PMC 20201220 pmc.key 4885502 NO-CC CODE no 0 0 Cryo-EM Studies with Glutamate Dehydrogenase 10.1124/mol.116.103382 4885502 27036132 MOL_103382 645 6 651 surname:Borgnia;given-names:Mario J. surname:Banerjee;given-names:Soojay surname:Milne;given-names:Jacqueline L. S. surname:Merk;given-names:Alan surname:Matthies;given-names:Doreen surname:Bartesaghi;given-names:Alberto surname:Rao;given-names:Prashant surname:Pierson;given-names:Jason surname:Earl;given-names:Lesley A. surname:Falconieri;given-names:Veronica surname:Subramaniam;given-names:Sriram TITLE front 89 2016 0 Using Cryo-EM to Map Small Ligands on Dynamic Metabolic Enzymes: Studies with Glutamate Dehydrogenase 0.9987944 experimental_method cleaner0 2023-07-27T15:28:14Z MESH: Cryo-EM 0.9987963 protein_type cleaner0 2023-07-27T15:32:06Z MESH: Glutamate Dehydrogenase ABSTRACT abstract 102 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. 0.9989001 experimental_method cleaner0 2023-07-27T15:28:19Z MESH: Cryo-electron microscopy 0.99874353 experimental_method cleaner0 2023-07-27T15:28:14Z MESH: cryo-EM 0.99726605 evidence cleaner0 2023-07-27T16:13:01Z DUMMY: structures 0.9116105 protein_type cleaner0 2023-07-27T15:32:06Z MESH: glutamate dehydrogenase 0.8540359 protein_type cleaner0 2023-07-27T15:32:11Z MESH: GDH 0.9758364 chemical cleaner0 2023-07-27T15:28:57Z CHEBI: glutamate 0.8643728 protein_type cleaner0 2023-07-27T15:32:11Z MESH: GDH 0.9988768 experimental_method cleaner0 2023-07-27T15:28:14Z MESH: cryo-EM 0.99851555 evidence cleaner0 2023-07-27T16:13:04Z DUMMY: structures 0.9978859 protein_type cleaner0 2023-07-27T15:32:11Z MESH: GDH 0.9963707 evidence cleaner0 2023-07-27T16:13:08Z DUMMY: crystal structures 0.999298 chemical cleaner0 2023-07-27T15:28:52Z CHEBI: NADH 0.9992781 chemical cleaner0 2023-07-27T15:29:01Z CHEBI: GTP 0.9991974 protein_state cleaner0 2023-07-27T15:28:41Z DUMMY: open 0.9992416 protein_state cleaner0 2023-07-27T15:28:46Z DUMMY: closed 0.99849856 evidence cleaner0 2023-07-27T16:13:11Z DUMMY: structure 0.9992193 chemical cleaner0 2023-07-27T15:28:52Z CHEBI: NADH 0.9989946 site cleaner0 2023-07-27T15:36:23Z SO: active site 0.9992312 protein_state cleaner0 2023-07-27T15:28:41Z DUMMY: open 0.99926895 protein_state cleaner0 2023-07-27T15:28:46Z DUMMY: closed 0.9990865 site cleaner0 2023-07-27T15:38:43Z SO: regulatory site 0.99889517 experimental_method cleaner0 2023-07-27T15:28:24Z MESH: X-ray crystallography 0.9988362 experimental_method cleaner0 2023-07-27T15:28:14Z MESH: cryo-EM INTRO title_1 1392 Introduction INTRO paragraph 1405 Recent advances in cryo-electron microscopy (cryo-EM) allow determination of structures of small protein complexes and membrane proteins at near-atomic resolution, marking a critical shift in the structural biology field. One specific area of broad general interest in drug discovery is the localization of bound ligands and cofactors under conditions in which efforts at crystallization have not been successful because of structural heterogeneity. Recent cryo-EM analyses have already demonstrated that it is now possible to use single-particle cryo-EM methods to localize small bound ligands or inhibitors on target proteins. Whether ligand binding can be visualized at high resolution is an important question, even in the more general case when multiple conformations are present simultaneously. Here, we address this question using mammalian glutamate dehydrogenase as an example. 0.9989047 experimental_method cleaner0 2023-07-27T15:28:19Z MESH: cryo-electron microscopy 0.9988249 experimental_method cleaner0 2023-07-27T15:28:14Z MESH: cryo-EM 0.99750286 evidence cleaner0 2023-07-27T16:13:13Z DUMMY: structures 0.9772683 experimental_method cleaner0 2023-07-27T16:11:30Z MESH: crystallization 0.99887514 experimental_method cleaner0 2023-07-27T15:28:14Z MESH: cryo-EM 0.99884266 experimental_method cleaner0 2023-07-27T15:29:24Z MESH: single-particle cryo-EM 0.9985678 taxonomy_domain cleaner0 2023-07-27T16:07:56Z DUMMY: mammalian 0.99871063 protein_type cleaner0 2023-07-27T15:32:05Z MESH: glutamate dehydrogenase INTRO paragraph 2292 Glutamate dehydrogenase (GDH) is a highly conserved enzyme expressed in most organisms. GDH plays a central role in glutamate metabolism by catalyzing the reversible oxidative deamination of glutamate to generate α-ketoglutarate and ammonia, with the concomitant transfer of a pair of electrons to either NAD+ or NADP+. Regulation of GDH is tightly controlled through multiple allosteric mechanisms. Extensive biochemical and crystallographic studies have characterized the enzymatic activity of GDH and its modulation by a chemically diverse group of compounds such as nucleotides, amino acids, steroid hormones, antipsychotic drugs, and natural products. X-ray crystallographic studies have shown that the functional unit of GDH is a homohexamer composed of a trimer of dimers, with a 3-fold axis and an equatorial plane that define its D3 symmetry (Fig. 1A). Each 56-kDa protomer consists of three domains. The first is located near the dimer interface and forms the core of the hexamer. The second, a nucleotide-binding domain (NBD) with a Rossmann fold, defines one face of the catalytic cleft bounded by the core domain. During the catalytic cycle, the NBD executes a large movement, hinged around a “pivot” helix, that closes the catalytic cleft, and drives a large conformational change in the hexamer from open to closed states (Fig. 1B). The third domain, dubbed the “antenna,” is an evolutionary acquisition in protista and animals. Antennae of adjacent protomers in each trimer intercalate to form a bundle, perpendicular to the pivot helices, that protrudes along the distal extremes of the 3-fold axis. When a protomer undergoes a conformational change, the rotation of its pivot helix is transferred through the antenna to the adjacent subunit. The influence of the antenna, present only in protozoan and metazoan enzymes, has been proposed to explain its cooperative behavior, which is absent in bacterial homologs. Deletion of this domain leads to loss of cooperativity. 0.99880797 protein_type cleaner0 2023-07-27T15:32:06Z MESH: Glutamate dehydrogenase 0.99915147 protein_type cleaner0 2023-07-27T15:32:11Z MESH: GDH 0.9990763 protein_state cleaner0 2023-07-27T15:36:33Z DUMMY: highly conserved 0.9990645 protein_type cleaner0 2023-07-27T15:32:11Z MESH: GDH 0.9654128 chemical cleaner0 2023-07-27T15:28:57Z CHEBI: glutamate 0.995365 chemical cleaner0 2023-07-27T15:28:57Z CHEBI: glutamate 0.99907213 chemical cleaner0 2023-07-27T15:32:16Z CHEBI: α-ketoglutarate 0.99903846 chemical cleaner0 2023-07-27T15:32:21Z CHEBI: ammonia 0.99906206 chemical cleaner0 2023-07-27T15:32:26Z CHEBI: NAD+ 0.9990859 chemical cleaner0 2023-07-27T15:32:31Z CHEBI: NADP+ 0.99913496 protein_type cleaner0 2023-07-27T15:32:11Z MESH: GDH 0.9988078 experimental_method cleaner0 2023-07-27T16:11:33Z MESH: biochemical and crystallographic studies 0.99890316 protein_type cleaner0 2023-07-27T15:32:11Z MESH: GDH 0.99894965 experimental_method cleaner0 2023-07-27T16:11:36Z MESH: X-ray crystallographic studies 0.985707 protein_type cleaner0 2023-07-27T15:32:11Z MESH: GDH 0.9984865 oligomeric_state cleaner0 2023-07-27T15:35:47Z DUMMY: homohexamer 0.9985979 oligomeric_state cleaner0 2023-07-27T15:35:50Z DUMMY: trimer 0.99865305 oligomeric_state cleaner0 2023-07-27T15:35:59Z DUMMY: dimers 0.99866486 oligomeric_state cleaner0 2023-07-27T15:35:55Z DUMMY: protomer 0.99872315 site cleaner0 2023-07-27T15:35:07Z SO: dimer interface 0.99862754 oligomeric_state cleaner0 2023-07-27T15:35:43Z DUMMY: hexamer 0.9992577 structure_element cleaner0 2023-07-27T15:34:50Z SO: nucleotide-binding domain 0.9992562 structure_element cleaner0 2023-07-27T15:34:54Z SO: NBD 0.9680246 structure_element cleaner0 2023-07-27T15:35:00Z SO: Rossmann fold 0.99891114 site cleaner0 2023-07-27T15:35:10Z SO: catalytic cleft 0.9994492 structure_element cleaner0 2023-07-27T15:34:55Z SO: NBD structure_element SO: cleaner0 2023-07-27T15:36:59Z “pivot” helix 0.9986644 site cleaner0 2023-07-27T15:35:11Z SO: catalytic cleft 0.9985611 oligomeric_state cleaner0 2023-07-27T15:35:44Z DUMMY: hexamer 0.9992925 protein_state cleaner0 2023-07-27T15:28:41Z DUMMY: open 0.99928766 protein_state cleaner0 2023-07-27T15:28:46Z DUMMY: closed 0.9992238 structure_element cleaner0 2023-07-27T15:38:09Z SO: antenna 0.99862075 taxonomy_domain cleaner0 2023-07-27T15:35:16Z DUMMY: protista 0.9982576 taxonomy_domain cleaner0 2023-07-27T15:35:22Z DUMMY: animals 0.9992933 structure_element cleaner0 2023-07-27T15:38:13Z SO: Antennae 0.99870396 oligomeric_state cleaner0 2023-07-27T16:06:37Z DUMMY: protomers 0.9986652 oligomeric_state cleaner0 2023-07-27T15:35:51Z DUMMY: trimer 0.998791 structure_element cleaner0 2023-07-27T15:37:50Z SO: pivot helices 0.9987753 oligomeric_state cleaner0 2023-07-27T15:35:55Z DUMMY: protomer 0.99898434 structure_element cleaner0 2023-07-27T15:37:54Z SO: pivot helix 0.9989784 structure_element cleaner0 2023-07-27T15:38:09Z SO: antenna 0.80029213 structure_element cleaner0 2023-07-27T16:06:57Z SO: subunit 0.99908864 structure_element cleaner0 2023-07-27T15:38:09Z SO: antenna 0.99850154 taxonomy_domain cleaner0 2023-07-27T15:35:27Z DUMMY: protozoan 0.99777126 taxonomy_domain cleaner0 2023-07-27T15:35:30Z DUMMY: metazoan 0.9988238 taxonomy_domain cleaner0 2023-07-27T15:35:34Z DUMMY: bacterial mol.116.103382f1.jpg F1 FIG fig_caption 4289 Structure and quaternary conformational changes in GDH. (A) Views of open (PDB ID 1NR7) and closed (PDB 3MW9) states of the GDH hexamer, shown in ribbon representation perpendicular to the 2-fold symmetry axis (side view, top) and 3-fold symmetry axis (top view, bottom). Only three protomers are shown in the top view for purposes of visual clarity. The dashed lines and arrows, respectively, highlight the slight extension in length, and twist in shape that occurs with transition from open to the closed state. The open state shown is for unliganded GDH, whereas the closed state has NADH, GTP, and glutamate bound. (B) Superposition of structures for closed and open conformations, along with a series of possible intermediate conformations along the trajectory that serve to illustrate the extent of change in structure across different regions of the protein. 