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Structure of the Human Cohesin Inhibitor Wapl

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Structure of the Human Cohesin Inhibitor Wapl Structure of the human cohesin inhibitor Wapl Zhuqing Ouyanga,1, Ge Zhenga,1, Jianhua Songb, Dominika M. Borekc, Zbyszek Otwinowskic, Chad A. Brautigamc, Diana R. Tomchickc, Susannah Rankinb, and Hongtao Yua,2 aHoward Hughes Medical Institute, Department of Pharmacology, and cDepartment of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX 75390; and bProgram in Cell Cycle and Cancer Biology, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104 Edited by Douglas Koshland, University of California, Berkeley, CA, and approved May 23, 2013 (received for review March 11, 2013) Cohesin, along with positive regulators, establishes sister-chromatid trigger cohesin release from chromosome arms (15, 18–21). A pool of cohesion by forming a ring to circle chromatin. The wings apart-like cohesin at centromeres is protected by the shugoshin (Sgo1)–protein protein (Wapl) is a key negative regulator of cohesin and forms phosphatase 2A (PP2A) complex (22, 23), which binds to cohesin, a complex with precocious dissociation of sisters protein 5 (Pds5) to dephosphorylates sororin, and protects cohesin from Wapl promote cohesin release from chromatin. Here we report the at centromeres (24). After all sister kinetochores attach properly crystal structure and functional characterization of human Wapl. to the mitotic spindle and are under tension, separase cleaves Wapl contains a flexible, variable N-terminal region (Wapl-N) and centromeric cohesin to initiate sister-chromatid separation. The a conserved C-terminal domain (Wapl-C) consisting of eight HEAT separated chromatids are evenly partitioned into the two daughter (Huntingtin, Elongation factor 3, A subunit, and target of rapamycin) cells through their attachment to microtubules originating from the repeats. Wapl-C folds into an elongated structure with two lobes. opposite spindle poles. Wapl inactivation alleviates both the re- Structure-based mutagenesis maps the functional surface of quirement for sororin in cohesion establishment in S phase and the Wapl-C to two distinct patches (I and II) on the N lobe and a localized need for Sgo1–PP2A in centromeric cohesion protection in mitosis patch (III) on the C lobe. Mutating critical patch I residues weaken (6, 7, 15, 24). Thus, Wapl is a critical negative regulator of cohesin. Wapl binding to cohesin and diminish sister-chromatid resolution Wapl-triggered cohesin release from chromatin requires the and cohesin release from mitotic chromosomes in human cells and opening of the cohesin ring at the junction between the Smc3 Xenopus egg extracts. Surprisingly,patchIIIontheClobedoes ATPase domain and the N-terminal WHD of Scc1 in budding not contribute to Wapl binding to cohesin or its known regula- yeast, fly, and humans (25–27). Furthermore, the structure of the tors. Although patch I mutations reduce Wapl binding to intact C-terminal domain of Wapl from the filamentous fungus Ashbya BIOPHYSICS AND cohesin, they do not affect Wapl–Pds5 binding to the cohesin sub- gossypii has recently been determined (28). The fungal Wapl COMPUTATIONAL BIOLOGY complex of sister chromatid cohesion protein 1 (Scc1) and stromal proteins bind to the isolated Smc3 ATPase domain. It has been antigen 2 (SA2) in vitro, which is instead mediated by Wapl-N. Thus, suggested that Wapl might trigger cohesin release from chro- – Wapl-N forms extensive interactions with Pds5 and Scc1 SA2. matin through stimulating the ATPase activity of cohesin, al- Wapl-C interacts with other cohesin subunits and possibly un- though this hypothesis remains to be biochemically tested. known effectors to trigger cohesin release from chromatin. In this study, we have determined the crystal structure of human Wapl (HsWapl). We have also systematically mapped the functional chromosome segregation | crystallography | genomic stability | mitosis | surface of Wapl using structure-based mutagenesis and performed – protein protein interaction in-depth functional and biochemical analyses of key Wapl mutants. Our results indicate that Wapl-mediated cohesin release from roper chromosome segregation during mitosis maintains chromatin requires extensive physical contacts among Wapl, mul- Pgenomic stability. Errors in this process cause aneuploidy, tiple cohesin subunits, and possibly an unknown effector. Our study which contributes to tumorigenesis under certain contexts (1). reveals both similarities and important differences between the Timely establishment and dissolution of sister-chromatid co- mechanisms of human and fungal Wapl proteins. hesion are critical for accurate chromosome segregation and require the cell-cycle–regulated interactions between cohesin Results and Discussion and its regulators (2–4). Crystal Structure of HsWapl. Wapl proteins from different species In human cells, cohesin consists of four core subunits: Structural each have a divergent N-terminal domain with variable lengths maintenance of chromosomes 1 (Smc1), Smc3, sister chromatid