Structural and biochemical characterization of DAXX-ATRX interaction

Structural and biochemical characterization of DAXX-ATRX interaction Zhuang Li, Dan Zhao, Bin Xiang, and Haitao Li* College of Life Sciences, Peking University, Beijing 100871, P.R. China MOE Key Laboratory of Protein Sciences, Tsinghua-Peking Joint Center for Life Sciences, Beijing Advanced Innovation Center for Structural Biology, Department of Basic Medical Sciences, School of Medicine, Tsinghua University, Beijing 100084, P.R. China


Molecular cloning, protein expression and purification
The DAXX-ATRX mini-complex for crystallization was produced using a co-expression strategy.
The truncated DID domain (ATRX 1244-1285 ) of ATRX (UniProt ID: P46100) were ligated into the first multiple cloning site (MCS) of the pRSFD-SUMO vector (modified from pRSFDuet-1, Novagen) with an N-terminal 6xHis-SUMO tag, and the DHB domain (DAXX 55-144 ) of DAXX (UniProt ID: Q9UER7) was ligated into the second MCS of pRSFD-SUMO without any tag. The transformed BL21 (DE3) cells (Invitrogen) were grown at 37°C in LB medium to reach an OD 600nm = 0.6-0.8, and then induced with 0.2 mM IPTG at 18°C overnight for recombinant protein expression. Centrifugation-collected cells were suspended in lysis buffer containing 500 mM NaCl, 20 mM imidazole and 50 mM Tris, pH 7.5, and then lysed by an EmulsiFlex-C3 high pressure homogenizer (AVESTIN). The cell lysates were further clarified by centrifugation and the 6xHis-SUMO-ATRX 1244-1285 -DAXX 55-144 mini-complex in supernatant was affinity purified through a HisTrap HP column (GE Healthcare). The 6xHis-SUMO tag was cleaved by the SUMO protease, Ulp1. After removal of imidazole from the digestion mixture by desalting column, the 6xHis-SUMO tag was affinity eliminated by an additional HisTrap HP column. The flow-through was then pooled, concentrated, and applied to a Superdex 75 10/300 GL (GE Healthcare) gel filtration column under the elution buffer containing 100 mM NaCl, 10 mM Tris, pH 7.5. The resultant peak was then pooled and concentrated to about 15 mg/ml for future use.
Free DHB domain of DAXX was constructed into the pGood6p vector (modified from pGEX-6p-1, GE Healthcare) with an N-terminal GST-tag. The recombinant protein was expressed in BL21 (DE3) cells. Cells were grown at 37°C and induced with 0.2 mM IPTG at 18°C overnight for protein expression and lysed in lysis buffer containing 500 mM NaCl, 50 mM Tris, pH 7.5. Procedures for protein purification include GST affinity purification, human rhinovirus 3C protease digestion, S cation exchange, and gel filtration. The purified DHB DAXX sample containing an N-terminal "GPLGS" uncleavable tag sequence was stored in ITC buffer containing 100 mM NaCl, 20 mM HEPES, pH 7.5 for future use.
Full length DID domain of ATRX (ATRX 1189-1326 ) was constructed into the pET28-b vector and expressed in BL21(DE3) cells. Cells were grown at 37°C and induced with 0.2 mM IPTG at 18°C overnight for protein expression and lysed in lysis buffer containing 500 mM NaCl, 20 mM imidazole, and 50 mM Tris, pH 7.5. Procedures for protein purification include Ni-NTA affinity purification, thrombin digestion, Q anion exchange, and gel filtration. The purified DID ATRX sample containing an N-terminal "GSHM" uncleavable tag sequence was stored in ITC buffer containing 100 mM NaCl, 20 mM HEPES, pH 7.5 for future use. reduction, phasing, and initial model building were done with HKL3000 suite (Minor et al., 2006). Further model building and refinement were done with COOT and PHENIX (Adams et al., 2010;Emsley and Cowtan, 2004). Detailed data processing and structural refinement statistics are summarized in table S1. Structural figures were created using UCSF Chimera (Pettersen et al., 2004) or PyMOL (https://pymol.org).

Isothermal titration calorimetry (ITC)
Calorimetric experiments were conducted at 25°C with a MicroCal iTC200 instrument (GE Healthcare). The DHB DAXX and full length DID ATRX samples were prepared in the buffer containing 100 mM NaCl and 20 mM HEPES, pH 7.5. Protein concentration was determined by absorbance spectroscopy at 280 nm. The DID ATRX truncation peptides were chemically synthesized by Scilight Biotechnology LLC (Beijing, China), and were quantified by weighing on a large scale. Acquired calorimetric titration data were analyzed using Origin 7.0 (GE Healthcare) using the "One Set of Binding Sites" fitting model. All the ITC experiments in this study used DID ATRX as the "ligand" in the syringe and the DHB DAXX in the sample cell.

Peptide competition electrophoretic mobility shift assay (EMSA)
In the peptide competition EMSA assay, purified protein complex stock of DAXX-ATRX as described above was prepared in the reaction buffer (100 mM NaCl and 20 mM HEPES, pH 7.5) at concentration of about 0.5 mM. The competitor peptides (Scilight Biotechnology LLC), Rassf1c 20-44 , p53 39-63 and Mdm2 293-317 , were dissolved in reaction buffer at a concentration of 4 mM. For each competition reaction, DAXX-ATRX complex was mixed with competitor peptide with or without reaction buffer to a total volume of 20 µL. The mixture was incubated on ice for 1 hour before subjected to native-PAGE (polyacrylamide gel electrophoresis) analysis. In an optimized native-PAGE system, the Tris buffer at pH 7.5 was used to prepare the polyacrylamide gel and Tris-MOPS, pH 8.0, was used as the running buffer.