0.718332 protein_type cleaner0 2023-07-27T15:32:11Z MESH: GDH 0.99922514 protein_state cleaner0 2023-07-27T15:28:41Z DUMMY: open 0.99928147 protein_state cleaner0 2023-07-27T15:28:46Z DUMMY: closed 0.89957345 protein_type cleaner0 2023-07-27T15:32:11Z MESH: GDH 0.99871457 oligomeric_state cleaner0 2023-07-27T15:35:44Z DUMMY: hexamer 0.9986778 oligomeric_state cleaner0 2023-07-27T16:07:04Z DUMMY: protomers 0.99914074 protein_state cleaner0 2023-07-27T15:28:41Z DUMMY: open 0.99912757 protein_state cleaner0 2023-07-27T15:28:46Z DUMMY: closed 0.9990472 protein_state cleaner0 2023-07-27T15:28:41Z DUMMY: open 0.9991609 protein_state cleaner0 2023-07-27T15:45:16Z DUMMY: unliganded 0.9177118 protein_type cleaner0 2023-07-27T15:32:11Z MESH: GDH 0.99912745 protein_state cleaner0 2023-07-27T15:28:46Z DUMMY: closed 0.99924785 chemical cleaner0 2023-07-27T15:28:52Z CHEBI: NADH 0.9992212 chemical cleaner0 2023-07-27T15:29:02Z CHEBI: GTP 0.9989837 chemical cleaner0 2023-07-27T15:28:57Z CHEBI: glutamate 0.9989303 protein_state cleaner0 2023-07-27T16:10:36Z DUMMY: bound 0.9985043 experimental_method cleaner0 2023-07-27T16:11:58Z MESH: Superposition 0.99673766 evidence cleaner0 2023-07-27T16:13:19Z DUMMY: structures 0.99926716 protein_state cleaner0 2023-07-27T15:28:46Z DUMMY: closed 0.9992894 protein_state cleaner0 2023-07-27T15:28:41Z DUMMY: open INTRO paragraph 5155 The transition between “closed” and “open” states of GDH is modulated by two allosteric sites in each protomer (Fig. 1A), which are differentially bound by GTP (an inhibitor) and ADP (an activator). These allosteric modulators tightly control GDH function in vivo. In the first site, which sits next to the pivot helix at the base of the antenna (the “GTP binding site”), GTP binding is known to act as an inhibitor, preventing release of the reaction product from the catalytic site by stabilizing the closed conformation of the catalytic cleft. In the second “regulatory site”, which is situated near the pivot helix between adjacent protomers, ADP acts as an activator of enzymatic activity, presumably by hastening the opening of the catalytic cleft that leads to the release of the reaction product. Interestingly, it has also been shown that the coenzyme NADH can bind to the regulatory site (also bound by the activator ADP), exerting a converse, inhibitory effect on GDH product release, although the role this may play in vivo is not entirely clear. 0.99919957 protein_state cleaner0 2023-07-27T15:28:46Z DUMMY: closed 0.99915993 protein_state cleaner0 2023-07-27T15:28:41Z DUMMY: open 0.99916553 protein_type cleaner0 2023-07-27T15:32:11Z MESH: GDH 0.99809766 site cleaner0 2023-07-27T15:38:47Z SO: allosteric sites 0.99857914 oligomeric_state cleaner0 2023-07-27T15:35:55Z DUMMY: protomer 0.91330147 protein_state cleaner0 2023-07-27T15:39:07Z DUMMY: bound by 0.9992711 chemical cleaner0 2023-07-27T15:29:02Z CHEBI: GTP 0.99927336 chemical cleaner0 2023-07-27T15:38:53Z CHEBI: ADP 0.9991462 protein_type cleaner0 2023-07-27T15:32:11Z MESH: GDH 0.9991275 structure_element cleaner0 2023-07-27T15:37:54Z SO: pivot helix 0.9787057 structure_element cleaner0 2023-07-27T15:38:09Z SO: antenna 0.9988017 site cleaner0 2023-07-27T15:38:36Z SO: GTP binding site 0.99916077 chemical cleaner0 2023-07-27T15:29:02Z CHEBI: GTP 0.99892986 site cleaner0 2023-07-27T16:09:05Z SO: catalytic site 0.99922884 protein_state cleaner0 2023-07-27T15:28:46Z DUMMY: closed 0.9989816 site cleaner0 2023-07-27T15:35:11Z SO: catalytic cleft 0.9832761 site cleaner0 2023-07-27T15:38:43Z SO: regulatory site 0.9990891 structure_element cleaner0 2023-07-27T15:37:54Z SO: pivot helix 0.998329 oligomeric_state cleaner0 2023-07-27T16:07:29Z DUMMY: protomers 0.9992016 chemical cleaner0 2023-07-27T15:38:54Z CHEBI: ADP 0.99899286 site cleaner0 2023-07-27T15:35:11Z SO: catalytic cleft 0.99929297 chemical cleaner0 2023-07-27T15:28:52Z CHEBI: NADH 0.9987782 site cleaner0 2023-07-27T15:38:43Z SO: regulatory site 0.9827585 protein_state cleaner0 2023-07-27T15:39:07Z DUMMY: bound by 0.99925977 chemical cleaner0 2023-07-27T15:38:54Z CHEBI: ADP 0.99798226 protein_type cleaner0 2023-07-27T15:32:11Z MESH: GDH INTRO paragraph 6230 Although there are numerous crystal structures available for GDH in complex with cofactors and nucleotides, they are limited to the combinations that have been amenable to crystallization. Nearly all X-ray structures of mammalian GDH are in the closed conformation, and the few structures that are in the open conformation are at lower resolution (Table 1). Of those structures in the closed conformation, most include NAD[P]H, GTP, and glutamate (or, alternately, NAD+, GTP, and α-ketoglutarate). However, the effects of coenzyme and GTP, bound alone or in concert in the absence of glutamate, have not been analyzed by crystallographic methods. Here, we report single-particle cryo-electron microscopy (cryo-EM) studies that show that under these conditions enzyme complexes coexist in both closed and open conformations. We show that the structures in both states can be resolved at near-atomic resolution, suggesting a molecular mechanism for synergistic inhibition of GDH by NADH and GTP (see Table 2 for detailed information on all cryo-EM-derived structures that we report in this work). 0.99880433 evidence cleaner0 2023-07-27T16:13:08Z DUMMY: crystal structures 0.9965873 protein_type cleaner0 2023-07-27T15:32:11Z MESH: GDH 0.99895054 protein_state cleaner0 2023-07-27T15:39:14Z DUMMY: in complex with 0.99693906 experimental_method cleaner0 2023-07-27T16:12:03Z MESH: crystallization experimental_method MESH: cleaner0 2023-07-27T16:13:37Z X-ray evidence DUMMY: cleaner0 2023-07-27T16:13:46Z structures 0.9984755 taxonomy_domain cleaner0 2023-07-27T16:08:02Z DUMMY: mammalian 0.983615 protein_type cleaner0 2023-07-27T15:32:11Z MESH: GDH 0.9992649 protein_state cleaner0 2023-07-27T15:28:46Z DUMMY: closed 0.9981117 evidence cleaner0 2023-07-27T16:13:50Z DUMMY: structures 0.9992205 protein_state cleaner0 2023-07-27T15:28:41Z DUMMY: open 0.9985306 evidence cleaner0 2023-07-27T16:13:53Z DUMMY: structures 0.99927074 protein_state cleaner0 2023-07-27T15:28:47Z DUMMY: closed 0.9990926 chemical cleaner0 2023-07-27T15:39:35Z CHEBI: NAD[P]H 0.9991685 chemical cleaner0 2023-07-27T15:29:02Z CHEBI: GTP 0.99859256 chemical cleaner0 2023-07-27T15:28:57Z CHEBI: glutamate 0.9992113 chemical cleaner0 2023-07-27T15:39:31Z CHEBI: NAD+ 0.9992071 chemical cleaner0 2023-07-27T15:29:02Z CHEBI: GTP 0.99916536 chemical cleaner0 2023-07-27T15:32:17Z CHEBI: α-ketoglutarate 0.99878365 chemical cleaner0 2023-07-27T15:29:02Z CHEBI: GTP 0.99912345 protein_state cleaner0 2023-07-27T16:10:45Z DUMMY: bound alone 0.99913263 protein_state cleaner0 2023-07-27T16:10:54Z DUMMY: absence of 0.9949268 chemical cleaner0 2023-07-27T15:28:57Z CHEBI: glutamate 0.99887645 experimental_method cleaner0 2023-07-27T15:39:25Z MESH: single-particle cryo-electron microscopy 0.99877906 experimental_method cleaner0 2023-07-27T15:28:14Z MESH: cryo-EM 0.9992718 protein_state cleaner0 2023-07-27T15:28:47Z DUMMY: closed 0.9992467 protein_state cleaner0 2023-07-27T15:28:41Z DUMMY: open 0.99840206 evidence cleaner0 2023-07-27T16:13:56Z DUMMY: structures 0.99060386 protein_type cleaner0 2023-07-27T15:32:11Z MESH: GDH 0.99922943 chemical cleaner0 2023-07-27T15:28:52Z CHEBI: NADH 0.99920636 chemical cleaner0 2023-07-27T15:29:02Z CHEBI: GTP 0.99880105 experimental_method cleaner0 2023-07-27T15:28:14Z MESH: cryo-EM 0.9982753 evidence cleaner0 2023-07-27T16:13:58Z DUMMY: structures T1.xml T1 TABLE table_caption 7329 X-ray structures of mammalian GDH reported in both the open and closed conformations experimental_method MESH: cleaner0 2023-07-27T16:14:11Z X-ray evidence DUMMY: cleaner0 2023-07-27T16:14:24Z structures 0.99856323 taxonomy_domain cleaner0 2023-07-27T16:08:06Z DUMMY: mammalian 0.9988951 protein_type cleaner0 2023-07-27T15:32:11Z MESH: GDH 0.9992053 protein_state cleaner0 2023-07-27T15:28:41Z DUMMY: open 0.9992336 protein_state cleaner0 2023-07-27T15:28:47Z DUMMY: closed T1.xml T1 TABLE table <?xml version="1.0" encoding="UTF-8"?