and a conserved C-terminal domain (Wapl-C) (Fig. 1A). To gain cohesion protein 1 (Scc1), and stromal antigen 1 or 2 (SA1/2). insight into the mechanism of Wapl-dependent cohesin release Smc1 and Smc3 are related ATPases, and each contains an ATPase from chromatin, we sought to analyze HsWapl biochemically and head domain, a long coiled-coil domain, and a hinge domain that structurally. The recombinant, purified full-length (FL) Wapl mediates Smc1–Smc3 heterodimerization. The Smc1–Smc3 and several N-terminal truncation mutants, including ΔN100–, heterodimer associates with the Scc1–SA1/2 heterodimer to ΔN200–, and ΔN300–Wapl, eluted from size-exclusion columns produce the intact cohesin. Specifically, the N- and C-terminal winged helix domains (WHDs) of Scc1 connect the ATPase domains of Smc3 and Smc1, respectively, forming a ring (4). Author contributions: Z. Ouyang, G.Z., S.R., and H.Y. designed research; Z. Ouyang, G.Z., J.S., Cohesin is loaded onto chromatin in telophase/G1, but the and C.A.B. performed research; Z. Ouyang, G.Z., J.S., D.M.B., Z. Otwinowski, C.A.B., D.R.T., S.R., and H.Y. analyzed data; and Z. Ouyang, G.Z., D.M.B., D.R.T., S.R., and H.Y. wrote chromatin-bound cohesin at this stage is highly dynamic and is ac- the paper. tively removed from chromatin by the cohesin inhibitor Wings The authors declare no conflict of interest. apart-like protein (Wapl) (5–7). During DNA replication in S This article is a PNAS Direct Submission. phase, the ATPase head domain of Smc3 is acetylated by the ace- Freely available online through the PNAS open access option. tyltransferase establishment of cohesion protein 1 (Eco1) (8–13). In Data deposition: The atomic coordinates and structure factors have been deposited in the vertebrates, replication-coupled Smc3 acetylation enables the Protein Data Bank, www.pdb.org (PDB ID code 4K6J). binding of precocious dissociation of sisters protein 5 (Pds5) and 1Z. Ouyang and G.Z. contributed equally to this work. sororin to cohesin (14–17). Sororin counteracts Wapl to stabilize 2To whom correspondence should be addressed. E-mail: hongtao.yu@utsouthwestern. cohesin on replicated chromatin and establishes sister-chromatid edu. cohesion (15). In prophase, polo-like kinase 1 (Plk1) and cyclin- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. dependent kinase 1 (Cdk1) phosphorylate cohesin and sororin and 1073/pnas.1304594110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1304594110 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 We next determined the structure of Wapl-C using X-ray crys- tallography (Fig. 1C and Table S1). Wapl-C has an elongated shape with two lobes and contains eight HEAT (Huntingtin, Elongation factor 3, A subunit, and target of rapamycin) repeats with variable lengths and a short N-terminal extension. HEAT2 and HEAT8 each have two helixes (αAandαB) (Fig. 1C and Fig. S1). All other HEAT repeats each have two long helixes (αAandαB) and a third short helix (αC). HEAT3 contains a helical insert between αAand αB. The αAandαB helices in HEAT1–3 are shorter than those in HEAT4–8. HEAT1–3 repeats and the HEAT3 insert form the N lobe of Wapl-C. The longer HEAT4–8 repeats form the C lobe. The N-terminal extension (residues 631–640) is likely unfolded in solution, but folds into a helix in our structure due to crystal packing interactions (see Fig. 5D below). During the course of our work, the structure of the C-terminal domain of Wapl from the filamentous fungus A. gossypii was reported (28). As expected, the structures of A. gossypii Wapl Fig. 1. Structure of HsWapl. (A) Schematic drawing of the Wapl proteins from different species (Xt, Xenopus tropicalis;Dr,Danio rerio;Dm,Drosophila (AgWapl) and HsWapl had similar folds (Fig. S2). Like HsWapl, melanogaster;Ce,Caenorhabditis elegans;Sc,Saccharomyces cerevisiae). AgWapl contains eight HEAT repeats, which form two lobes. Wapl-C, the C-terminal domain of Wapl. The boundaries of human Wapl-C are AgWapl also contains a helical insert between helices αAandαB indicated. (B)SEC-MALSprofiles of human Wapl-C and ΔN200–Wapl. (C) of HEAT3. A major difference between AgWapl and HsWapl is Cartoon drawing of the crystal structure of human Wapl-C in two different the relative orientation between their N and C lobes, suggesting orientations. The H3 insert and the N-terminal extension helix are colored gray the intriguing possibility that the connection between HEAT3 and and orange, respectively. The rest of the protein is colored green. The N and C HEAT4 is flexible, and these two repeats can rotate relative to – lobes are labeled. The positions of the HEAT repeats 1 8 are indicated in Right. each other. All structure figures were made with PyMOL (www.pymol.org). Mapping the Functional Surface of Wapl-C. We failed to detect (SECs) with apparent molecular masses much larger than the ex- binding of human Wapl-C to known cohesin subunits and regu- pected molecular masses for their respective monomers. By contrast, lators in vitro, which prohibited us from determining the structure of Wapl-C bound to cohesin or its regulators.
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