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. With glutamate dehydrogenase, a highly conserved enzyme expressed in most organisms, we showed that have shown that NADH binding results in a mixture of complexes co-exist in both closed and open conformations. We show that the structures in both states can be resolved at near-atomic resolution, with our studies suggesting a molecular mechanism for synergistic inhibition of GDH by NADH and GTP. Our structural studies have established that whether or not GTP is bound, NADH binding is detectable at catalytic and regulatory sites, in both the open and closed conformational states. While the orientation in which NADH binds at the catalytic site is similar for both conformations, the orientation of the nicotinamide portion of NADH in the regulatory site is different between the open and closed conformations. In the closed state, the nicotinamide moiety of NADH is inserted into a well-defined cavity at the interface between two adjacent protomers in the trimer. This cavity is much narrower in the open state, suggesting that this cavity may be unavailable to the NADH nicotinamide moiety when the enzyme is in the open conformation. These structural features have led us to propose a structural explanation of the mechanism by which NADH binding inhibits the activity of the enzyme by stabilizing the closed conformation state.These studies were published in the journal Molecular Pharmacology and featured on the cover of the June 2016 issue of the journal. We are now beginning to extend these studies to isocitrate dehydrogenase where we also reported that inhibitor binding results in conformational changes. The development of effective inhibitors against IDH1 is of interest both at the NCI and in several other cancer centers, but in many cases, it has not been possible to obtain high resolution structures of the complexes of the relevant lead compounds with crystallography. Cryo-EM methods now offer an opportunity to bridge this gap and help the identification of the interactions between selected compounds and the active site of the enzyme. In a similar vein, we are also evaluating a variety of compounds that target the AAA ATPase p97. Last year, we reported an atomic resolution model for p97 bound to a phenyl indole derivative that defined the regions of this lead compound that bind to p97. In continued efforts to find better inhibitors, we are now exploring structures of complexes of p97 with other compounds that inhibit its activity, which help definition of the relevant inhibition mechanisms.