> <table frame="hsides" rules="groups"><col width="17.06%" span="1"/><col width="33.34%" span="1"/><col width="13.13%" span="1"/><col width="20.31%" span="1"/><col width="16.16%" span="1"/><thead><tr><th valign="top" align="center" scope="col" rowspan="1" colspan="1">GDH</th><th valign="top" align="center" scope="col" rowspan="1" colspan="1">Ligands</th><th valign="top" align="center" scope="col" rowspan="1" colspan="1">PDB ID</th><th valign="top" align="center" scope="col" rowspan="1" colspan="1">Conformation</th><th valign="top" align="center" scope="col" rowspan="1" colspan="1">Resolution</th></tr></thead><tbody><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">WT</td><td valign="top" align="left" rowspan="1" colspan="1">NADH + GLU + GTP</td><td valign="top" align="left" rowspan="1" colspan="1">3MW9</td><td valign="top" align="left" rowspan="1" colspan="1">Closed</td><td valign="top" align="center" rowspan="1" colspan="1">2.4</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">WT</td><td valign="top" align="left" rowspan="1" colspan="1">Glu, GTP, NADPH, and Bithionol</td><td valign="top" align="left" rowspan="1" colspan="1">3ETD</td><td valign="top" align="left" rowspan="1" colspan="1">Closed</td><td valign="top" align="center" rowspan="1" colspan="1">2.5</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">WT</td><td valign="top" align="left" rowspan="1" colspan="1">Glu, NADPH, GTP + GW5074</td><td valign="top" align="left" rowspan="1" colspan="1">3ETG</td><td valign="top" align="left" rowspan="1" colspan="1">Closed</td><td valign="top" align="center" rowspan="1" colspan="1">2.5</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">WT</td><td valign="top" align="left" rowspan="1" colspan="1">apo</td><td valign="top" align="left" rowspan="1" colspan="1">1L1F</td><td valign="top" align="left" rowspan="1" colspan="1">Open</td><td valign="top" align="center" rowspan="1" colspan="1">2.7</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">WT</td><td valign="top" align="left" rowspan="1" colspan="1">NADPH, glutamate, and GTP</td><td valign="top" align="left" rowspan="1" colspan="1">1HWZ</td><td valign="top" align="left" rowspan="1" colspan="1">Closed</td><td valign="top" align="center" rowspan="1" colspan="1">2.8</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">WT</td><td valign="top" align="left" rowspan="1" colspan="1">NADPH + GLU + GTP + Zinc</td><td valign="top" align="left" rowspan="1" colspan="1">3MVQ</td><td valign="top" align="left" rowspan="1" colspan="1">Closed</td><td valign="top" align="center" rowspan="1" colspan="1">2.94</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">WT</td><td valign="top" align="left" rowspan="1" colspan="1">NADPH, Glu, GTP, Hexachlorophene</td><td valign="top" align="left" rowspan="1" colspan="1">3ETE</td><td valign="top" align="left" rowspan="1" colspan="1">Closed</td><td valign="top" align="center" rowspan="1" colspan="1">3</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">WT</td><td valign="top" align="left" rowspan="1" colspan="1">NAD, PO4, and 2-oxoglutarate</td><td valign="top" align="left" rowspan="1" colspan="1">1HWY</td><td valign="top" align="left" rowspan="1" colspan="1">Closed</td><td valign="top" align="center" rowspan="1" colspan="1">3.2</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">WT</td><td valign="top" align="left" rowspan="1" colspan="1">NADPH + GLU + Eu</td><td valign="top" align="left" rowspan="1" colspan="1">3MVO</td><td valign="top" align="left" rowspan="1" colspan="1">Closed</td><td valign="top" align="center" rowspan="1" colspan="1">3.23</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">R463A mutant</td><td valign="top" align="left" rowspan="1" colspan="1">apo</td><td valign="top" align="left" rowspan="1" colspan="1">1NR1</td><td valign="top" align="left" rowspan="1" colspan="1">Open</td><td valign="top" align="center" rowspan="1" colspan="1">3.3</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">WT</td><td valign="top" align="left" rowspan="1" colspan="1">apo</td><td valign="top" align="left" rowspan="1" colspan="1">1NR7</td><td valign="top" align="left" rowspan="1" colspan="1">Open</td><td valign="top" align="center" rowspan="1" colspan="1">3.3</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">WT</td><td valign="top" align="left" rowspan="1" colspan="1">ADP</td><td valign="top" align="left" rowspan="1" colspan="1">1NQT</td><td valign="top" align="left" rowspan="1" colspan="1">Open</td><td valign="top" align="center" rowspan="1" colspan="1">3.5</td></tr><tr><td valign="top" align="left" scope="row" rowspan="1" colspan="1">WT</td><td valign="top" align="left" rowspan="1" colspan="1">NADPH and Epicatechin-3-gallate (Ecg)</td><td valign="top" align="left" rowspan="1" colspan="1">3QMU</td><td valign="top" align="left" rowspan="1" colspan="1">Open</td><td valign="top" align="center" rowspan="1" colspan="1">3.62</td></tr></tbody></table> 7414 GDH Ligands PDB ID Conformation Resolution WT NADH + GLU + GTP 3MW9 Closed 2.4 WT Glu, GTP, NADPH, and Bithionol 3ETD Closed 2.5 WT Glu, NADPH, GTP + GW5074 3ETG Closed 2.5 WT apo 1L1F Open 2.7 WT NADPH, glutamate, and GTP 1HWZ Closed 2.8 WT NADPH + GLU + GTP + Zinc 3MVQ Closed 2.94 WT NADPH, Glu, GTP, Hexachlorophene 3ETE Closed 3 WT NAD, PO4, and 2-oxoglutarate 1HWY Closed 3.2 WT NADPH + GLU + Eu 3MVO Closed 3.23 R463A mutant apo 1NR1 Open 3.3 WT apo 1NR7 Open 3.3 WT ADP 1NQT Open 3.5 WT NADPH and Epicatechin-3-gallate (Ecg) 3QMU Open 3.62 protein PR: cleaner0 2023-07-27T16:14:40Z GDH protein_state DUMMY: cleaner0 2023-07-27T15:40:17Z WT chemical CHEBI: cleaner0 2023-07-27T15:40:36Z NADH chemical CHEBI: cleaner0 2023-07-27T15:44:08Z GLU chemical CHEBI: cleaner0 2023-07-27T15:40:42Z GTP protein_state DUMMY: cleaner0 2023-07-27T15:40:30Z Closed protein_state DUMMY: cleaner0 2023-07-27T15:40:17Z WT chemical CHEBI: cleaner0 2023-07-27T15:44:08Z Glu chemical CHEBI: cleaner0 2023-07-27T15:40:42Z GTP chemical CHEBI: cleaner0 2023-07-27T15:40:57Z NADPH protein_state DUMMY: cleaner0 2023-07-27T15:40:30Z Closed protein_state DUMMY: cleaner0 2023-07-27T15:40:17Z WT chemical CHEBI: cleaner0 2023-07-27T15:44:07Z Glu chemical CHEBI: cleaner0 2023-07-27T15:40:57Z NADPH chemical CHEBI: cleaner0 2023-07-27T15:40:42Z GTP protein_state DUMMY: cleaner0 2023-07-27T15:40:30Z Closed protein_state DUMMY: cleaner0 2023-07-27T15:40:17Z WT protein_state DUMMY: cleaner0 2023-07-27T15:41:05Z apo protein_state DUMMY: cleaner0 2023-07-27T15:40:26Z Open protein_state DUMMY: cleaner0 2023-07-27T15:40:17Z WT chemical CHEBI: cleaner0 2023-07-27T15:40:57Z NADPH chemical CHEBI: cleaner0 2023-07-27T15:43:30Z glutamate chemical CHEBI: cleaner0 2023-07-27T15:40:42Z GTP protein_state DUMMY: cleaner0 2023-07-27T15:40:30Z Closed protein_state DUMMY: cleaner0 2023-07-27T15:40:17Z WT chemical CHEBI: cleaner0 2023-07-27T15:40:57Z NADPH chemical CHEBI: cleaner0 2023-07-27T15:44:08Z GLU chemical CHEBI: cleaner0 2023-07-27T15:40:42Z GTP chemical CHEBI: cleaner0 2023-07-27T15:43:06Z Zinc protein_state DUMMY: cleaner0 2023-07-27T15:40:30Z Closed protein_state DUMMY: cleaner0 2023-07-27T15:40:17Z WT chemical CHEBI: cleaner0 2023-07-27T15:40:57Z NADPH chemical CHEBI: cleaner0 2023-07-27T15:44:08Z Glu chemical CHEBI: cleaner0 2023-07-27T15:40:42Z GTP protein_state DUMMY: cleaner0 2023-07-27T15:40:30Z Closed protein_state DUMMY: cleaner0 2023-07-27T15:40:17Z WT chemical CHEBI: cleaner0 2023-07-27T15:41:33Z NAD chemical CHEBI: cleaner0 2023-07-27T15:41:44Z PO4 chemical CHEBI: cleaner0 2023-07-27T15:42:32Z 2-oxoglutarate protein_state DUMMY: cleaner0 2023-07-27T15:40:30Z Closed protein_state DUMMY: cleaner0 2023-07-27T15:40:17Z WT chemical CHEBI: cleaner0 2023-07-27T15:40:57Z NADPH chemical CHEBI: cleaner0 2023-07-27T15:44:08Z GLU protein_state DUMMY: cleaner0 2023-07-27T15:40:30Z Closed protein_state DUMMY: cleaner0 2023-07-27T15:41:21Z mutant protein_state DUMMY: cleaner0 2023-07-27T15:41:05Z apo protein_state DUMMY: cleaner0 2023-07-27T15:40:26Z Open protein_state DUMMY: cleaner0 2023-07-27T15:40:17Z WT protein_state DUMMY: cleaner0 2023-07-27T15:41:05Z apo protein_state DUMMY: cleaner0 2023-07-27T15:40:26Z Open protein_state DUMMY: cleaner0 2023-07-27T15:40:17Z WT chemical CHEBI: cleaner0 2023-07-27T15:42:21Z ADP protein_state DUMMY: cleaner0 2023-07-27T15:40:26Z Open protein_state DUMMY: cleaner0 2023-07-27T15:40:17Z WT chemical CHEBI: cleaner0 2023-07-27T15:40:57Z NADPH protein_state DUMMY: cleaner0 2023-07-27T15:40:26Z Open T2.xml T2 TABLE table_caption 7991 Cryo-EM structures of mammalian GDH determined for this study 0.9988289 experimental_method cleaner0 2023-07-27T15:28:14Z MESH: Cryo-EM 0.99778193 evidence cleaner0 2023-07-27T16:14:46Z DUMMY: structures 0.9982822 taxonomy_domain cleaner0 2023-07-27T16:08:08Z DUMMY: mammalian 0.9989058 protein_type cleaner0 2023-07-27T15:32:11Z MESH: GDH T2.xml T2 TABLE table <?xml version="1.0" encoding="UTF-8"?