Structure of the YSPTSPS repeat containing two SPXX motifs in the CTD of RNA polymerase II: NMR studies of cyclic model peptides reveal that the SPTS turn is more stable than SPSY in water

Abstract

The carboxyl-terminal domain of RNA polymerase II, which is rich in phosphorylation sites, contains 17–52 tandem repeats with the consensus sequence of the heptapeptide, YSPTSPS. The repeat unit of the heptapeptide has two SPXX motifs showing potential β-turns, SPTS and SPSY. NMR studies were performed in water at pH 4.0 for two cyclic peptides containing one and two repeat units, cyclo-[C 1R 2D 3Y 4S 5P 6T 7S 8P 9S 10Y 11S 12R 13D 14C 15] (peptide 1) and cyclo-[C 1R 2D 3Y 4S 5P 6T 7S 8P 9S 10Y 11S 12P 13T 14S 15P 16N 17Y 18S 19R 20D 21C 22] (peptide 2), which are cyclized with a disulfide bridge of two Cys residues at the N- and C-termini. SP in 1 and 2 are predominantly in trans form. The following NMR parameters were detected: (1) lower temperature coefficients of amide proton chemical shifts of T7 and S8 in 1, and Tx (T7 or T14), Sx (S8 or S15), Tz (T14 or T7) and Sz (S15 or S8) in 2, (2) significantly large deviation of H α chemical shifts from its random coil value (ΔH α) of Pro preceding the Thr (P6 in 1, and Px and Pz in 2), (3) relatively large 3J HNHα coupling constants (>8.7 Hz) of T7 in 1 and Tx and Tz in 2, and (4) NOE (d NN ( i, i+1)) connectivities between the amide protons of T7-S8 and S10-Y11 in 1, and Tx-Sx, S10-Y11, Tz-Sz, and N17-Y18 in 2, although two Pro-Thr-Ser segments in 2 (each of these are annotated by ‘x’ and ‘z’) in the first and second repeat units were not distinguishable. Comparison of the NMR parameters between the cyclic peptides and the corresponding linear peptides indicates that cyclization promotes structural stabilization in water. The present NMR data were consistent with the presence of a β-turn at both SPTS and SPSY: S 5P 6T 7S 8 and S 8P 9S 10Y 11 in 1, and SPxTxSx, SPzTzSz, SP 9S 10Y 11, SP 16N 17Y 18 in 2. However, the structure of the SPTS segment is more stable than that of the SPSY segment. Conformations consistent with NMR parameters including NOE distances were obtained through molecular dynamics and energy minimization methods. These calculations yielded two stable conformers for the SPTS segment. One of the two corresponds to a type I β-turn.