3D architecture and structural flexibility revealed in the subfamily of large glutamate dehydrogenases by a mycobacterial enzyme.

Glutamate dehydrogenases (GDHs) are widespread metabolic enzymes that play key roles in nitrogen homeostasis. Large glutamate dehydrogenases composed of 180 kDa subunits (L-GDHs180) contain long N- and C-terminal segments flanking the catalytic core. Despite the relevance of L-GDHs180 in bacterial physiology, the lack of structural data for these enzymes has limited the progress of functional studies. Here we show that the mycobacterial L-GDH180 (mL-GDH180) adopts a quaternary structure that is radically different from that of related low molecular weight enzymes. Intersubunit contacts in mL-GDH180 involve a C-terminal domain that we propose as a new fold and a flexible N-terminal segment comprising ACT-like and PAS-type domains that could act as metabolic sensors for allosteric regulation. These findings uncover unique aspects of the structure-function relationship in the subfamily of L-GDHs.

Allosteric regulation of glutamate dehydrogenase deamination activity.

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.

Energy landscape of domain motion in glutamate dehydrogenase deduced from cryo-electron microscopy.

Analysis of the conformational changes of protein is important to elucidate the mechanisms of protein motions correlating with their function. Here, we studied the spontaneous domain motion of unliganded glutamate dehydrogenase from Thermococcus profundus using cryo-electron microscopy and proposed a novel method to construct free-energy landscape of protein conformations. Each subunit of the homo-hexameric enzyme comprises nucleotide-binding domain (NAD domain) and hexamer-forming core domain. A large active-site cleft is situated between the two domains and varies from open to close according to the motion of a NAD domain. A three-dimensional map reconstructed from all cryo-electron microscopy images displayed disordered volumes of NAD domains, suggesting that NAD domains in the collected images adopted various conformations in domain motion. Focused classifications on NAD domain of subunits provided several maps of possible conformations in domain motion. To deduce what kinds of conformations appeared in EM images, we developed a novel analysis method that describe the EM maps as a linear combination of representative conformations appearing in a 200-ns molecular dynamics simulation as reference. The analysis enabled us to estimate the appearance frequencies of the representative conformations, which illustrated a free-energy landscape in domain motion. In the open/close domain motion, two free-energy basins hindered the direct transformation from open to closed state. Structure models constructed for representative EM maps in classifications demonstrated the correlation between the energy landscape and conformations in domain motion. Based on the results, the domain motion in glutamate dehydrogenase and the analysis method to visualize conformational changes and free-energy landscape were discussed. DATABASE: The EM maps of the four conformations were deposited to Electron Microscopy Data Bank (EMDB) as accession codes EMD-9845 (open), EMD-9846 (half-open1), EMD-9847 (half-open2), and EMD-9848 (closed), respectively. In addition, the structural models built for the four conformations were deposited to the Protein Data Bank (PDB) as accession codes 6JN9 (open), 6JNA (half-open1), 6JNC (half-open2), and 6JND (closed), respectively.

Routine determination of ice thickness for cryo-EM grids.

Recent advances in instrumentation and automation have made cryo-EM a popular method for producing near-atomic resolution structures of a variety of proteins and complexes. Sample preparation is still a limiting factor in collecting high quality data. Thickness of the vitreous ice in which the particles are embedded is one of the many variables that need to be optimized for collection of the highest quality data. Here we present two methods, using either an energy filter or scattering outside the objective aperture, to measure ice thickness for potentially every image collected. Unlike geometrical or tomographic methods, these can be implemented directly in the single particle collection workflow without interrupting or significantly slowing down data collection. We describe the methods as implemented into the Leginon/Appion data collection workflow, along with some examples from test cases. Routine monitoring of ice thickness should prove helpful for optimizing sample preparation, data collection, and data processing.

Breaking Cryo-EM Resolution Barriers to Facilitate Drug Discovery.

Recent advances in single-particle cryoelecton microscopy (cryo-EM) are enabling generation of numerous near-atomic resolution structures for well-ordered protein complexes with sizes ≥ ∼200 kDa. Whether cryo-EM methods are equally useful for high-resolution structural analysis of smaller, dynamic protein complexes such as those involved in cellular metabolism remains an important question. Here, we present 3.8 Å resolution cryo-EM structures of the cancer target isocitrate dehydrogenase (93 kDa) and identify the nature of conformational changes induced by binding of the allosteric small-molecule inhibitor ML309. We also report 2.8-Å- and 1.8-Å-resolution structures of lactate dehydrogenase (145 kDa) and glutamate dehydrogenase (334 kDa), respectively. With these results, two perceived barriers in single-particle cryo-EM are overcome: (1) crossing 2 Å resolution and (2) obtaining structures of proteins with sizes < 100 kDa, demonstrating that cryo-EM can be used to investigate a broad spectrum of drug-target interactions and dynamic conformational states.

