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

Nature volume 467, pages 729–733 (2010)Cite this article

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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)1,2,3,4; it also participates in transcription initiation and termination by RNA polymerase II (Pol II)5,6. Symplekin mediates interactions between many different proteins in this machinery1,2,7,8,9, 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 phosphatase18,19, 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 conformations20,21. 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.

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Figure 1: Structure of the human symplekin–Ssu72–CTD phosphopeptide ternary complex.
Figure 2: Recognition of the CTD phosphopeptide by human Ssu72.
Figure 3: Structural and biochemical characterizations of the symplekin–Ssu72 interface.
Figure 4: Functional characterization of the symplekin–Ssu72 interaction.

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Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates have been deposited at the Protein Data Bank (accession codes 3O2Q, 3O2S, 3O2T, 3ODR and 3ODS).

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Acknowledgements

We thank F. Forouhar and J. Seetharaman for help with data collection; T. Auperin for initial experiments with symplekin; R. Abramowitz and J. Schwanof for setting up the X4C beamline; S. Myers for setting up the X29A beamline at the National Synchrotron Light Source; M. Heidemann and D. Eick for providing the 3E10 antibody; N. Rao and C. Logan for HeLa nuclear extract and help with in vitro assays; and J. Decatur for characterizing the phosphopeptide by NMR. This research is supported in part by grants from the National Institutes of Health to L.T. (GM077175) and J.L.M. (GM028983).

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Author notes

  1. Song Xiang

    Present address: Present address: Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.,

  2. Takashi Nagaike and Song Xiang: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biological Sciences, Columbia University, New York, 10027, New York, USA

    Kehui Xiang, Takashi Nagaike, Song Xiang, Turgay Kilic, Maia M. Beh, James L. Manley & Liang Tong

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Contributions

K.X., S.X., T.K. and M.M.B. performed protein expression, purification and crystallization experiments. K.X., S.X. and L.T. conducted crystallographic data collection, structure determination and refinement. T.N. and K.X. performed polyadenylation experiments. K.X. performed Ssu72 phosphatase assays. All authors commented on the manuscript. L.T. and J.L.M. designed the experiments, analysed the data and wrote the paper.

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Correspondence to Liang Tong.

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Xiang, K., Nagaike, T., Xiang, S. et al. Crystal structure of the human symplekin–Ssu72–CTD phosphopeptide complex. Nature 467, 729–733 (2010). https://doi.org/10.1038/nature09391

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