Cofactor-dependent conformational heterogeneity of GAD65 and its role in autoimmunity and neurotransmitter homeostasis

Abstract

The human neuroendocrine enzyme glutamate decarboxylase (GAD) catalyses the synthesis of the inhibitory neurotransmitter gammaaminobutyric acid (GABA) using pyridoxal 5′-phosphate as a cofactor. GAD exists as two isoforms named according to their respective molecular weights: GAD65 and GAD67. Although cytosolic GAD67 is typically saturated with the cofactor (holoGAD67) and constitutively active to produce basal levels of GABA, the membrane-associated GAD65 exists mainly as the inactive apo form. GAD65, but not GAD67, is a prevalent autoantigen, with autoantibodies to GAD65 being detected at high frequency in patients with autoimmune (type 1) diabetes and certain other autoimmune disorders. The significance of GAD65 autoinactivation into the apo form for regulation of neurotransmitter levels and autoantibody reactivity is not understood. We have used computational and experimental approaches to decipher the nature of the holo → apo conversion in GAD65 and thus, its mechanism of autoinactivation. Molecular dynamics simulations of GAD65 reveal coupling between the C-terminal domain, catalytic loop, and pyridoxal 5′-phosphate-binding domain that drives structural rearrangement, dimer opening, and autoinactivation, consistent with limited proteolysis fragmentation patterns. Together with small-angle X-ray scattering and fluorescence spectroscopy data, our findings are consistent with apoGAD65 existing as an ensemble of conformations. Antibody-binding kinetics suggest a mechanism of mutually induced conformational changes, implicating the flexibility of apoGAD65 in its autoantigenicity. Although conformational diversity may provide a mechanism for cofactor-controlled regulation of neurotransmitter biosynthesis, it may also come at a cost of insufficient development of immune self-tolerance that favors the production of GAD65 autoantibodies.conformational dynamics | normal mode analysis | GABA biosynthesis | immunogenicity | autoepitopes T he two mammalian isoforms of glutamate decarboxylase (GAD; EC 4.1.1.15), GAD65 and GAD67, are responsible for the biosynthesis of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) (1, 2). GAD65 and GAD67 belong to the group II pyridoxal 5′-phosphate (PLP)-dependent enzymes of fold type I, which comprise two other evolutionarily related human enzymes: aromatic L-amino acid decarboxylase (AADC; synonym: L-dopa decarboxylase; EC 4.1.1.28) and histidine decarboxylase (EC 4.1.1.22) (3). Expression of these enzymes in the brain is responsible for the synthesis of the biogenic amines GABA, dopamine, serotonin, and histamine. Because they are implicated in a wide range of biological activities from central homeostatic functions to cognitive phenomena, it is not surprising that their activities need to be finely regulated. In this respect, the binding of the cofactor PLP to the apo forms of these enzymes might represent an important mechanism for regulation (4). The interaction of apoGAD with PLP is a major factor in the short-term regulation of GAD activity (5). Although GAD67 i...

How pyridoxal 5′‐phosphate differentially regulates human cytosolic and mitochondrial serine hydroxymethyltransferase oligomeric state

“…In the apo‐enzyme, the two large domains have twisted 10° and moved apart to a maximum distance of 10 Å. Such opening of the dimer, which has been shown to occur in DOPA and glutamate decarboxylases , had not been proposed for SHMT. Moreover, while holo‐SHMT2 displays a well‐structured and buried active site, the PLP‐binding site in the apo‐form is solvent‐exposed and partially unstructured.…”

Conformational Equilibrium of CDK/Cyclin Complexes by Molecular Dynamics with Excited Normal Modes

“…The effect of loop conformations within the overall dynamics on proteins or complexes is of general interest. We recently showed the importance of the catalytic loop conformation in the context of opening/closing events taking place on the glutamate decarboxylase protein (47). We anticipate that this conformational flexibility may enable such complexes to adapt their conformation upon interaction with other partners, or may occur and be prompted upon substrate binding to achieve optimal phosphoryl transfer reaction.…”

Extracellular HMGB1 Modulates Glutamate Metabolism Associated with Kainic Acid-Induced Epilepsy-Like Hyperactivity in Primary Rat Neural Cells

“…GAD67 tightly binds the coenzyme pyridoxal 5-phosphate (PLP) and is constitutively active (holoenzyme form), robustly producing GABA in the brain [51, 52]. In contrast, GAD65 depends on the interconversion between an active PLP-bound holoenzyme and an inactive apoenzyme [53], requiring the activation of extra GABA synthesis. Moreover, GAD67 is more ubiquitously distributed within neurons and associated with the synthesis of GABA released.…”