> <table frame="hsides" rules="groups"><col width="7.86%" span="1"/><col width="15.31%" span="1"/><col width="18.73%" span="1"/><col width="14.12%" span="1"/><col width="16.09%" span="1"/><col width="12.62%" span="1"/><col width="15.27%" span="1"/><thead><tr><th valign="top" align="center" scope="col" rowspan="1" colspan="1">GDH</th><th valign="top" align="center" scope="col" rowspan="1" colspan="1">Ligands</th><th valign="top" align="center" scope="col" rowspan="1" colspan="1">EMDB ID</th><th valign="top" align="center" scope="col" rowspan="1" colspan="1">PDB ID</th><th valign="top" align="center" scope="col" rowspan="1" colspan="1">Conformation</th><th valign="top" align="center" scope="col" rowspan="1" colspan="1">Resolution</th><th valign="top" align="center" scope="col" rowspan="1" colspan="1">Particles</th></tr></thead><tbody><tr><td valign="top" align="center" scope="row" rowspan="1" colspan="1">WT</td><td valign="top" align="left" rowspan="1" colspan="1">apo</td><td valign="top" align="left" rowspan="1" colspan="1">EMD-6630</td><td valign="top" align="center" rowspan="1" colspan="1">3JCZ</td><td valign="top" align="left" rowspan="1" colspan="1">Open</td><td valign="top" align="center" rowspan="1" colspan="1">3.26</td><td valign="top" align="center" rowspan="1" colspan="1">22462</td></tr><tr><td valign="top" align="center" scope="row" rowspan="1" colspan="1">WT</td><td valign="top" align="left" rowspan="1" colspan="1">GTP</td><td valign="top" align="left" rowspan="1" colspan="1">EMD-6631</td><td valign="top" align="center" rowspan="1" colspan="1">3JD0</td><td valign="top" align="left" rowspan="1" colspan="1">Open</td><td valign="top" align="center" rowspan="1" colspan="1">3.47</td><td valign="top" align="center" rowspan="1" colspan="1">39439</td></tr><tr><td valign="top" align="center" scope="row" rowspan="1" colspan="1">WT</td><td valign="top" align="left" rowspan="1" colspan="1">NADH</td><td valign="top" align="left" rowspan="1" colspan="1">EMD-6635</td><td valign="top" align="center" rowspan="1" colspan="1">3JD2</td><td valign="top" align="left" rowspan="1" colspan="1">Open</td><td valign="top" align="center" rowspan="1" colspan="1">3.27</td><td valign="top" align="center" rowspan="1" colspan="1">34716</td></tr><tr><td valign="top" align="center" scope="row" rowspan="1" colspan="1">WT</td><td valign="top" align="left" rowspan="1" colspan="1">NADH</td><td valign="top" align="left" rowspan="1" colspan="1">EMD-6634</td><td valign="top" align="center" rowspan="1" colspan="1">3JD1</td><td valign="top" align="left" rowspan="1" colspan="1">Closed</td><td valign="top" align="center" rowspan="1" colspan="1">3.27</td><td valign="top" align="center" rowspan="1" colspan="1">34926</td></tr><tr><td valign="top" align="center" scope="row" rowspan="1" colspan="1">WT</td><td valign="top" align="left" rowspan="1" colspan="1">NADH + GTP</td><td valign="top" align="left" rowspan="1" colspan="1">EMD-6632</td><td valign="top" align="center" rowspan="1" colspan="1">3JD3</td><td valign="top" align="left" rowspan="1" colspan="1">Open</td><td valign="top" align="center" rowspan="1" colspan="1">3.55</td><td valign="top" align="center" rowspan="1" colspan="1">14793</td></tr><tr><td valign="top" align="center" scope="row" rowspan="1" colspan="1">WT</td><td valign="top" align="left" rowspan="1" colspan="1">NADH + GTP</td><td valign="top" align="left" rowspan="1" colspan="1">EMD-6633</td><td valign="top" align="center" rowspan="1" colspan="1">3JD4</td><td valign="top" align="left" rowspan="1" colspan="1">Closed</td><td valign="top" align="center" rowspan="1" colspan="1">3.40</td><td valign="top" align="center" rowspan="1" colspan="1">20429</td></tr></tbody></table> 8053 GDH Ligands EMDB ID PDB ID Conformation Resolution Particles WT apo EMD-6630 3JCZ Open 3.26 22462 WT GTP EMD-6631 3JD0 Open 3.47 39439 WT NADH EMD-6635 3JD2 Open 3.27 34716 WT NADH EMD-6634 3JD1 Closed 3.27 34926 WT NADH + GTP EMD-6632 3JD3 Open 3.55 14793 WT NADH + GTP EMD-6633 3JD4 Closed 3.40 20429 0.98749566 protein_type cleaner0 2023-07-27T15:32:11Z MESH: GDH protein_state DUMMY: cleaner0 2023-07-27T15:40:17Z WT 0.9988914 protein_state cleaner0 2023-07-27T15:41:05Z DUMMY: apo 0.9940724 protein_state cleaner0 2023-07-27T15:28:41Z DUMMY: Open 0.99706405 protein_state cleaner0 2023-07-27T15:40:17Z DUMMY: WT 0.99901605 chemical cleaner0 2023-07-27T15:29:02Z CHEBI: GTP 0.9962424 protein_state cleaner0 2023-07-27T15:28:41Z DUMMY: Open 0.99640036 protein_state cleaner0 2023-07-27T15:40:17Z DUMMY: WT 0.9986166 chemical cleaner0 2023-07-27T15:28:52Z CHEBI: NADH 0.99558604 protein_state cleaner0 2023-07-27T15:28:41Z DUMMY: Open 0.9958423 protein_state cleaner0 2023-07-27T15:40:17Z DUMMY: WT 0.9985378 chemical cleaner0 2023-07-27T15:28:52Z CHEBI: NADH 0.9955289 protein_state cleaner0 2023-07-27T15:28:47Z DUMMY: Closed 0.99510795 protein_state cleaner0 2023-07-27T15:40:17Z DUMMY: WT 0.9025693 chemical cleaner0 2023-07-27T15:28:52Z CHEBI: NADH 0.6816098 chemical cleaner0 2023-07-27T15:29:02Z CHEBI: GTP 0.99488443 protein_state cleaner0 2023-07-27T15:28:41Z DUMMY: Open 0.9968978 protein_state cleaner0 2023-07-27T15:40:17Z DUMMY: WT 0.965978 chemical cleaner0 2023-07-27T15:28:52Z CHEBI: NADH 0.70165646 chemical cleaner0 2023-07-27T15:29:02Z CHEBI: GTP 0.9965994 protein_state cleaner0 2023-07-27T15:28:47Z DUMMY: Closed METHODS title_1 8371 Materials and Methods METHODS title_3 8393 Specimen Preparation. METHODS paragraph 8415 Bovine glutamate dehydrogenase (Enzyme Commission 1.4.1.3; Sigma-Aldrich/MilliporeSigma, St. Louis, MO) was dialyzed overnight against fractionation buffer (100 mM potassium phosphate, pH 6.8) prior to fractionation by size-exclusion chromatography using a Superdex 200 10/30 column connected to an ÄKTA FPLC apparatus (GE Healthcare Bio-Sciences, Piscataway, NJ ). The concentration of GDH was adjusted to ∼2 mg/ml by rapid mixing with potassium phosphate buffer containing the concentration of ligand as necessary and with n-octyl glucopyranoside at a final concentration of 0.1%. The final concentration of each ligand was 20 mM. Small volumes of sample, typically 3 µl, were deposited on 200 mesh Quantifoil R2/2 grids (Quantifoil Micro Tools, Großlöbichaum, Germany), blotted, and plunge-frozen in liquid ethane using an FEI Vitrobot Mark IV (FEI Company, Hillsboro, OR). Frozen grids were mounted into autoloader cartridges and transferred to the microscope. METHODS title_3 9386 Cryo-Electron Microscopy. METHODS paragraph 9412 Specimens were imaged on an FEI Titan Krios microscope (FEI Company) aligned for parallel illumination and operated at 300 kV. The instrument was furnished with a Gatan K2 Summit camera placed at the end of a GIF Quantum energy filter (Gatan Inc., Pleasanton, CA), operated in zero-energy-loss mode with a slit width of 20 eV. Images were collected manually at a dose rate of ∼5 e– pixel−1 s−1, i.e., in the linear range of the detector. The physical pixel size at the plane of the specimen was 1.275 Å, corresponding to a super-resolution pixel size of 0.6375 Å. The total exposure time was 15.2 s, and intermediate frames were recorded every 0.4 s, giving an accumulated dose of ∼45 e–/Å2 and a total of 38 frames per image. The majority of images were collected at under-focus values between 1 μm and 3 μm. METHODS title_3 10239 Data Processing. METHODS paragraph 10256 Drift and beam-induced motion were compensated by whole-frame alignment of movies, and CTF was estimated as described in. Integrated frames were manually examined and selected on the basis of the quality of the CTF estimation, astigmatism, drift, and particle distribution. Molecular images were automatically identified from selected integrated micrographs by detecting the local maxima of correlation of each image with a Gaussian disk of 150 Å in radius. Individual particle projections were extracted from integrated super-resolution images using a binning factor of 4 and a box size of 96 × 96 pixels and assigned into 20–100 groups by iterative reference free two-dimensional classification as implemented in EMAN2. Following 8 iterations of classification, a subset of classes depicting intact particles were used to build symmetric (D3) density maps using the program e2initialmodel.py from the EMAN2 suite. One or more maps were selected as reference for further processing on the basis of consistency between projections and the original classes. These “initial models” were refined to ∼15–20 Å using e2refine_easy.py. Unbinned particles were then re-extracted from the original super-resolution images using a binning of 2 and a box size of 384 × 384, and subject to classification in three dimensions using the maximum likelihood method implemented in RELION (; MRC Laboratory, Cambridge, UK) (regularization parameter of T=4). Unless noted otherwise, D3 symmetry was imposed for three-dimensional classification runs, the number of classes was initially determined on the basis of the number of particles included in the analysis and later adjusted on the basis of the number of conformations detected in the sample (see Supplemental Table 1 for details). Iteration over classification in three dimensions was continued until convergence as judged by resolution and distribution of particles among the classes. Particles belonging to “good” three-dimensional classes were pooled into one