Crystal structure of the human symplekin-Ssu72-CTD phosphopeptide complex

Abstract

Symplekin (Pta1 in yeast) is a scaffold in the large protein complex that is required for 3'-end cleavage and polyadenylation of eukaryotic messenger RNA precursors (pre-mRNAs); it also participates in transcription initiation and termination by RNA polymerase II (Pol II). Symplekin mediates interactions between many different proteins in this machinery, although the molecular basis for its function is not known. Here we report the crystal structure at 2.4 Å resolution of the amino-terminal domain (residues 30-340) of human symplekin in a ternary complex with the Pol II carboxy-terminal domain (CTD) Ser 5 phosphatase Ssu72 (refs 7, 10-17) and a CTD Ser 5 phosphopeptide. The N-terminal domain of symplekin has the ARM or HEAT fold, with seven pairs of antiparallel α-helices arranged in the shape of an arc. The structure of Ssu72 has some similarity to that of low-molecular-mass phosphotyrosine protein phosphatase, although Ssu72 has a unique active-site landscape as well as extra structural features at the C terminus that are important for interaction with symplekin. Ssu72 is bound to the concave face of symplekin, and engineered mutations in this interface can abolish interactions between the two proteins. The CTD peptide is bound in the active site of Ssu72, with the pSer 5-Pro 6 peptide bond in the cis configuration, which contrasts with all other known CTD peptide conformations. Although the active site of Ssu72 is about 25 Å from the interface with symplekin, we found that the symplekin N-terminal domain stimulates Ssu72 CTD phosphatase activity in vitro. Furthermore, the N-terminal domain of symplekin inhibits polyadenylation in vitro, but only when coupled to transcription. Because catalytically active Ssu72 overcomes this inhibition, our results show a role for mammalian Ssu72 in transcription-coupled pre-mRNA 3'-end processing.

Figure 1. Structure of the human symplekin-Ssu72-CTD phosphopeptide ternary complex

(a). Domain organization of human symplekin and S. cerevisiae Pta1. The domains are suggested by secondary structure predictions, and the middle region of Pta1 is suggested by functional studies . (b). Schematic drawing of the structure of human symplekin-Ssu72-CTD phosphopeptide ternary complex, in two views. The N-terminal domain of symplekin is shown in cyan, and Ssu72 in yellow. The CTD phosphopeptide is shown as a stick model (in green for carbon atoms). (c). Overlay of the structures of the N-terminal domain of human (in cyan) and Drosophila (in gray) symplekin . Drosophila symplekin is missing the last two pairs of helical repeats (boxed). All structure figures were produced with PyMOL (www.pymol.org).

Figure 2. Recognition of the CTD phosphopeptide by human Ssu72

(a). Schematic drawing of the structure of human Ssu72-CTD phosphopeptide complex. (b). Overlay of the structures of Ssu72 (yellow) and low-molecular-weight phosphotyrosine protein phosphatase (gray) ,. Arrows point to unique structural features in Ssu72. For stereo version of a and b, please see Supplementary Fig. 4. (c). Two views of the omit F_o–F_c electron density at 2.4 Å resolution for the CTD phosphopeptide, contoured at 3σ. (d). Detailed interactions between the CTD phosphopeptide and the active site of Ssu72. Ion-pair and hydrogen-bonding interactions are indicated with the dashed lines in red. (e). Molecular surface of the active site region of Ssu72. The CTD phosphopeptide is shown as a stick model.

Figure 3. Structural and biochemical characterizations of the symplekin-Ssu72 interface

(a). Detailed interactions between symplekin (in cyan) and Ssu72 (in yellow) in the interface. The molecular surface of symplekin is also shown (in cyan). Side chains making important contributions to the interface are shown as stick models. Residues labeled in red were selected for mutagenesis. (b). The activity of Ssu72, measured by the hydrolysis of pNPP, as a function of the molar ratio of symplekin is shown. TA/VA/FA: T190A/V191A/F193A mutant of Ssu72 (stimulation by wild-type symplekin). The error bars are ±SD, from three independent experiments. (c). Stimulation of the CTD Ser⁵ phosphatase activity of Ssu72 by symplekin. The levels of pSer⁵ and total CTD were determined using the H14 and 8WG16 antibodies, respectively. (d). Gel-filtration profiles for wild-type symplekin N-terminal domain alone, wild-type human Ssu72 (full-length) alone, and their mixture (with Ssu72 present in roughly 2-fold molar excess). (e). Gel-filtration profiles for wild-type Ssu72 alone, K185A mutant of symplekin alone, and their mixture.

Figure 4. Functional characterization of the symplekin-Ssu72 interaction

(a). Transcription-coupled polyadenylation is inhibited in a dose-dependent fashion by symplekin N-terminal domain. Transcription-processing was performed in HeLa nuclear extract, RNAs purified and separated into poly(A)-and poly(A)+ fractions, and resolved by denaturing PAGE. Positions of unprocessed “run-off” RNA (pre-mRNA) and cleaved and polyadenylated RNA (poly(A) RNA) are indicated. (b). Symplekin N-terminal domain does not inhibit transcription. Poly(A)- RNAs (2%) from the transcription-coupled polyadenylation assays in the presence of increasing concentrations of symplekin N-terminal domain are shown. (c). Ssu72 overcomes the inhibition of transcription-coupled polyadenylation by symplekin. The effects of the K185A mutant of symplekin and the C12S and T190A/V191A/F193A (labeled TA/VA/FA) mutants of Ssu72 on polyadenylation are also shown. (d). Polyadenylation of SV40 late pre-mRNA, uncoupled to transcription, is not inhibited by symplekin N-terminal domain.