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 -Phe(2)-Pro(3)-Pro(4)-Val(5)-Pro (6)-Gly(7)-Pro(8)) corresponding to the conserved proline-rich motif(P RM) of the intracellular domain of the prolactin receptor (PRL-R) was studied by one- and two-dimensional (1D and 2D) proton NMR spectroscop y in water and DMSO in order to characterize its conformational dynami cs, The purified PRL-R PRM peptide eluted as two partially resolved pe aks in equilibrium on reverse-phase HPLC (RP-HPLC) at 20 degrees C wit h a ratio of 60:40, At 30 degrees C, the two peaks coalesced into a si ngle peak. The two RP-HPLC peaks correspond to two peptide conformers resulting from the slow cis-trans isomerization of one of the four pro line amide bonds. Although the peptide has only three amide (NH) proto ns, its 1D NMR spectrum in water contains approximately 15 discernible NH region peaks, providing evidence for multiple conformers. The amid e resonances were assigned on the basis of 2D-COSY spectra, chemical s hift values, resonance splitting patterns and temperature coefficients . The cis:trans ratio for each proline in water, calculated from integ rated intensities and/or peak heights of the appropriate resonances, w ere Phe(2)-Pro(3) (35:65), Pro(3)-Pro(4) (40:60), Val(5)-Pro(6) (70:30 ), and Gly(7)-Pro(8) (30:70). Temperature studies (25-70 degrees C) we re used to semi-quantitatively estimate the rates of isomerization for the different prolines, In water, Pro(8) undergoes rapid isomerizatio n; Pro(3) isomerizes at an intermediate rate; while Pro(4) and Pro(6) both appear to isomerize very slowly since no coalescence of amide res onances was observed. In DMSO, only Pro(4) displayed slow isomerizatio n. Slow kinetics combined with a similar 60:40 ratio of conformers det ermined by RP-HPLC and NMR suggests that isomerization of the Pro(3)-P ro(4) bond generates the two RP-HPLC peaks. Both proximal and distal p roline isomerization effects were observed in NMR experiments. All of the 16 theoretical (2(4) = 16) proline configurations appear to exist in equilibrium in water. The predominant (19%,) conformation, trans(3) - trans(4)-cis(6)-trans(8), may reflect the configuration of the PRM p rolines in the native PRL-R. Isomerization of Pro(6) from cis to trans generates an interaction between the peptide N-and C-termini, suggest ing an overall pseudo-cyclic conformation. This all-trans proline conf iguration may play an important biochemical role in the function of cy tokine/haematopoietin receptors. A model is proposed which suggests th at isomerization of the PRM by an immunophilin such as the FK506-bindi ng protein (FKBP) serves as an on-off switch for cytokine receptor act ivation.