or more classes, depending on the conformational landscape of the complex, and refined using the “gold standard” method in RELION. The refined maps were corrected for the MTF of the camera and for B-factor in the framework of RELION. Figures were generated using UCSF Chimera and Maxon Cinema4D (Maxon Computer Inc., Newbury Park, CA), and two-dimensionally composited in Adobe Photoshop and Illustrator. METHODS title_3 12684 Building of Atomic Models. METHODS paragraph 12711 The deposited models for unliganded GDH (1NR7), the binary complex with ADP (1NQT), and the quaternary complex with NADH, GTP, and Glu (3MW9) were used to derive models from the six structures reported here. Conflicts in sequence between the deposited models were solved by conforming to the primary sequence as reported in 3MW9. The models were placed in the corresponding map (open or closed) by rigid body fitting as implemented in Chimera. In the closed state, in which all relative orientations of the ligands are known, only the ligands known to be present in each structure were retained, all other nonstandard residues were deleted. For the open state, ligands were initially placed on the basis of their orientation relative to the corresponding binding site. The models were refined against a map derived from one-half of the dataset using Rosetta as described (https://faculty.washington.edu/dimaio/files/density_tutorial.pdf), followed by real space refinement in PHENIX (; PHENIX, Berkeley, CA) and evaluated by calculating the Fourier shell correlation between the model and the map derived from the second half of the dataset. For each complex, ten best scoring instances on the basis of the Fourier shell correlation were selected among one hundred runs, visually examined, and the one deemed best interactively corrected in Coot. Each corrected model was subject to a final instance of real space refinement using PHENIX. RESULTS title_1 14150 Results and Discussion RESULTS paragraph 14173 To explore the conformational landscape of apo-GDH, we first determined its structure in the absence of any added ligands (Supplemental Fig. 1, Fig. 2, A–C). The density map, refined to an average resolution of ∼3.0 Å (Supplemental Fig. 2), is in the open conformation and closely matches the model of unliganded GDH derived by X-ray crystallography at 3.3 Å resolution (PDB ID 1NR7). The variation in local resolution from the core to the periphery, as reported by ResMap (Supplemental Fig. 3D), is consistent with the B-factor gradient observed in the crystal structure (Supplemental Fig. 3A). Extensive classification without imposing symmetry yielded only open structures and failed to detect any closed catalytic cleft in the unliganded enzyme, suggesting that all six protomers are in the open conformation. Consistent with this conclusion, the loops connecting the β-strands of the Rossmann fold are well-defined (Fig. 2B), implying that there is little movement at the NBD, as the transition between closed and open states is associated with NBD movement (Fig. 1B). 0.99936336 protein_state cleaner0 2023-07-27T15:41:05Z DUMMY: apo 0.99453545 protein cleaner0 2023-07-27T15:44:42Z PR: GDH 0.981202 evidence cleaner0 2023-07-27T16:14:51Z DUMMY: structure 0.99872416 protein_state cleaner0 2023-07-27T16:10:54Z DUMMY: absence of 0.9985163 evidence cleaner0 2023-07-27T16:14:54Z DUMMY: density map 0.9992907 protein_state cleaner0 2023-07-27T15:28:42Z DUMMY: open 0.99930954 protein_state cleaner0 2023-07-27T15:45:16Z DUMMY: unliganded 0.8116277 protein cleaner0 2023-07-27T15:44:50Z PR: GDH 0.9986926 experimental_method cleaner0 2023-07-27T15:28:24Z MESH: X-ray crystallography 0.9988036 experimental_method cleaner0 2023-07-27T16:12:09Z MESH: ResMap 0.845573 evidence cleaner0 2023-07-27T16:15:00Z DUMMY: B-factor gradient 0.9979139 evidence cleaner0 2023-07-27T16:15:07Z DUMMY: crystal structure 0.9992725 protein_state cleaner0 2023-07-27T15:28:42Z DUMMY: open 0.9983443 evidence cleaner0 2023-07-27T16:15:03Z DUMMY: structures 0.9992125 protein_state cleaner0 2023-07-27T15:28:47Z DUMMY: closed 0.99904025 site cleaner0 2023-07-27T15:35:11Z SO: catalytic cleft 0.999288 protein_state cleaner0 2023-07-27T15:45:16Z DUMMY: unliganded 0.997468 oligomeric_state cleaner0 2023-07-27T16:07:35Z DUMMY: protomers 0.99926406 protein_state cleaner0 2023-07-27T15:28:42Z DUMMY: open 0.9990702 structure_element cleaner0 2023-07-27T15:44:55Z SO: loops 0.9990347 structure_element cleaner0 2023-07-27T15:44:58Z SO: β-strands 0.99909985 structure_element cleaner0 2023-07-27T15:35:00Z SO: Rossmann fold 0.99953437 structure_element cleaner0 2023-07-27T15:34:55Z SO: NBD 0.99922514 protein_state cleaner0 2023-07-27T15:28:47Z DUMMY: closed 0.9991897 protein_state cleaner0 2023-07-27T15:28:42Z DUMMY: open 0.99954224 structure_element cleaner0 2023-07-27T15:34:55Z SO: NBD mol.116.103382f2.jpg F2 FIG fig_caption 15254 Cryo-EM structures of GDH in unliganded and NADH-bound states. (A) Refined cryo-EM map of unliganded GDH at ∼3 Å resolution. (B, C) Illustration of density map in the regions that contain the Rossmann nucleotide binding fold (B), pivot and antenna helices (C) in the unliganded GDH map. (D) Cryo-EM-derived density maps for two coexisting conformations that are present when GDH is bound to the cofactor NADH. Each protomer is shown in a different color and densities for NADH bound in both regulatory (red) and catalytic (purple) sites on one protomer are indicated. The overall quaternary structures of the two conformations are essentially the same as that of the open and closed states observed by X-ray crystallography. 0.9984913 experimental_method cleaner0 2023-07-27T15:28:14Z MESH: Cryo-EM 0.9980799 evidence cleaner0 2023-07-27T16:15:13Z DUMMY: structures 0.94350004 protein cleaner0 2023-07-27T16:15:45Z PR: GDH 0.99929535 protein_state cleaner0 2023-07-27T15:45:16Z DUMMY: unliganded 0.9991093 protein_state cleaner0 2023-07-27T15:45:12Z DUMMY: NADH-bound 0.9986468 experimental_method cleaner0 2023-07-27T15:28:14Z MESH: cryo-EM 0.998569 evidence cleaner0 2023-07-27T16:15:15Z DUMMY: map 0.9992817 protein_state cleaner0 2023-07-27T15:45:15Z DUMMY: unliganded 0.9956453 protein cleaner0 2023-07-27T16:15:53Z PR: GDH 0.9983629 evidence cleaner0 2023-07-27T16:15:17Z DUMMY: density map 0.9992018 structure_element cleaner0 2023-07-27T15:45:29Z SO: Rossmann nucleotide binding fold 0.9991868 structure_element cleaner0 2023-07-27T15:45:30Z SO: pivot and antenna helices 0.99929273 protein_state cleaner0 2023-07-27T15:45:16Z DUMMY: unliganded 0.9963735 protein cleaner0 2023-07-27T16:16:08Z PR: GDH 0.9970074 evidence cleaner0 2023-07-27T16:15:29Z DUMMY: map 0.9986481 experimental_method cleaner0 2023-07-27T15:28:14Z MESH: Cryo-EM 0.9985701 evidence cleaner0 2023-07-27T16:15:19Z DUMMY: density maps 0.99651164 protein cleaner0 2023-07-27T16:16:01Z PR: GDH 0.99913245 protein_state cleaner0 2023-07-27T15:45:34Z DUMMY: bound to 0.9992711 chemical cleaner0 2023-07-27T15:28:52Z CHEBI: NADH 0.998767 oligomeric_state cleaner0 2023-07-27T15:35:55Z DUMMY: protomer 0.9957604 evidence cleaner0 2023-07-27T16:15:21Z DUMMY: densities 0.99877113 chemical cleaner0 2023-07-27T15:28:52Z CHEBI: NADH 0.88400507 protein_state cleaner0 2023-07-27T15:45:39Z DUMMY: bound in 0.9983589 site cleaner0 2023-07-27T16:09:12Z SO: regulatory 0.9984016 site cleaner0 2023-07-27T16:09:16Z SO: catalytic 0.9940574 site cleaner0 2023-07-27T16:09:18Z SO: sites 0.99874145 oligomeric_state cleaner0 2023-07-27T15:35:55Z DUMMY: protomer 0.9992987 protein_state cleaner0 2023-07-27T15:28:42Z DUMMY: open 0.99929917 protein_state cleaner0 2023-07-27T15:28:47Z DUMMY: closed 0.99866635 experimental_method cleaner0 2023-07-27T15:28:24Z MESH: X-ray crystallography RESULTS paragraph 15982 When GDH is bound to NADH, GTP, and glutamate, the enzyme adopts a closed conformation; this “abortive complex” has been determined to 2.4-Å resolution by X-ray crystallography (PDB 3MW9). However, crystal structures of GDH bound only to NADH or to GTP have not yet been reported. To test the effect of NADH binding on GDH conformation in solution, we determined the structure of this binary complex using cryo-EM methods combined with three-dimensional classification. Two dominant conformational states, in an all open or all closed conformation were detected, segregated (Fig. 2D), and further refined to near-atomic resolution (∼3.3 Å; Supplemental Fig. 2). Densities for 12 molecules of bound NADH were identified in maps of both open and closed states (Supplemental Fig. 4). The NADH-bound closed conformation matches the structure of the quaternary complex observed by X-ray crystallography, with the exception that density corresponding to GTP and glutamate was absent in the cryo-EM-derived map. 0.96874183 protein cleaner0 2023-07-27T16:16:20Z PR: GDH 0.9990741 protein_state cleaner0 2023-07-27T15:45:35Z DUMMY: bound to 0.9990263 chemical cleaner0 2023-07-27T15:28:52Z CHEBI: NADH 0.998454 chemical cleaner0 2023-07-27T15:29:02Z CHEBI: GTP 0.9938263 chemical cleaner0 2023-07-27T15:28:57Z CHEBI: glutamate 0.9992925 protein_state cleaner0 2023-07-27T15:28:47Z DUMMY: closed 0.99878615 experimental_method