Using Cryo-EM to Map Small Ligands on Dynamic Metabolic Enzymes: Studies with Glutamate Dehydrogenase.

Cryo-electron microscopy (cryo-EM) methods are now being used to determine structures at near-atomic resolution and have great promise in molecular pharmacology, especially in the context of mapping the binding of small-molecule ligands to protein complexes that display conformational flexibility. We illustrate this here using glutamate dehydrogenase (GDH), a 336-kDa metabolic enzyme that catalyzes the oxidative deamination of glutamate. Dysregulation of GDH leads to a variety of metabolic and neurologic disorders. Here, we report near-atomic resolution cryo-EM structures, at resolutions ranging from 3.2 Å to 3.6 Å for GDH complexes, including complexes for which crystal structures are not available. We show that the binding of the coenzyme NADH alone or in concert with GTP results in a binary mixture in which the enzyme is in either an "open" or "closed" state. Whereas the structure of NADH in the active site is similar between the open and closed states, it is unexpectedly different at the regulatory site. Our studies thus demonstrate that even in instances when there is considerable structural information available from X-ray crystallography, cryo-EM methods can provide useful complementary insights into regulatory mechanisms for dynamic protein complexes.

Conformational flexibility of a model protein upon immobilization on self-assembled monolayers.

The present study reports on the retention of conformational flexibility of a model allosteric protein upon immobilization on self-assembled monolayers (SAMs) on gold. Organothiolated SAMs of different compositions were utilized for adsorptive and covalent attachment of bovine liver glutamate dehydrogenase (GDH), a well-characterized allosteric enzyme. Sensitive fluorimetric assays were developed to determine immobilization capacity, specific activity, and allosteric properties of the immobilized preparations as well as the potential for repeated use and continuous catalytic transformations. The allosteric response of the free and immobilized forms towards ADP, L-leucine and high concentrations of NAD(+), some of the well-known activators for this enzyme, were determined and compared. The enzyme immobilized by adsorption or chemical binding responded similarly to the activators with a greater degree of activation, as compared to the free form. Also loss of activity involving the two immobilization procedures were similar, suggesting that residues essential for catalytic activity or allosteric properties of GDH remained unchanged in the course of chemical modification. A recently established method was used to predict GDH orientation upon immobilization, which was found to explain some of the experimental results presented. The general significance of these observations in connection with retention of native properties of protein structures upon immobilization on SAMs is discussed.

Quantitative ultrastructural localization of glutamate dehydrogenase in the rat cerebellar cortex.

Glutamate dehydrogenase is one of the main enzymes involved in the formation and metabolism of the neurotransmitter glutamate. In the present study we investigated the enzyme ultrastructurally in the cerebellar cortex, a region rich in well defined glutamatergic neurons, by pre-embedding immunocytochemical staining (peroxidase-antiperoxidase), as well as by post-embedding immunogold labelling employing a new system for quantitation and for specificity testing under the conditions of the immunocytochemical procedure. A new antiserum against immunologically purified bovine liver glutamate dehydrogenase or antibodies isolated from this by affinity chromatography were used in rats fixed by perfusion with aldehydes. The pre-embedding method displayed peroxidase reaction preferentially in mitochondria of astroglial cells (including the Bergmann glia). Mitochondria of neuronal tissue elements were usually free of peroxidase-reaction product. Extra-mitochondrial staining was not observed. The post-embedding immunogold method was employed to overcome penetration problems and allow semiquantitative analysis of localization and specificity. The highest densities of gold particles were found over the mitochondria in astroglial cell elements (including the Bergmann glia). Mitochondria in cell bodies of Bergmann glia had a lower particle density than those in astrocytic processes. In the latter, analysis of frequency distribution revealed no evidence of a population of mitochondria lacking glutamate dehydrogenase, but suggested the presence of populations with different levels of immunoreactivity. Comparison with the labelling of embedded bovine liver glutamate dehydrogenase indicated that the enzyme constitutes a high proportion (10%) of the total matrix protein of these mitochondria. A weaker but significant labelling was found in oligodendrocytes of the white matter. The labelling of mitochondria in neuronal elements including glutamatergic mossy fibre terminals was of the order of 15% of that in astroglial mitochondria. No difference was detected between glutamatergic neurons (mossy and parallel fibres, granular cells) and non-glutamatergic neurons (Purkinje cells). The particle density over non-mitochondrial areas was very close to background over empty resin. The results, obtained with different methods of tissue and antibody preparation, agree to show that the present form of glutamate dehydrogenase is restricted to mitochondria and preferentially localized in astrocytes.

