resolve its amide I' band from that of the receptor in the IR spectrum of the complex. By comparing the IR spectra of the isotope-labeled G-CSF and the receptor with that of the complex, we have provided spectral evidence that the AB loop region involving the unique 310 helix segment of G-CSF likely undergoes a conformational change to a regular alpha-helix upon binding to the receptor. The IR data also indicate a possible minor increase in alpha-helical conformation for the receptor in the complex. Furthermore, FTIR spectra of G-CSF, the receptor, and their complex demonstrate clearly that protein conformations of both G-CSF and the receptor have been dramatically stabilized by complex formation. Specifically, the melting transition (Tm value) of the alpha-helix in G-CSF is increased by nearly 30 degrees C and that of the beta-strand in the receptor by nearly 15 degrees C in the G-CSF/receptor complex. We estimate from the current FTIR data that the native conformations of approximately 15% of all receptor residues are stabilized by G-CSF binding. On the other hand, the entire alpha-helical content of G-CSF appears to be stabilized in the complex. Together, these results indicate that formation of the ligand/receptor complex results in not only conformational changes in the receptor but also significant structural changes in the ligand. This adds insight to the general consensus that binding of ligand to cytokine receptors induces mostly structural changes in the receptor which lead to receptor oligomerization and signal transduction. The current data also suggest a possible physiological role of the 310 helix present in G-CSF for its receptor binding activity.