Multiple cis-trans conformers of the prolactin receptor proline-rich motif (PRM) peptide detected by reverse-phase HPLC, CD and NMR spectroscopy

An eight-amino-acid synthetic peptide (Ile¹-Phe²-Pro³-Pro⁴-Val⁵-Pro⁶-Gly⁷-Pro⁸) corresponding to the conserved proline-rich motif (PRM) of the intracellular domain of the prolactin receptor (PRL-R) was studied by one- and two-dimensional (1D and 2D) proton NMR spectroscopy in water and DMSO in order to characterize its conformational dynamics. The purified PRL-R PRM peptide eluted as two partially resolved peaks in equilibrium on reverse-phase HPLC (RP-HPLC) at 20 °C with a ratio of 60:40. At 30 °C, the two peaks coalesced into a single peak. The two RP-HPLC peaks correspond to two peptide conformers resulting from the slow cis–trans isomerization of one of the four proline amide bonds. Although the peptide has only three amide (NH) protons, its 1D NMR spectrum in water contains approximately 15 discernible NH region peaks, providing evidence for multiple conformers. The amide resonances were assigned on the basis of 2D-COSY spectra, chemical shift values, resonance splitting patterns and temperature coefficients. The cis:trans ratio for each proline in water, calculated from integrated intensities and/or peak heights of the appropriate resonances, were Phe²-Pro³ (35:65), Pro³-Pro⁴ (40:60), Val⁵-Pro⁶ (70:30), and Gly⁷-Pro⁸ (30:70). Temperature studies (25–70 °C) were used to semi-quantitatively estimate the rates of isomerization for the different prolines. In water, Pro⁸ undergoes rapid isomerization; Pro³ isomerizes at an intermediate rate; while Pro⁴ and Pro⁶ both appear to isomerize very slowly since no coalescence of amide resonances was observed. In DMSO, only Pro⁴ displayed slow isomerization. Slow kinetics combined with a similar 60:40 ratio of conformers determined by RP-HPLC and NMR suggests that isomerization of the Pro³-Pro⁴ bond generates the two RP-HPLC peaks. Both proximal and distal proline isomerization effects were observed in NMR experiments. All of the 16 theoretical (2⁴ = 16) proline configurations appear to exist in equilibrium in water. The predominant (19%) conformation, trans³-trans⁴-cis⁶-trans⁸, may reflect the configuration of the PRM prolines in the native PRL-R. Isomerization of Pro⁶ from cis to trans generates an interaction between the peptide N-and C-termini, suggesting an overall pseudo-cyclic conformation. This all-trans proline configuration may play an important biochemical role in the function of cytokine/haematopoietin receptors. A model is proposed which suggests that isomerization of the PRM by an immunophilin such as the FK506-binding protein (FKBP) serves as an on–off switch for cytokine receptor activation.