Structural Studies of Metabolic Enzymes and Their Regulation

Division Of Basic Sciences - Nci

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Abstract

Key central metabolites such as ATP, ADP, GTP, GDP, NAD+, NADH and many other small molecules play critical roles in metabolism by modulating the activity of a variety of protein complexes. Metabolic misregulation can result in diseases such as neurodegeneration, cancer and immune disorders. Thousands of enzymes and enzyme isoforms have been biochemically characterized; X-ray crystallographic and NMR spectroscopic analyses have resulted in structural insights into the effects of metabolite binding on protein structure in many instances. To better understand the structural origins of fundamental regulatory mechanisms in allosteric enzymes, we have continued our focus on cryo-EM analysis of a set of metabolic enzymes such as glutamate dehydrogenase and isocitrate dehydrogenase as well as other key cancer targets such as p97 which are involved in protein degradation and other cellular regulatory activities. While these studies have continued, we have extended our studies to work with CRISPR systems that are well known for their role in DNA editing - but these complexes evolved in bacteria as a way for the cells to defend themselves against invading viruses. Although these systems are highly diverse, CRISPR complexes generally use similar mechanisms to specifically recognize and eventually degrade invading viral genetic material. We contributed to a 2017 study published in Cell, which reported the structure of the Pseudomonas aeruginosa Type I-F CRISPR surveillance complex (Csy) bound to a double-stranded DNA template. Our structure revealed the path of the target DNA strand, starting from the recognition site (where the two DNA strands are separated) and continuing its path along the backbone of the Csy complex bound to the complementary CRISPR RNA. At the DNA fork, we found a hook that turns down to hold the DNA in place. A comparison of this structure with the unbound form shows that the DNA bound form of the Csy complex is stretched along its axis, which might allow access to nucleases that can degrade the DNA. In the same study, we also imaged Csy with several inhibitors, which are employed by viruses to overcome the CRISPR system. Each inhibitor worked in a unique way to interfere with the ability of the CRISPR system to bind with the target DNA strand: some bound to the belly of the complex, blocking the DNA interaction with the CRISPR RNA, while others bind closer to the fork, blocking the interaction between the DNA and the Csy hook.