1H, 13C and 15N resonance assignments of TbBDF5-bromo1 domain from Trypanosoma brucei

1H, 13C and 15N resonance assignments are presented for the first bromo domain of TbBDF5 (TbBDF5-bromo1) from Trypanosoma brucei. TbBDF5 is localized in the nucleus and plays a potential role in transcription regulation. Bromo domains can recognize acetylated histone through a conserved binding pocket. Here we report the NMR resonance assignments of TbBDF5-bromo1 domain for further studies of the relationship between its structure and function.

Bromo domain has been identified to play an essential role in gene expression in all kinds of eukaryotes including protozoan parasites. Bromodomain-containing proteins, which can exist alone or as part of a larger protein complex, have a variety of physiological functions. These proteins participate in histone modifications, act as components of chromatin remodeling complexes and serve as scaffolds that control the recruitment of other transcriptional regulators to chromatin (Fujisawa and Filippakopoulos 2017). Bromo domain is a kind of motif recognizing acetylated lysine of histones that occur in the N-terminal regions of proteins (Zeng and Zhou 2002). It is well known that histone lysine acetylation can neutralize the positive charge of histone, increase hydrophobicity and bind to the conserved amino acid residues in the hydrophobic pocket of bromo domain. The bromo domain is evolutionarily conserved, consists of about 110 amino acids and shares a globular fold that comprises a lefthanded bundle of four α-helices (αZ, αA, αB, αC) linked by two variable-loop regions (ZA-loop, BC-loop), which is conserved across all bromo domains (Filippakopoulos et al. 2012). In many typical bromo domains, ZA-loop contains a short single-turn helix (called the ZA helix) (Marmorstein and Berger 2001). A number of studies have provided evidence that bromodomain-containing protein can be used as targets for the treatment of cancer and other diseases (Schulz et al. 2015;Alonso et al. 2019).
Trypanosoma brucei (T. brucei) is a single-cell eukaryote, which is different from other eukaryotic cells and has unique characteristics in gene transcription. There are seven bromodomain-containing proteins in the T. brucei (Fleck et al. 2021), but only TbBDF5 contains a tandem bromo domain, which happens mostly in the BET (bromodomain and extra-terminal) protein family. TbBDF1-6 are enriched at transcription start sites to promote RNAPII-mediated transcription and TbBDF7 is enriched at the transcription termination (Staneva et al. 2021). TbBDF5 interacts with other uncharacterized proteins to form a complex at transcription initiation sites. The bromo domain of TbBDF5 may guide HAT2 (histone acetyltransferase 2) to acetylation modification at the transcription initiation site to maintain the acetylation state required for effective transcription.
To gain critical insights into the structure and function of TbBDF5-bromo1 domain, we analyzed the NMR data of TbBDF5-bromo1 domain to establish its structural information. In this article, the backbone and side-chain 1 H, 13 C and 15 N resonance assignments of TbBDF5-bromo1 domain from T. brucei are determined.

Methods and experiments
The DNA fragment encoding the first bromo domain (residue 1-122) of TbBDF5 (Tb927.11.13400) from T. brucei was amplified by polymerase chain reaction (PCR) from T. brucei genomic DNA. The forward and reverse primers were 5′-GGA GAT ATA CAT ATG AGT CAA AAC CGG CAG -3′ and 5′-GTG GTG GTG CTC GAG GCC ACC CAC TTT CTG -3′, respectively. PCR products were then cloned into the vector pET-22(b) (Novagen) cleaved with NdeI/XhoI by infusion method. The recombinant vectors were transformed into Escherichia coli strain BL21 (DE3). The recombinant TbBDF5-bromo1 domain possesses 130 amino acids with a His-tag in the C-terminus (LEHHH-HHH). To obtain isotopically labeled TbBDF5-bromo1 domain, the domain was overproduced in Escherichia coli BL21 (DE3) using minimal medium containing 2.5 g/L 99% 13 C-glucose and 0.5 g/L 99% 15 NH 4 Cl as the sole carbon and nitrogen source, respectively. Cells were cultured at 37℃ until OD 600 = 0.8, then induced with 0.5 mM IPTG at 16 °C for 20 h. The induced cells were harvested and resuspended in 35 mL lysis buffer (20 mM Tris, 500 mM NaCl, pH 7.8), then lysed by sonication on ice. The lysate was centrifuged at 11,000×g and 4 °C for 30 min to remove precipitate. The protein was purified by Ni 2+ -NTA column. The labeled protein was eluted with 15 mL Tris NaCl buffer containing 500 mM imidazole. The eluted protein was further purified using a Superdex 75 column on an AKTA purification system. The purified proteins were dialyzed with buffer containing 25 mM NaH 2 PO 4 , 100 mM NaCl, 1 mM EDTA and 1 mM DL-Dithiothreitol (DTT) (pH 6.8) for 4 times and then concentrated to 500 μL with a concentration of 0.8 mM. The final NMR buffer contains 25 mM phosphate (pH 6.8), 100 mM NaCl, 1 mM EDTA, 1 mM DTT in 10:90% D 2 O: H 2 O.
All NMR data were collected at 293 K on a Bruker DMX 600 spectrometer. The following spectra were recorded to obtain backbone and side chain resonance assignments: 1 H-15 N HSQC, CBCA(CO)NH, CBCANH, HC(CO)NH, HBHA(CO)NH, C(CO)NH, 3D 15 N-edited and 13 C-edited NOESY. NMR data was processed using NMRpipe and NMRDraw software (Delaglio et al. 1995), and then analyzed with Sparky 3 (Goddard and Kneller 1993).

Assignment and data deposition
The 1 H-15 N-HSQC spectrum of TbBDF5-bromo1 is shown in Fig. 1. Except three non-proline residues (Ser2, Gly68 and Thr75), 1 H and 15 N backbone chemical shifts were assigned for almost all residues (97%). In addition, 95% of NH and 15 N, 97% of 13 C, and 90% of 1 H chemical shifts were assigned. 90% of side-chain resonance assignments were completed. The data sets from spectra CBCANH, CBCA(CO)NH and HC(CO)NH contained the 13 C signals for the proline residues.
The consensus chemical shift index (CSI) derived from 1 Hα, 13 Cα and 13 Cβ chemical shifts (Wishart and Sykes 1994) is shown in Fig. 2. This data indicates the TbBDF5-bromo1 domain contains five α-helices without β-sheet. A variable-loop (ZA-loop) is located between the first and third α-helices. The second small α-helix is located in the long ZA-loop region. Another variable-loop (BC-loop) is located between the fourth and fifth α-helices.