cleaner0 2023-07-27T15:28:24Z MESH: X-ray crystallography 0.9983753 evidence cleaner0 2023-07-27T16:13:08Z DUMMY: crystal structures 0.9649907 protein cleaner0 2023-07-27T16:16:27Z PR: GDH 0.9990327 protein_state cleaner0 2023-07-27T15:45:45Z DUMMY: bound only to 0.9990496 chemical cleaner0 2023-07-27T15:28:52Z CHEBI: NADH 0.71146953 protein_state cleaner0 2023-07-27T15:45:51Z DUMMY: to 0.9990275 chemical cleaner0 2023-07-27T15:29:02Z CHEBI: GTP 0.99858487 chemical cleaner0 2023-07-27T15:28:52Z CHEBI: NADH 0.8397284 protein cleaner0 2023-07-27T16:16:34Z PR: GDH 0.996267 evidence cleaner0 2023-07-27T16:16:39Z DUMMY: structure 0.9987752 experimental_method cleaner0 2023-07-27T15:28:14Z MESH: cryo-EM 0.99817914 experimental_method cleaner0 2023-07-27T16:12:18Z MESH: three-dimensional classification protein_state DUMMY: cleaner0 2023-07-27T15:28:42Z open protein_state DUMMY: cleaner0 2023-07-27T15:28:47Z closed 0.9983753 evidence cleaner0 2023-07-27T16:16:42Z DUMMY: Densities 0.99764234 protein_state cleaner0 2023-07-27T15:46:04Z DUMMY: bound 0.99921286 chemical cleaner0 2023-07-27T15:28:52Z CHEBI: NADH 0.998577 evidence cleaner0 2023-07-27T16:16:44Z DUMMY: maps 0.999295 protein_state cleaner0 2023-07-27T15:28:42Z DUMMY: open 0.9992986 protein_state cleaner0 2023-07-27T15:28:47Z DUMMY: closed 0.99908704 protein_state cleaner0 2023-07-27T15:45:12Z DUMMY: NADH-bound 0.9989404 protein_state cleaner0 2023-07-27T15:28:47Z DUMMY: closed 0.9974841 evidence cleaner0 2023-07-27T16:16:46Z DUMMY: structure 0.9987444 experimental_method cleaner0 2023-07-27T15:28:24Z MESH: X-ray crystallography 0.9972446 evidence cleaner0 2023-07-27T16:16:48Z DUMMY: density 0.9980788 chemical cleaner0 2023-07-27T15:29:02Z CHEBI: GTP 0.99235004 chemical cleaner0 2023-07-27T15:28:57Z CHEBI: glutamate 0.99871415 experimental_method cleaner0 2023-07-27T15:28:14Z MESH: cryo-EM 0.99860364 evidence cleaner0 2023-07-27T16:16:51Z DUMMY: map RESULTS paragraph 16995 Comparison of the NADH-bound closed conformation to the NADH-bound open conformation shows that, as expected, the catalytic cleft is closed and the NBDs are displaced toward the equatorial plane, accompanied by a rotation of the pivot helix by ∼7°, concomitant with a large conformational change in the antennae domains (Figs. 1 and 2D). A comparison between NADH-bound open and closed conformations also involves a displacement of helix 5 (residues 171–186), as well as a tilt of the core β-sheets relative to the equatorial plane of the enzyme (residues 57–97, 122–130) and α-helix 2 (residues 36–54), and a bending of the N-terminal helix. Thus, closure of the catalytic cleft is accompanied by a quaternary structural change that can be described as a global bending of the structure about an axis that runs parallel to the pivot helix, accompanied by an expansion of the core (Figs. 1A and 2D). 0.9990828 protein_state cleaner0 2023-07-27T15:45:12Z DUMMY: NADH-bound 0.9991443 protein_state cleaner0 2023-07-27T15:28:47Z DUMMY: closed 0.9990894 protein_state cleaner0 2023-07-27T15:45:12Z DUMMY: NADH-bound 0.9990368 protein_state cleaner0 2023-07-27T15:28:42Z DUMMY: open 0.99876136 site cleaner0 2023-07-27T15:35:11Z SO: catalytic cleft 0.9992411 protein_state cleaner0 2023-07-27T15:28:47Z DUMMY: closed 0.99957377 structure_element cleaner0 2023-07-27T15:46:45Z SO: NBDs 0.99929595 structure_element cleaner0 2023-07-27T15:37:54Z SO: pivot helix structure_element SO: cleaner0 2023-07-27T15:38:14Z antennae 0.999078 protein_state cleaner0 2023-07-27T15:45:12Z DUMMY: NADH-bound 0.9991357 protein_state cleaner0 2023-07-27T15:28:42Z DUMMY: open 0.9993374 protein_state cleaner0 2023-07-27T15:28:47Z DUMMY: closed 0.9993145 structure_element cleaner0 2023-07-27T15:46:42Z SO: helix 5 0.9978593 residue_range cleaner0 2023-07-27T16:06:18Z DUMMY: 171–186 0.9982042 structure_element cleaner0 2023-07-27T15:46:35Z SO: β-sheets 0.99780947 residue_range cleaner0 2023-07-27T16:06:22Z DUMMY: 57–97 0.997643 residue_range cleaner0 2023-07-27T16:06:25Z DUMMY: 122–130 0.99933445 structure_element cleaner0 2023-07-27T15:46:40Z SO: α-helix 2 0.9977677 residue_range cleaner0 2023-07-27T16:06:28Z DUMMY: 36–54 0.9845567 structure_element cleaner0 2023-07-27T15:46:38Z SO: helix 0.99878156 site cleaner0 2023-07-27T15:35:11Z SO: catalytic cleft 0.9939615 evidence cleaner0 2023-07-27T16:16:57Z DUMMY: structure 0.9993472 structure_element cleaner0 2023-07-27T15:37:54Z SO: pivot helix RESULTS paragraph 17908 Detailed analysis of the GDH/NADH structures shows that both the adenosine and nicotinamide moieties of NADH bind to the catalytic site within the NBD in nearly the same orientation in both the open and the closed states, and display closely comparable interactions with the Rossmann fold (Fig. 3, A and B). At the regulatory site, where either ADP can bind as an activator or NADH can bind as an inhibitor, the binding of the adenine moiety of NADH is nearly identical between the two conformers. However, there is a significant difference in the orientation of the nicotinamide and phosphate moieties in the two conformational states (Fig. 3, C and D). In the closed state, the nicotinamide group is oriented toward the center of the hexamer, inserted into a narrow cavity between two adjacent subunits in the trimer. There are extensive interactions between NADH and the residues lining this cavity, which may explain the well-defined density of this portion of NADH in the closed state. In contrast, in the open conformation, the cavity present in the closed state becomes too narrow for the nicotinamide group; instead, the group is oriented in the opposite direction, parallel to the pivot helix with the amido group extending toward the C-terminal end of the helix. 0.9989229 complex_assembly cleaner0 2023-07-27T15:47:00Z GO: GDH/NADH 0.9978829 evidence cleaner0 2023-07-27T16:17:02Z DUMMY: structures 0.99905795 chemical cleaner0 2023-07-27T15:28:52Z CHEBI: NADH 0.99909294 site cleaner0 2023-07-27T16:09:23Z SO: catalytic site 0.99957985 structure_element cleaner0 2023-07-27T15:34:55Z SO: NBD 0.99925643 protein_state cleaner0 2023-07-27T15:28:42Z DUMMY: open 0.99927026 protein_state cleaner0 2023-07-27T15:28:47Z DUMMY: closed 0.9992144 structure_element cleaner0 2023-07-27T15:35:00Z SO: Rossmann fold 0.99911773 site cleaner0 2023-07-27T15:38:43Z SO: regulatory site 0.9991086 chemical cleaner0 2023-07-27T15:38:54Z CHEBI: ADP 0.99898523 chemical cleaner0 2023-07-27T15:28:52Z CHEBI: NADH 0.9990736 chemical cleaner0 2023-07-27T15:28:52Z CHEBI: NADH 0.99927324 protein_state cleaner0 2023-07-27T15:28:47Z DUMMY: closed 0.9986626 oligomeric_state cleaner0 2023-07-27T15:35:44Z DUMMY: hexamer 0.996176 site cleaner0 2023-07-27T16:09:28Z SO: cavity 0.99576217 structure_element cleaner0 2023-07-27T16:07:46Z SO: subunits 0.998681 oligomeric_state cleaner0 2023-07-27T15:35:51Z DUMMY: trimer 0.9987847 chemical cleaner0 2023-07-27T15:28:52Z CHEBI: NADH 0.9952844 site cleaner0 2023-07-27T16:09:30Z SO: cavity 0.99668294 evidence cleaner0 2023-07-27T16:17:11Z DUMMY: density 0.99878377 chemical cleaner0 2023-07-27T15:28:52Z CHEBI: NADH 0.99926203 protein_state cleaner0 2023-07-27T15:28:47Z DUMMY: closed 0.99923563 protein_state cleaner0 2023-07-27T15:28:42Z DUMMY: open 0.998295 site cleaner0 2023-07-27T16:09:32Z SO: cavity 0.9992661 protein_state cleaner0 2023-07-27T15:28:47Z DUMMY: closed 0.9983104 structure_element cleaner0 2023-07-27T15:37:54Z SO: pivot helix 0.9987325 structure_element cleaner0 2023-07-27T16:08:59Z SO: helix mol.116.103382f3.jpg F3 FIG fig_caption 19181 Detailed view of NADH conformation in catalytic and regulatory sites. (A, B) NADH density (purple) and interactions in the catalytic sites of closed (A) and open (B) states. (C, D) NADH density (red) and interactions in the regulatory sites of closed (C) and open (D) states. 0.9990859 chemical cleaner0 2023-07-27T15:28:52Z CHEBI: NADH 0.99858665 site cleaner0 2023-07-27T16:09:37Z SO: catalytic and regulatory sites 0.9988092 chemical cleaner0 2023-07-27T15:28:52Z CHEBI: NADH 0.9816166 evidence cleaner0 2023-07-27T16:17:15Z DUMMY: density 0.9990692 site cleaner0 2023-07-27T16:09:41Z SO: catalytic sites 0.9993414 protein_state cleaner0 2023-07-27T15:28:47Z DUMMY: closed 0.9993337 protein_state cleaner0 2023-07-27T15:28:42Z DUMMY: open 0.99882585 chemical cleaner0 2023-07-27T15:28:52Z CHEBI: NADH 0.98555994 evidence cleaner0 2023-07-27T16:17:17Z DUMMY: density 0.9990089 site cleaner0 2023-07-27T16:09:43Z SO: regulatory sites 0.99933225 protein_state cleaner0 2023-07-27T15:28:47Z DUMMY: closed 0.9993401 protein_state cleaner0 2023-07-27T15:28:42Z DUMMY: open RESULTS paragraph 19457 Although there is a difference in orientation of the nicotinamide moiety between the closed and open states in the regulatory site, in both structures the adenine portion of NADH has a similar binding pocket and is located in almost exactly the same position as ADP, a potent activator of GDH function (Supplemental Fig. 5). In the open state, the binding of ADP or NADH is further stabilized by His209, a residue that undergoes a large movement during the transition from open to closed conformation (Fig. 3, C and D). In the open conformation, the distance between His209 and the α-phosphate of NADH is ∼4.4 Å, which is comparable with the corresponding distance in the ADP-bound conformation. In the closed conformation, however, this key histidine residue is >10.5 Å away from the nearest phosphate group on NADH, altering a critical stabilization point within the regulatory site. This suggests that although the conformation of NADH in the open state regulatory site more closely mimics the binding of ADP, the conformation of NADH in the closed state regulatory site is significantly different; these differences may contribute to the opposite effects of NADH and ADP on GDH enzymatic activity. 