Expression and purification of an NSD3-GB1 fusion protein as a way to study the structure of complex formation with Brd4 ET.
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Date
2020-05
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The Ohio State University
Abstract
The BRD4 protein belongs to the bromodomain and extraterminal domain (BET) family of eukaryotic transcription factors, and is linked to several types of cancer, inflammation, and obesity. Bromodomain inhibitors are currently in clinical trials for treating several types of cancer however, bromodomains are not exclusive to the BET family. The ET domain is exclusive to the BET family and is responsible for recruiting chromatin-modifying factors that result in transcriptional co-activation. One example of these factors is nuclear receptor binding SET domain protein 3 (NSD3), which promotes cell cycle progression through methylation of a lysine residue on histone H3. Due to its role in cell cycle regulation, NSD3 is essential in maintaining acute myeloid leukemia.
Previous studies using fragments of a 645-residue short isoform of NSD3 showed that residues 100-263 were sufficient to maintain binding with the ET domain with an affinity of 2.1 μM. Subsequent studies using isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) indicated that residues 152-163 of NSD3 form a complex with the ET domain and reported a Kd of 140 μM. However, unpublished work from our laboratory using NMR showed that titration of the same peptide into ET yielded a Kd of 3mM, this suggests that more residues are important in the interaction. Thus, the goal of my research is to define the interacting region between NSD3 and the BRD4 ET domain.
Prior experiments in the Foster lab showed that NSD3 residues 100-263 are not soluble at concentrations required to characterize its structure. These residues are soluble when fused to glutathione-S-transferase (GST); however, removal of this GST 'tag' resulted in protein precipitation. To overcome this solubility problem, we fused NSD3 100-263 to a GB1 solubility tag. GB1 has a well-defined NMR spectrum and has shown to significantly increase the solubility of small peptides to which it is fused. There is no need to remove the GB1 tag as long as its signals don't overlap or convolute the spectrum of the NSD3-BRD4 complex.
Two-dimensional 1H-15N NMR spectra will be recorded of free GB1, the NSD3-GB1 fusion and the BRD4 ET domain, these will serve as reference spectra. To characterize the interaction between NSD3 and BRD4 ET, NMR spectra will be recorded of labeled ET domain in the presence of unlabeled NSD3 and labeled NSD3 in the presence of unlabeled ET. Because signals in the spectra can be assigned to individual amino acids, overlaying the interaction spectra with their reference spectra will show shifted peaks for the residues involved in the interaction. Based on previous data, it is expected that more than just residues 152-163 of NSD3 will be shifted, indicating that they are involved in the interaction with the ET domain.
Overall, elucidating the mechanism of interaction between the BRD4 ET domain and NSD3 will give a clearer picture into the specific interactions of the ET domain. Specifically, a conserved mechanism of interaction between ET and its binding partners would be of interest and serve as a guide to treatments specifically targeting the BET family.
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Keywords
protein expression, cancer research, protein interactions, NMR