[Study of the quaternary structure of glutamate carboxylase from Escherichia coli]. / Issledovanie chetvertichnoi struktury glutamatdekarboksilazy iz Escherichia coli.

It was shown by electron microscopy, that the native molecule of glutamate decarboxylase is a hexamer with dihedral symmetry; the subunits are situated at the apices of an octahedron. Apoenzyme at pH 6.0 is dissociated form. It were found s20.w - 12.8 +/- 0.54S and 5.51 +/- 0.43S for the native hexamer and a dissociated form, respectively. By column gel-filtration the molecular mass of the dissociated form was estimated as 105-106 kDa, this value corresponds to a dimer. There were 10 buried SH-groups per subunit in the hexamer, after dimer formation 8 of them became accessible. The reversible hexamer-dimer dissociation depends on pH and PLP. The pH dependences of the enzyme dissociation and activity are very similar. In the result of adding of 6 PLP equivalents to the dimers the reactivation and hexamer assembly were reached, the SH-groups burying preceded both these reactions. Effect of pH and PLP on the quaternary structure is known for some other PLP-enzymes. It may be the additional proof for the idea of a common ancestor for PLP-enzymes.

Conformational flexibility in glutamate dehydrogenase. Role of water in substrate recognition and catalysis.

We have solved the structure of the binary complex of the glutamate dehydrogenase from Clostridium symbiosum with glutamate to 1.9 A resolution. In this complex, the glutamate side-chain lies in a pocket on the enzyme surface and a key determinant of the enzymic specificity is an interaction of the substrate gamma-carboxyl group with the amino group of Lys89. In the apo-enzyme, Lys113 from the catalytic domain forms an important hydrogen bond to Asn373, in the NAD(+)-binding domain. On glutamate binding, the side-chain of this lysine undergoes a significant movement in order to optimize its hydrogen bonding to the alpha-carboxyl group of the substrate. Despite this shift, the interaction between Lys113 and Asn373 is maintained by a large-scale conformational change that closes the cleft between the two domains. Modelling studies indicate that in this "closed" conformation the C-4 of the nicotinamide ring and the alpha-carbon atom of the amino acid substrate are poised for efficient hydride transfer. Examination of the structure has led to a proposal for the catalytic activity of the enzyme, which involves Asp165 as a general base, and an enzyme-bound water molecule, hydrogen-bonded to an uncharged lysine residue, Lys125, as an attacking nucleophile in the reaction.

Crystallization of the NADP(+)-dependent glutamate dehydrogenase from Escherichia coli.

The NADP(+)-dependent hexameric glutamate dehydrogenase from Escherichia coli has been crystallized as the apo-enzyme and also in the presence of its substrates 2-oxoglutarate, glutamate or NADP+, using either pulsed equilibrium microdialysis, or the hanging drop method of vapour diffusion. Three non-isomorphous, but related, crystal forms have been obtained, all of which belong to the orthorhombic system and are most likely to be in space group P2(1)2(1)2(1). One crystal form is grown from ammonium sulphate, includes the apoenzyme and the binary complexes with 2-oxoglutarate or NADP+, and has cell dimensions a = 157.5 A, b = 212.5 A, c = 101.0 A with a hexamer in the asymmetric unit. Crystallizations using glutamate as the precipitant produced two further crystal forms, which show significant changes in the b and c cell dimensions with respect to the apo-enzyme crystals, with parameters a = 160.0 A, b = 217.5 A c = 92.4 A and a = 160.0 A, b = 223.0 A c = 92.4 A, respectively. X-ray diffraction photographs taken with synchrotron radiation show measurable reflections to beyond 3.0 A resolution.

Effect of additives on the crystallization of glutamate dehydrogenase from Clostridium symbiosum. Evidence for a ligand-induced conformational change.

A new crystal form of the hexameric NAD(+)-linked glutamate dehydrogenase (GDH) from Clostridium symbiosum has been grown using the hanging drop method of vapour diffusion. The crystals are obtained either by using high concentrations of the amino acid substrate of the enzyme, glutamate, as the precipitant or by co-crystallization from ammonium sulphate in the presence of either p-chloromercuribenzene sulphonate or potassium tetracyanoplatinate. The crystals diffract well and X-ray photographs have established that they are in the space group R32. Considerations of the values of Vm indicate that the asymmetric unit of the R32 crystals contains a single subunit. Packing considerations based on the structure of the native enzyme determined from a different crystal form suggest that the molecule must undergo a significant conformational change in order to be accommodated in the new cell. Such a conformational rearrangement may represent an important step in the catalytic cycle.