0.99932384 protein_state cleaner0 2023-07-27T15:28:47Z DUMMY: closed 0.999311 protein_state cleaner0 2023-07-27T15:28:42Z DUMMY: open 0.99911374 site cleaner0 2023-07-27T15:38:43Z SO: regulatory site 0.9983247 evidence cleaner0 2023-07-27T16:17:21Z DUMMY: structures 0.99916303 chemical cleaner0 2023-07-27T15:28:52Z CHEBI: NADH 0.99900854 site cleaner0 2023-07-27T16:09:47Z SO: binding pocket 0.9990884 chemical cleaner0 2023-07-27T15:38:54Z CHEBI: ADP 0.9989911 protein cleaner0 2023-07-27T15:58:10Z PR: GDH 0.9992756 protein_state cleaner0 2023-07-27T15:28:42Z DUMMY: open 0.99897265 chemical cleaner0 2023-07-27T15:38:54Z CHEBI: ADP 0.9990429 chemical cleaner0 2023-07-27T15:28:52Z CHEBI: NADH 0.9995297 residue_name_number cleaner0 2023-07-27T16:00:21Z DUMMY: His209 0.99926263 protein_state cleaner0 2023-07-27T15:28:42Z DUMMY: open 0.999252 protein_state cleaner0 2023-07-27T15:28:47Z DUMMY: closed 0.99930274 protein_state cleaner0 2023-07-27T15:28:42Z DUMMY: open 0.9995384 residue_name_number cleaner0 2023-07-27T16:00:21Z DUMMY: His209 0.99889994 chemical cleaner0 2023-07-27T15:28:52Z CHEBI: NADH 0.9991041 protein_state cleaner0 2023-07-27T15:57:49Z DUMMY: ADP-bound 0.99930465 protein_state cleaner0 2023-07-27T15:28:47Z DUMMY: closed 0.9826623 residue_name cleaner0 2023-07-27T16:06:11Z SO: histidine 0.9988972 chemical cleaner0 2023-07-27T15:28:52Z CHEBI: NADH 0.99906135 site cleaner0 2023-07-27T15:38:43Z SO: regulatory site 0.9986971 chemical cleaner0 2023-07-27T15:28:52Z CHEBI: NADH 0.9992798 protein_state cleaner0 2023-07-27T15:28:42Z DUMMY: open 0.999044 site cleaner0 2023-07-27T15:38:43Z SO: regulatory site 0.9988551 chemical cleaner0 2023-07-27T15:38:54Z CHEBI: ADP 0.9987978 chemical cleaner0 2023-07-27T15:28:53Z CHEBI: NADH 0.999305 protein_state cleaner0 2023-07-27T15:28:47Z DUMMY: closed 0.99903715 site cleaner0 2023-07-27T15:38:43Z SO: regulatory site 0.998934 chemical cleaner0 2023-07-27T15:28:53Z CHEBI: NADH 0.99881065 chemical cleaner0 2023-07-27T15:38:54Z CHEBI: ADP 0.99901974 protein cleaner0 2023-07-27T15:58:21Z PR: GDH RESULTS paragraph 20664 In the absence of NADH, GTP binds weakly to GDH with a dissociation constant of ∼20 μM. Cryo-EM analysis of GDH incubated with GTP resulted in a structure at an overall resolution of 3.5 Å, showing that it is in an open conformation (Supplemental Fig. 6), with all NBDs in the open state. The density for GTP is not very well defined, suggesting considerable wobble in the binding site. Subtraction of the GTP-bound map with that of the apo state shows that GTP binding can nevertheless be visualized specifically in the GTP binding site (Supplemental Fig. 6). Importantly, the binding of GTP alone does not appear to drive the transition from the open to the closed state of GDH. 0.9991131 protein_state cleaner0 2023-07-27T16:10:54Z DUMMY: absence of 0.99894017 chemical cleaner0 2023-07-27T15:28:53Z CHEBI: NADH 0.99902225 chemical cleaner0 2023-07-27T15:29:02Z CHEBI: GTP 0.99219173 protein cleaner0 2023-07-27T15:58:55Z PR: GDH 0.9977647 evidence cleaner0 2023-07-27T16:17:25Z DUMMY: dissociation constant 0.9987583 experimental_method cleaner0 2023-07-27T15:28:14Z MESH: Cryo-EM 0.9932561 protein cleaner0 2023-07-27T15:59:03Z PR: GDH 0.8448001 protein_state cleaner0 2023-07-27T16:11:08Z DUMMY: incubated with 0.9988732 chemical cleaner0 2023-07-27T15:29:02Z CHEBI: GTP 0.99858713 evidence cleaner0 2023-07-27T16:17:28Z DUMMY: structure 0.99923015 protein_state cleaner0 2023-07-27T15:28:42Z DUMMY: open 0.99954456 structure_element cleaner0 2023-07-27T15:46:45Z SO: NBDs 0.99920124 protein_state cleaner0 2023-07-27T15:28:42Z DUMMY: open 0.99864095 evidence cleaner0 2023-07-27T16:17:30Z DUMMY: density 0.9989548 chemical cleaner0 2023-07-27T15:29:02Z CHEBI: GTP 0.9989122 site cleaner0 2023-07-27T16:09:54Z SO: binding site 0.9963775 experimental_method cleaner0 2023-07-27T16:12:27Z MESH: Subtraction 0.9990127 protein_state cleaner0 2023-07-27T15:58:35Z DUMMY: GTP-bound 0.99843186 evidence cleaner0 2023-07-27T16:17:32Z DUMMY: map 0.9993505 protein_state cleaner0 2023-07-27T15:41:05Z DUMMY: apo 0.9970625 chemical cleaner0 2023-07-27T15:29:02Z CHEBI: GTP 0.99904937 site cleaner0 2023-07-27T15:38:36Z SO: GTP binding site 0.9990978 chemical cleaner0 2023-07-27T15:29:02Z CHEBI: GTP 0.9992861 protein_state cleaner0 2023-07-27T15:28:42Z DUMMY: open 0.99928904 protein_state cleaner0 2023-07-27T15:28:47Z DUMMY: closed 0.9957417 protein cleaner0 2023-07-27T15:59:21Z PR: GDH RESULTS paragraph 21349 To further dissect the roles of NADH and GTP in the transition from the open to closed conformations, we next determined structures of GDH in complex with both NADH and GTP, but without glutamate. When NADH and GTP are both present, classification reveals the presence of both closed and open GDH conformations, similar to the condition when only NADH is present (Fig. 4, A and B). Reconstruction without classification, however, yields a structure clearly in the closed conformation, suggesting that, in coordination with NADH, GTP may further stabilize the closed conformation. The location of GTP in the open and closed states of the GDH/NADH/GTP complex is similar to that in the crystal structure observed in the presence of NADH, GTP, and glutamate. Likewise, the position of NADH in the open and closed states closely resembles the position of NADH in the GDH/NADH open and closed structures. One key difference between the open and closed states of these structures is the position of the His209 residue: As mentioned above, His209 swings away from the adenine moiety of NADH in the closed state. When GTP is present in the GTP binding site, His209 instead interacts with GTP, probably stabilizing the closed conformation (Fig. 4, C and D). Thus, GTP binding to GDH appears synergistic with NADH and displaces the conformational landscape toward the closed state. 0.9990538 chemical cleaner0 2023-07-27T15:28:53Z CHEBI: NADH 0.99909663 chemical cleaner0 2023-07-27T15:29:02Z CHEBI: GTP 0.9992446 protein_state cleaner0 2023-07-27T15:28:42Z DUMMY: open 0.99927586 protein_state cleaner0 2023-07-27T15:28:47Z DUMMY: closed 0.8281136 experimental_method cleaner0 2023-07-27T16:12:31Z MESH: determined 0.99192166 evidence cleaner0 2023-07-27T16:17:36Z DUMMY: structures 0.98979175 protein cleaner0 2023-07-27T15:59:30Z PR: GDH protein_state DUMMY: cleaner0 2023-07-27T15:39:15Z in complex with 0.99887913 chemical cleaner0 2023-07-27T15:28:53Z CHEBI: NADH 0.9989507 chemical cleaner0 2023-07-27T15:29:02Z CHEBI: GTP 0.9970987 protein_state cleaner0 2023-07-27T15:59:32Z DUMMY: without 0.6332602 chemical cleaner0 2023-07-27T15:28:57Z CHEBI: glutamate 0.99893636 chemical cleaner0 2023-07-27T15:28:53Z CHEBI: NADH 0.9988495 chemical cleaner0 2023-07-27T15:29:02Z CHEBI: GTP 0.995776 experimental_method cleaner0 2023-07-27T16:12:37Z MESH: classification protein_state DUMMY: cleaner0 2023-07-27T16:11:14Z presence of 0.9992207 protein_state cleaner0 2023-07-27T15:28:47Z DUMMY: closed 0.99923444 protein_state cleaner0 2023-07-27T15:28:42Z DUMMY: open 0.8307806 protein cleaner0 2023-07-27T16:00:04Z PR: GDH 0.99833554 chemical cleaner0 2023-07-27T15:28:53Z CHEBI: NADH 0.9285469 experimental_method cleaner0 2023-07-27T16:12:40Z MESH: Reconstruction without classification 0.9980426 evidence cleaner0 2023-07-27T16:17:38Z DUMMY: structure 0.9992361 protein_state cleaner0 2023-07-27T15:28:47Z DUMMY: closed 0.9989649 chemical cleaner0 2023-07-27T15:28:53Z CHEBI: NADH 0.99884725 chemical cleaner0 2023-07-27T15:29:02Z CHEBI: GTP 0.9992543 protein_state cleaner0 2023-07-27T15:28:47Z DUMMY: closed 0.99891984 chemical cleaner0 2023-07-27T15:29:02Z CHEBI: GTP 0.9992256 protein_state cleaner0 2023-07-27T15:28:42Z DUMMY: open 0.9992687 protein_state cleaner0 2023-07-27T15:28:47Z DUMMY: closed 0.9991662 complex_assembly cleaner0 2023-07-27T15:59:53Z GO: GDH/NADH/GTP 0.9985813 evidence cleaner0 2023-07-27T16:17:42Z DUMMY: crystal structure 0.9991308 protein_state cleaner0 2023-07-27T16:11:14Z DUMMY: presence of 0.9989949 chemical cleaner0 2023-07-27T15:28:53Z CHEBI: NADH 0.9986966 chemical cleaner0 2023-07-27T15:29:02Z CHEBI: GTP 0.95512336 chemical cleaner0 2023-07-27T15:28:57Z CHEBI: glutamate 0.9990609 chemical cleaner0 2023-07-27T15:28:53Z CHEBI: NADH 0.9992643 protein_state cleaner0 2023-07-27T15:28:42Z DUMMY: open 0.99928856 protein_state cleaner0 2023-07-27T15:28:47Z DUMMY: closed 0.9990212 chemical cleaner0 2023-07-27T15:28:53Z CHEBI: NADH 0.99880105 complex_assembly cleaner0 