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 SPTSturn is more stable than SPSY in water

The carboxyl-terminal domain of RNA polymerase IT, which is rich in phospho rylation sites, contains 17-52 tandem repeats with the consensus sequence o f the heptapeptide, YSPTSPS. The repeat unit of the heptapeptide has two SP XX motifs showing potential beta -turns, SPTS and SPSY. NMR studies were pe rformed in water at pH 4.0 for two cyclic peptides containing one and two r epeat units, cyclo-[(CRDYPTSPSYSRDC15)-R-1-D-2-Y-3-P-45-T-6-S-7-P-8-S-9-Y-1 0-S-11-R-12-D-13-C-14] (peptide 1) and cyclo[(CRDYSPTSPSYSPTSPNYSRDC22)-R-1 -D-2-Y-3-S-4-P-5-T-6-S-7-P-8-S-9-Y-10-S-11-P-12-T-13-S-14-P-15-N-16-Y-17-S- 18-R-19-D-20-C-21] (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 predominantl y in trans form. The following NMR parameters were detected: (1) lower temp erature coefficients of amide proton chemical shifts of T7 and S8 in I, and Tx (T7 or T14), Sr (S8 or S15), Tz (T14 or T7) and St (S15 or S8) in 2, (2 ) significantly large deviation of H-alpha chemical shifts from its random coil value DeltaH(alpha)) of Pro preceding the Thr (P6 in I, and Pr and Pt in 2), (3) relatively large (3)J(HNH alpha) 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, SIO-YI I, T z-Sz, and N17-Y18 in 2, although two Pro-Thr-Ser segments in 2 teach of the se are annotated by 'x' and 'z') in the first and second repeat units were not distinguishable. Comparison of the NMR parameters between the cyclic pe ptides and the corresponding linear peptides indicates that cyclization pro motes structural stabilization in water. The present NMR data were consiste nt with the presence of a beta -turn at both SPTS and SPSY: (SPTS8)-P-5-T-6 -S-7 and (SPSD11)-P-8-S-9-D-10 i, I, and SPxTxSx, SPzTzSz, (SPSY11)-S-9-Y-1 0, Sp(16)N(17)Y(18) in 2. However, the structure of the SPTS segment is mor e stable than that of the SPSY segment. Conformations consistent with NMR p arameters including NOE distances were obtained through molecular dynamics and energy minimization methods. These calculations yielded two stable conf ormers for the SPTS segment. One of the two corresponds to a type I beta -t urn. (C) 2001 Published by Elsevier Science B.V.