Eukaryotic transcriptional repressors function by recruiting large co-regulatory complexes that target histone deacetylase enzymes to gene promoters/enhancers. Transcriptional repression complexes, assembled by the co-repressor NCoR, and its homologue SMRT, play critical roles in many processes including development and metabolic physiology. The core repression complex involves the recruitment of three proteins: HDAC3, GPS2 and TBL1 to a highly conserved repression domain within SMRT and NCoR. We have used a variety of structural and functional approaches to gain insight into the assembly, stoichiometry and biological role of this complex. We report the crystal structure of the tetrameric oligomerization domain of TBL1, which interacts with both SMRT and GPS2, and the NMR structure of the interface complex between GPS2 and SMRT. These structures, together with computational docking, mutagenesis and functional assays, reveal the assembly mechanism and stoichiometry of the co-repressor complex.The regulated repression of transcription plays a key role in many biological processes. These include cell fate decisions during development and cellular differentiation, as well as the maintenance of homeostasis. SMRT and NCoR are large homologous co-repressor proteins that were identified through their role in transcriptional repression by many 5 Corresponding author: john.schwabe@le.ac.uk. 4 These authors should be considered co-first authors. Author ContributionsThe contributions of J.O., L.F. & P.W. were critical to the final manuscript and these authors should be considered co-first authors. J.O. (assisted by J.Gooch) performed most of the protein cloning, expression, purification and interaction mapping, although important preliminary experiments were performed by B.K. J.O. prepared the GPS2-SMRT complex for NMR structure determination which was carried out by J-C.Y. and D.N. Crystallizations were performed primarily by J.O., with some later trials by J.Greenwood and L.F. Crystal structure determinations were performed by L.F., J.O. and J.W.R.S. The interaction motifs in GPS2 and SMRT were identified by J.W.R.S. and tested by pull-down experiments by J.O. Fluorescence polarization, co-immunoprecipitation and co-transfection/ purification assays were performed by P.W. The two-hybrid assays, gel filtrations and NMR comparisons of WT and MT TBL1 were performed by P.W., Z.C. and B.G. In silico docking experiments were performed by T.K. The laboratories of L.N. and D.N. provided experimental expertise for transfection and NMR studies respectively. J.W.R.S. planned and supervised the project and prepared the manuscript with assistance from the other authors. Accession Codes1H, 13C and 15N NMR resonance assignments for the SMRT(167-207) -GPS2(53-90) complex have been deposited at the BioMagResBank under accession code 17271, and the coordinates have been deposited under the pdb accession code 215G. The TBL1 X-ray structures have been deposited with the PDB codes (2XTC, 2XTD & 2XTE). When purified from HeLa cell extr...
Cellular stress in early mitosis activates the antephase checkpoint, resulting in the decondensation of chromosomes and delayed mitotic progression. Checkpoint with forkheadassociated and RING domains (CHFR) is central to this checkpoint, and its activity is ablated in many tumors and cancer cell lines through promoter hypermethylation or mutation. The interaction between the PAR-binding zinc finger (PBZ) of CHFR and poly(ADP-ribose) (PAR) is crucial for a functional antephase checkpoint. We determined the crystal structure of the cysteine-rich region of human CHFR (amino acids 425-664) to 1.9 Å resolution, which revealed a multizinc binding domain of elaborate topology within which the PBZ is embedded. The PBZ of CHFR closely resembles the analogous motifs from aprataxin-like factor and CG1218-PA, which lie within unstructured regions of their respective proteins. Based on co-crystal structures of CHFR bound to several different PARlike ligands (adenosine 5-diphosphoribose, adenosine monophosphate, and P 1 P 2 -diadenosine 5-pyrophosphate), we made a model of the CHFR-PAR interaction, which we validated using site-specific mutagenesis and surface plasmon resonance. The PBZ motif of CHFR recognizes two adenine-containing subunits of PAR and the phosphate backbone that connects them. More generally, PBZ motifs may recognize different numbers of PAR subunits as required to carry out their functions.
The serine/threonine phosphatase protein phosphatase 5 (PP5) regulates hormone-and stress-induced cellular signaling by association with the molecular chaperone heat shock protein 90 (Hsp90). PP5-mediated dephosphorylation of the cochaperone Cdc37 is essential for activation of Hsp90-dependent kinases. However, the details of this mechanism remain unknown. We determined the crystal structure of a Cdc37 phosphomimetic peptide bound to the catalytic domain of PP5. The structure reveals PP5 utilization of conserved elements of phosphoprotein phosphatase (PPP) structure to bind substrate and provides a template for many PPP-substrate interactions. Our data show that, despite a highly conserved structure, elements of substrate specificity are determined within the phosphatase catalytic domain itself. Structure-based mutations in vivo reveal that PP5-mediated dephosphorylation is required for kinase and steroid hormone receptor release from the chaperone complex. Finally, our data show that hyper-or hypoactivity of PP5 mutants increases Hsp90 binding to its inhibitor, suggesting a mechanism to enhance the efficacy of Hsp90 inhibitors by regulation of PP5 activity in tumors.Hsp90 | PP5 | Cdc37 | chaperone | phosphatase P rotein phosphatase 5 (PP5) has pleiotropic roles in cellular signaling, including DNA damage repair, proliferation of breast cancer cells, circadian cycling, response to cytotoxic stresses, Rac-dependent potassium ion channel activity, and activation of steroid hormone receptors [e.g., glucocorticoid receptor (GR) and estrogen receptor] (1, 2). It is a member of the phosphoprotein phosphatase (PPP) family of serine/threonine phosphatases, which has members that share a highly conserved catalytic core and catalytic mechanism dependent on two metal ions, commonly Mn 2+ . Most PPP family members exhibit high, nonspecific phosphatase activity. Specificity is provided by a large cohort of regulatory and other interacting proteins that function to inhibit basal activity and recruit substrates, thereby finely tuning the enzymes (3). This combinatorial approach enables a small number of catalytic subunits to have the breadth of specificity equivalent to that seen in kinases, which are greater in number by an order of magnitude. Structures of complexes between regulatory and catalytic domains have illuminated the importance of regulatory subunits in facilitating substrate recruitment (3). However, to date, there is no structural information describing how a substrate binds at the active site of a PPP; therefore, a central question remains of how local interactions between the substrate and the catalytic domain contribute to the molecular basis of dephosphorylation.PP5 is unique among the PPP family because it has a low basal activity caused by an autoinhibitory N-terminal tetratricopeptide (TPR) domain (4). Its activity is promoted by a number of cellular factors, including fatty acids and the molecular chaperone heat shock protein 90 (Hsp90) (5), both of which release autoinhibition by interacting with the TPR...
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