2023-07-27T15:47:00Z GO: GDH/NADH 0.9992926 protein_state cleaner0 2023-07-27T15:28:42Z DUMMY: open 0.9993414 protein_state cleaner0 2023-07-27T15:28:47Z DUMMY: closed 0.9984408 evidence cleaner0 2023-07-27T16:17:46Z DUMMY: structures 0.9992549 protein_state cleaner0 2023-07-27T15:28:42Z DUMMY: open 0.9992506 protein_state cleaner0 2023-07-27T15:28:47Z DUMMY: closed 0.99817526 evidence cleaner0 2023-07-27T16:17:49Z DUMMY: structures 0.999451 residue_name_number cleaner0 2023-07-27T16:00:21Z DUMMY: His209 0.99944216 residue_name_number cleaner0 2023-07-27T16:00:21Z DUMMY: His209 0.99909997 chemical cleaner0 2023-07-27T15:28:53Z CHEBI: NADH 0.99925214 protein_state cleaner0 2023-07-27T15:28:47Z DUMMY: closed 0.9987779 chemical cleaner0 2023-07-27T15:29:02Z CHEBI: GTP 0.99891776 site cleaner0 2023-07-27T15:38:36Z SO: GTP binding site 0.999456 residue_name_number cleaner0 2023-07-27T16:00:21Z DUMMY: His209 0.99750525 chemical cleaner0 2023-07-27T15:29:02Z CHEBI: GTP 0.9992095 protein_state cleaner0 2023-07-27T15:28:47Z DUMMY: closed 0.99874985 chemical cleaner0 2023-07-27T15:29:02Z CHEBI: GTP 0.9704605 protein cleaner0 2023-07-27T16:00:33Z PR: GDH 0.9989549 chemical cleaner0 2023-07-27T15:28:53Z CHEBI: NADH 0.99925166 protein_state cleaner0 2023-07-27T15:28:47Z DUMMY: closed mol.116.103382f4.jpg F4 FIG fig_caption 22721 Cryo-EM structure of GDH bound to both NADH and GTP. (A, B) Observation of co-existing open (A) and closed (B) conformations in the GDH-NADH-GTP ternary complex. Densities for GTP (yellow) as well as NADH bound to both catalytic (purple) and regulatory (red) sites in each protomer are shown. (C, D) Detailed inspection of the interactions near the regulatory site show that the orientation of His209 switches between the two states, which may allow interactions with bound GTP in the closed (D), but not open (C) conformation. 0.99880934 experimental_method cleaner0 2023-07-27T15:28:14Z MESH: Cryo-EM 0.9970041 evidence cleaner0 2023-07-27T16:17:54Z DUMMY: structure 0.66216755 protein cleaner0 2023-07-27T16:01:08Z PR: GDH 0.9991266 protein_state cleaner0 2023-07-27T15:45:35Z DUMMY: bound to 0.9992132 chemical cleaner0 2023-07-27T15:28:53Z CHEBI: NADH 0.999258 chemical cleaner0 2023-07-27T15:29:02Z CHEBI: GTP 0.9993253 protein_state cleaner0 2023-07-27T15:28:42Z DUMMY: open 0.99934715 protein_state cleaner0 2023-07-27T15:28:47Z DUMMY: closed 0.9992854 complex_assembly cleaner0 2023-07-27T16:00:47Z GO: GDH-NADH-GTP 0.99802315 evidence cleaner0 2023-07-27T16:17:56Z DUMMY: Densities 0.99926347 chemical cleaner0 2023-07-27T15:29:02Z CHEBI: GTP 0.9991916 chemical cleaner0 2023-07-27T15:28:53Z CHEBI: NADH 0.9990061 protein_state cleaner0 2023-07-27T15:45:35Z DUMMY: bound to 0.99908483 site cleaner0 2023-07-27T16:10:01Z SO: catalytic 0.99892175 site cleaner0 2023-07-27T16:10:03Z SO: regulatory 0.99871826 site cleaner0 2023-07-27T16:10:05Z SO: sites 0.99875784 oligomeric_state cleaner0 2023-07-27T15:35:55Z DUMMY: protomer 0.9990251 site cleaner0 2023-07-27T15:38:43Z SO: regulatory site 0.9995421 residue_name_number cleaner0 2023-07-27T16:00:21Z DUMMY: His209 0.9986702 protein_state cleaner0 2023-07-27T16:11:19Z DUMMY: bound 0.9990889 chemical cleaner0 2023-07-27T15:29:02Z CHEBI: GTP 0.99931157 protein_state cleaner0 2023-07-27T15:28:47Z DUMMY: closed 0.99916506 protein_state cleaner0 2023-07-27T15:28:42Z DUMMY: open RESULTS paragraph 23249 Our structural studies thus establish that whether or not GTP is bound, NADH binding is detectable at catalytic and regulatory sites, in both the open and closed conformational states. Whereas 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 (Figs. 3 and 4). In the closed state, the nicotinamide moiety is inserted into a well-defined cavity at the interface between two adjacent protomers in the trimer. As mentioned above, 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 provide a potential explanation of the weaker density for the nicotinamide moiety of NADH in the open state, and may account for the higher reported affinity of NADH for the closed state. The role of the nicotinamide moiety in acting as a wedge that prevents the transition to the open conformation also suggests a structural explanation of the mechanism by which NADH binding inhibits the activity of the enzyme by stabilizing the closed conformation state. 0.9984971 experimental_method cleaner0 2023-07-27T16:12:47Z MESH: structural studies 0.9985751 chemical cleaner0 2023-07-27T15:29:02Z CHEBI: GTP 0.9976909 protein_state cleaner0 2023-07-27T16:11:23Z DUMMY: bound 0.99857664 chemical cleaner0 2023-07-27T15:28:53Z CHEBI: NADH 0.99882007 site cleaner0 2023-07-27T16:10:09Z SO: catalytic and regulatory sites 0.9992549 protein_state cleaner0 2023-07-27T15:28:42Z DUMMY: open 0.9992772 protein_state cleaner0 2023-07-27T15:28:47Z DUMMY: closed 0.99857247 chemical cleaner0 2023-07-27T15:28:53Z CHEBI: NADH 0.99903965 site cleaner0 2023-07-27T16:10:13Z SO: catalytic site 0.99883884 chemical cleaner0 2023-07-27T15:28:53Z CHEBI: NADH 0.9990834 site cleaner0 2023-07-27T15:38:43Z SO: regulatory site 0.9992575 protein_state cleaner0 2023-07-27T15:28:42Z DUMMY: open 0.9992855 protein_state cleaner0 2023-07-27T15:28:47Z DUMMY: closed 0.9992781 protein_state cleaner0 2023-07-27T15:28:47Z DUMMY: closed 0.96565443 site cleaner0 2023-07-27T16:10:17Z SO: cavity 0.9969772 site cleaner0 2023-07-27T16:10:20Z SO: interface 0.9985948 oligomeric_state cleaner0 2023-07-27T16:07:50Z DUMMY: protomers 0.9988117 oligomeric_state cleaner0 2023-07-27T15:35:51Z DUMMY: trimer 0.99401975 site cleaner0 2023-07-27T16:10:23Z SO: cavity 0.9992507 protein_state cleaner0 2023-07-27T15:28:42Z DUMMY: open 0.975312 site cleaner0 2023-07-27T16:10:25Z SO: cavity 0.9984908 chemical cleaner0 2023-07-27T15:28:53Z CHEBI: NADH 0.99924904 protein_state cleaner0 2023-07-27T15:28:42Z DUMMY: open 0.9779223 evidence cleaner0 2023-07-27T16:18:00Z DUMMY: density 0.9989441 chemical cleaner0 2023-07-27T15:28:53Z CHEBI: NADH 0.99927527 protein_state cleaner0 2023-07-27T15:28:42Z DUMMY: open 0.998892 chemical cleaner0 2023-07-27T15:28:53Z CHEBI: NADH 0.9992582 protein_state cleaner0 2023-07-27T15:28:47Z DUMMY: closed 0.9988876 protein_state cleaner0 2023-07-27T15:28:42Z DUMMY: open 0.99809366 chemical cleaner0 2023-07-27T15:28:53Z CHEBI: NADH 0.9991322 protein_state cleaner0 2023-07-27T15:28:47Z DUMMY: closed RESULTS paragraph 24506 The rapid emergence of cryo-EM as a tool for near-atomic resolution structure determination provides new opportunities for complementing atomic resolution information from X-ray crystallography, as illustrated here with GDH. Perhaps the most important contribution of these methods is the prospect that when there are discrete subpopulations present, the structure of each state can be determined at near-atomic resolution. What we demonstrate here with GDH is that by employing three-dimensional image classification approaches, we not only can isolate distinct, coexisting conformations, but we can also localize small molecule ligands in each of these conformations. These kinds of approaches will probably become increasingly important in molecular pharmacology, especially in the context of better understanding drug-target interactions in dynamic protein complexes. 0.99890405 experimental_method cleaner0 2023-07-27T15:28:14Z MESH: cryo-EM 0.83094627 experimental_method cleaner0 2023-07-27T16:12:51Z MESH: structure determination 0.998904 experimental_method cleaner0 2023-07-27T15:28:25Z MESH: X-ray crystallography 0.79175967 protein cleaner0 2023-07-27T16:01:19Z PR: GDH 0.9978859 evidence cleaner0 2023-07-27T16:18:02Z DUMMY: structure 0.9639614 protein cleaner0 2023-07-27T16:01:27Z PR: GDH 0.98971945 experimental_method cleaner0 2023-07-27T16:12:55Z MESH: three-dimensional image classification approaches SUPPL title_1 25378 Supplementary Material SUPPL footnote 25401 This research was supported by funds from the National Cancer Institute Center for Cancer Research, the IATAP program at NIH, and the NIH-FEI Living Laboratory for Structural Biology (S.S., J.L.S.M.). This work was supported by the Intramural Research Program of the National Institutes of Health National Cancer Institute. SUPPL footnote 25726 dx.doi.org/10.1124/mol.116.103382. SUPPL footnote 25761 This article has supplemental material available at molpharm.aspetjournals.org. ABBR title 25841 Abbreviations ABBR paragraph 25855 cryo-EM ABBR paragraph 25863 cryo-electron microscopy ABBR paragraph 25888 GDH ABBR paragraph 25892 glutamate dehydrogenase ABBR paragraph 25916 NBD ABBR paragraph 25920 nucleotide binding domain AUTH_CONT title 25946 Authorship Contributions AUTH_CONT paragraph 25971 Participated in research design: Borgnia, Banerjee, Merk, Subramaniam, Milne. AUTH_CONT paragraph 26049 Conducted experiments: Borgnia, Banerjee, Merk, Rao, Pierson. AUTH_CONT paragraph 26111 Performed data analysis: Borgnia, Banerjee, Merk, Matthies, Bartesaghi, Earl, Falconieri, Subramaniam, Milne. AUTH_CONT paragraph 26221 Wrote or contributed to the writing of the manuscript: Borgnia, Banerjee, Earl, Falconieri, Subramaniam, Milne. 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