Preparation data of the bromodomains BRD3(1), BRD3(2), BRD4(1), and BRPF1B and crystallization of BRD4(1)-inhibitor complexes

This article presents detailed purification procedures for the bromodomains BRD3(1), BRD3(2), BRD4(1), and BRPF1B. In addition we provide crystallization protocols for apo BRD4(1) and BRD4(1) in complex with numerous inhibitors. The protocols described here were successfully applied to obtain affinity data by isothermal titration calorimetry (ITC) and by differential scanning fluorimetry (DSF) as well as structural characterizations of BRD4(1) inhibitor complexes (PDB codes: PDB: 4LYI, PDB: 4LZS, PDB: 4LYW, PDB: 4LZR, PDB: 4LYS, PDB: 5D24, PDB: 5D25, PDB: 5D26, PDB: 5D3H, PDB: 5D3J, PDB: 5D3L, PDB: 5D3N, PDB: 5D3P, PDB: 5D3R, PDB: 5D3S, PDB: 5D3T). These data have been reported previously and are discussed in more detail elsewhere [1], [2].


a b s t r a c t
This article presents detailed purification procedures for the bromodomains BRD3(1), BRD3(2), BRD4 (1), and BRPF1B. In addition we provide crystallization protocols for apo BRD4(1) and BRD4 (1) in complex with numerous inhibitors. The protocols described here were successfully applied to obtain affinity data by isothermal titration calorimetry (ITC) and by differential scanning fluorimetry (DSF) as well as structural characterizations of BRD4 (1)   All proteins were produced heterologously in E. coli.

Experimental features
Proteins were isolated by IMAC via His 6 -tag followed by tag cleavage with TEV and polishing via size exclusion chromatography. Proteins were concentrated for crystallization to 10 mg/mL and supplied with ligands at a concentration of 2 mM.

Value of the data
Provides details on purification and crystallization of BRD4(1). Provides details on co-crystallization strategies of BRD4(1) with inhibitors. Provides details on purification of BRD3(1), BRD3(2), and BRPF1B. Data shown here may serve as benchmarks for other groups working with bromodomains.

Data
We present a detailed strategy for the heterologous overproduction and purification of BRD4 (1) including chromatograms and SDS-gels, shown in Figs. 1 and 2. The data allowed for the characterization of 25 inhibitors for bromodomains with a special focus on BRD4(1), published in [1] and [2]. The protocol shown here was also used for the preparation of BRPF1B, BRD3(1) and BRD3(2) to determine their affinities to various novel inhibitors (see [1] for original affinity data). In addition, we provide photographs of crystals of apo BRD4(1) (Fig. 4) and BRD4(1) in complex with several inhibitors (Fig. 3).
For 14 inhibitors we obtained high resolution X-ray structures in complex with BRD4(1) [1,2]. Structure formulas, SMILES strings and pdb ID codes are summarized in Supplementary Table 1.

Protein preparation
The plasmids for protein production are gifts from Nicola Burgess-Brown, purchasable via Addgene (Addgene plasmids # 38941, # 53620, # 38940, # 38943). All constructs share the vector backbone pNIC28-Bsa4 with a Kanamycin resistance gene. They encode for an N-terminal His 6 -tag followed by a TEV-cleavage site N-terminal of the target protein.

Protein crystallization
Prior to crystallization the protein solutions were supplemented with inhibitor from a 100 mM stock solution in DMSO to a final inhibitor concentration of 2 mM Subsequently, precipitated protein was removed by centrifugation (16,000xg, 4°C, 5 min, Centrifuge 5415 R, Eppendorf, Germany). Cocrystals with the described inhibitors were obtained in the Index HT Screen (Hampton Research, Aliso Viejo, USA) using 38% protein (10 mg/ml) and 62% reservoir solution. The drops (400 nl) were pipetted into an Intelli-Plate 96 low profile (Hampton Research, USA) with an Oryx Nano crystallization robot (Douglas Instruments, UK). Single crystals formed after one to five days at 20°C (Fig. 3). Note, that BRD4(1) crystallizes spontaneously after two weeks in the final crystallization buffer at 10-12°C in a reaction tube, if the concentration exceeds 20 mg/ml (Fig. 4).