title: 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 beta-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(1)R(2)D(3)Y(4)S(5)P(6)T(7)S(8)P(9)S(10)Y(11)S(12)R(13)D(14)C(15)] (peptide 1) and cyclo-[C(1)R(2)D(3)Y(4)S(5)P(6)T(7)S(8)P(9)S(10)Y(11)S(12)P(13)T(14)S(15)P(16)N(17)Y(18)S(19)R(20)D(21)C(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(alpha) chemical shifts from its random coil value (Delta H(alpha)) of Pro preceding the Thr (P6 in 1, and Px and Pz 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, 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 beta-turn at both SPTS and SPSY: S(5)P(6)T(7)S(8) and S(8)P(9)S(10)Y(11) in 1, and SPxTxSx, SPzTzSz, SP(9)S(10)Y(11), SP(16)N(17)Y(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 beta-turn.
authors.affiliation: High-Resolution NMR Laboratory, Graduate School of Science, Hokkaido University, Sapporo, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Kumaki
authors.forename: N
authors.initials: N
authors.lastname: Matsushima
authors.forename: H
authors.initials: H
authors.lastname: Yoshida
authors.forename: K
authors.initials: K
authors.lastname: Nitta
authors.forename: K
authors.initials: K
authors.lastname: Hikichi
mesh_terms: D000595:Amino Acid Sequence; D016384:Consensus Sequence; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008969:Molecular Sequence Data; D010456:Peptides, Cyclic / Q000737:chemistry*; D011487:Protein Conformation; D012319:RNA Polymerase II / Q000737:chemistry*; D020871:RNA Stability; D013237:Stereoisomerism; D020080:Tandem Repeat Sequences; D013696:Temperature
chemical_list: D010456:Peptides, Cyclic; D012319:RNA Polymerase II