Abstract
Glutamate dehydrogenase (GDH) is a key enzyme interlinking carbon and nitrogen metabolism. Recent discoveries of the GDH specific role in breast cancer, hyperinsulinism/hyperammonemia (HI/HA) syndrome, and neurodegenerative diseases have reinvigorated interest on GDH regulation, which remains poorly understood despite extensive and long standing studies. Notwithstanding the growing evidence of the complexity of allosteric network behind GDH regulation, identifications of allosteric factors and associated mechanisms are paramount to deepen our understanding of the complex dynamics that regulate GDH enzymatic activity. Combining structural analyses of cryo-electron microscopy data with molecular dynamic simulations, here we show that the cofactor NADH is a key player in the GDH regulation process. Our structural analysis indicates that, binding to the regulatory sites in proximity of the antenna region, NADH acts as a positive allosteric modulator by enhancing both the affinity of the inhibitor GTP binding and inhibition of GDH catalytic activity. We further show that the binding of GTP to the NADH-bound GDH activates a triangular allosteric network, interlinking the inhibitor with regulatory and catalytic sites. This allostery produces a local conformational rearrangement that triggers an anticlockwise rotational motion of interlinked alpha-helices with specific tilted helical extension. This structural transition is a fundamental switch in the GDH enzymatic activity. It introduces a torsional stress, and the associated rotational shift in the Rossmann fold closes the catalytic cleft with consequent inhibition of the deamination process. In silico mutagenesis examinations further underpin the molecular basis of HI/HA dominant mutations and consequent over-activity of GDH through alteration of this allosteric communication network. These results shed new light on GDH regulation and may lay new foundation in the design of allosteric agents.
Highlights
GDH provides an essential link between carbon and nitrogen m etabolisms[8]
Using a structural superimposition with root means square deviations (RMSDs) fingerprint of individual residues, we have focused on the detail characterization of the differences between the open and closed structures
GDH has a homohexameric structure composed of a trimer of dimers, where each monomer consists of three identified d omains[18] (i) Catalytic domain, (ii) Nucleotide-binding domain (NBD), and (iii) the Antennae
Summary
GDH provides an essential link between carbon and nitrogen m etabolisms[8]. Despite extensive studies for the last 40 years, its regulation remains e lusive[9,10]. The molecular basis of the GTP inhibition and the associated GTP insensitivity are one of the key remaining open questions of the GDH regulatory system. The role of the cofactor NADH in the regulation of GDH activity has emerged only very recently. Single-particle cryo-electron microscopy (cryo-EM) studies have further revealed that the GTP binding affinity is increased in presence of NADH at the regulatory site, and GTP synergistically displaces the complex towards the closed conformational state[17]. The results presented in this study indicate that GTP activates a triangular allosteric network interlinking distant GTP binding sites, regulatory NADH, and catalytic sites. This network controls the Nucleotide-binding domain motion that eventually blocks the catalytic cleft in the GTP-induced inhibition dynamics. Using in silico dominant mutational analysis, we show that impeding the allostery network leads to GTP-insensitivity, resulting in GDH over-activity
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