Determinants for dephosphorylation of the RNA polymerase II C-terminal domain by Scp1

Abstract

Phosphorylation and dephosphorylation of the C-terminal domain (CTD) of RNA polymerase II (Pol II) represent a critical regulatory checkpoint for transcription. Transcription initiation requires Fcp1/Scp1-mediated dephosphorylation of phospho-CTD. Fcp1 and Scp1 belong to a family of Mg2+ -dependent phosphoserine (P.Ser)/phosphothreonine (P.Thr)-specific phosphatases. We recently showed that Scp1 is an evolutionarily conserved regulator of neuronal gene silencing. Here, we present the X-ray crystal structures of a dominant-negative form of human Scp1 (D96N mutant) bound to mono- and diphosphorylated peptides encompassing the CTD heptad repeat (Y1S2P3T4S5P6S7). Moreover, kinetic and thermodynamic analyses of Scp1-phospho-CTD peptide complexes support the structures determined. This combined structure-function analysis discloses the residues in Scp1 involved in CTD binding and its preferential dephosphorylation of P.Ser5 of the CTD heptad repeat. Moreover, these results provide a template for the design of specific inhibitors of Scp1 for the study of neuronal stem cell development.

Figure 1. Family of Human CTD Phosphatases

(A) Dendrogram of human CTD phosphatases. The bootstrap values within the dendrogram are shown at nodes on the dendrogram. The NCBI accession numbers for the proteins are Fcp1 (AAC64549), Scp1 (AAH12977), Scp2 (AAH65920), Scp3 (NP005799), HSPC 129 (AAF29093), Dullard (AAH09295), TIMM50 (NP001001563), and MGC10067 (AAH13425). (B) Domain structure of CTD phosphatases. Each domain is represented by a colored block. The catalytic domain is red. An insertion domain is partially conserved and is gray. (C) Phosphopeptides used for the experiments. Phosphoresidues (blue) are highlighted in parentheses. Residue numbers are shown as subscripts.

Figure 2. Structures of Human Scp1 Complexed to CTD Phosphopeptides

(A) Stereo ribbon diagram of human Scp1 bound to a CTD phosphopeptide with helices as red coils and b strands as blue arrows. The three-stranded insert is labeled βID1-3. The CTD peptide is a stick diagram with color-coded bonds. Yellow is carbon, red is oxygen, blue is nitrogen, and magenta is phosphorus. The Mg²⁺ ion is shown as a magenta van der Waals sphere. (B) Model of the monophosphorylated CTD peptide complex (P.Ser₅) as a half-colored bond diagram with the blue SIGMAA weighted 2F_o – F_c electron-density map contoured at 1σ. An intramolecular hydrogen bond between the Ser₂ and Thr₄ is shown as red cylinders. (C) Accessible surface of Scp1 bound to monophosphorylated P.Ser₅ CTD peptide. Surfaces conserved between human Fcp1 and human Scp1 are orange, and chemically similar residues are pink. Phe106 is yellow. The peptide is shown as half-colored bonds with carbon atoms light green. (D) Model of the doubly phosphorylated 14-mer CTD peptide complex (P.Ser₅-P.Ser₅) as a half-colored bond diagram with the blue SIGMAA-weighted 2F_o – F_c electron-density map contoured at 1σ.

Figure 3. DXDX Family of Mg²⁺-Dependent Enzymes

(A) β-phosphoglucomutase, a member of the DXDX family, shares its three-dimensional architecture with Scp1 (PDB code 1o08). The conserved core is merlot, and the elements that do not align structurally with Scp1 are light gray. The Mg²⁺ is depicted as a van der Waals sphere. (B) Three-dimensional architecture of Scp1 shown in the same orientation. (C) Active sites of Scp1 and β-phosphoglucomutase. The β-phosphoglucomutase ribbons are gold, and the Scp1 ribbons are lavender. Labels are in italic for β-phosphoglucomutase. (D) Mg²⁺ coordination of the phospho-CTD peptide with bonds as green cylinders. The peptide is shown as half-colored bonds. Residues essential for the dephosphorylation reaction (Hausmann and Shuman, 2003) are highlighted as color-coded half bonds.

Figure 4. Reaction and Binding Mechanisms of Scp1

(A) Reaction mechanism of Scp1. The active site geometry, water coordination, and position of Asp206 supports its participation as a general base for activating a water for breakdown of the mixed anhydride intermediate formed after nucleophilic attack of Asp96 on P.Ser₅. Alternatively, the residue equivalent to Asp98 may function as the general base for this final catalytic step (Wang et al., 2002). (B) Pro₃ binding pocket of Scp1 and comparison with Fcp1. The lavender ribbon underlies a transparent surface used to illustrate the steric volume surrounding the Pro₃ moiety. Peptide and side chains are shown as half-colored bonds with green highlighting carbon for the peptide and yellow highlighting carbon for Scp1. The equivalent residues in human Fcp1 are labeled blue in italic. (C) Scp1 residues involved in the binding of the phospho-CTD. Intermolecular hydrogen bonds are shown as rendered green cylinders, and an intramolecular hydrogen bond in the CTD peptide is shown as rendered red cylinders.

Figure 5. CTD-Binding Proteins

(A) Superimposition of the Pin1 WW domain and the Scp1 insertion domain. The connectivity pattern of β strands for both Pin1 (lavender) and Scp1 (cyan) is shown schematically as an inset. (B) Comparison of the bound citrate molecule previously observed in apo Scp1 (Kamenski et al., 2004) and the bound phospho-CTD peptides reported here. The citrate molecule and the phospho-CTD peptide are shown as rendered bonds (CTD peptide is yellow and the citrate molecule is cyan). Citrate superimposes with the CTD peptide backbone and the Pro₃ and Thr₄ side chains.