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-edi ted FTIR spectroscopy. To determine unambiguously the protein conforma tions of G-CSF and the receptor in the complex, we have prepared unifo rmly C-13/N-15 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 r egion involving the unique 3(10) helix segment of G-CSF likely undergo es a conformational change to a regular alpha-helix upon binding to th e receptor. The IR data also indicate a possible minor increase in alp ha-helical conformation for the receptor in the complex. Furthermore, FTIR spectra of G-CSF, the receptor, and their complex demonstrate cle arly that protein conformations of both G-CSF and the receptor have be en dramatically stabilized by complex formation. Specifically, the mel ting transition (T-m value) of the alpha-helix in G-CSF is increased b y nearly 30 degrees C and that of the beta-strand in the receptor by n early 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 stabili zed in the complex. Together, these results indicate that formation of the ligand/receptor complex results in not only conformational change s in the receptor but also significant structural changes in the ligan d. This adds insight to the general consensus that binding of ligand t o cytokine receptors induces mostly structural changes in the receptor which lead to receptor oligomerization and signal transduction. The c urrent data also suggest a possible physiological role of the 3(10) he lix present in G-CSF for its receptor binding activity.