Saturday, April 26, 2025
4/26/2025
Structurally Defining rBH3 Interactions - Project Summary This project will utilize protein purification and structure techniques to determine how a novel protein motif, known as the reverse Bcl-2 homology 3 (rBH3) motif binds to the anti-apoptotic protein MCL1 (myeloid cell leukemia 1). MCL1 is a key regulator of cell fate decisions, with MCL1 levels and activity affecting cellular processes that include apoptosis, cell cycle progression, and transcriptional regulation. Recent work from my lab has demonstrated MCL1's direct role in the regulation of many of these processes, and most recently, data from our lab indicates that MCL1 can directly bind to and activate the apical metabolic enzyme hexokinase 2 (HK2). However, despite increasing functional evidence, the structural determinants for MCL1 binding to the rBH3 motif remain unknown. Recent data in our lab brings even more interest to how MCL1 binds to and affects rBH3 containing proteins. MCL1 binding to Hexokinase 2 leads to a 30% increase in hexokinase 2 activity. However, the putative rBH3 motif of hexokinase 2 is located within the active site. Further, while only two residues of the rBH3 motif are currently known to be necessary, one of these residues is also a catalytically essential residue in the HK2 catalytic cleft. Therefore, a structure of the complex is necessary for understanding how MCL1 binding can lead to an increase in activity, and this discovery will provide novel understanding about the function of HK2 and MCL1. Finally, to determine the structural changes that occur in HK2 upon MCL1 binding, I must first understand the structure of HK2. Previous structure studies of HK2 have used X-ray crystallography and have relied on the use of the binding of both substrate and product to HK2 to induce crystal formation. However, the unbound structure of HK2 as it would be within the cellular environment has not been observed. Therefore, before solving the structure of the HK2-MCL1 complex, I will solve the structure of apo HK2 utilizing cryo-electron microscopy (cryo- EM). This will provide the first unbound, native state structure of a bi-domain hexokinase. At the conclusion of the proposed project, I will have propelled my chosen field of structural biology forward, while simultaneously preparing myself to be a successful independent investigator of protein structure. Utilizing cryo-EM to solve the structure of the 104 kDa HK2 protein will be technically innovative as a cryo-EM structure of a small protein, with structures in the 100 kDa range only being recently achieved by cryo-EM. Further, my work utilizing binding assays to determine key residues in the rBH3 motif will provide me with a strong background in protein binding assay design and execution. Finally, my NMR studies, both in regards to binding and in utilizing NMR to complement the cryo-EM complex structure by solving the structure of the MCL1-rBH3 peptide complex will complete my training as a structural biologist prepared to take on the questions of the future.
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