title: Using Cryo-EM to Map Small Ligands on Dynamic Metabolic Enzymes: Studies with Glutamate Dehydrogenase.
abstract: 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.
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland (M.J.B., S.B., A.M., D.M., A.B., P.R., L.A.E., V.F., S.S., J.L.S.M.) and FEI Company, Hillsboro, Oregon (J.P.).
authors.forename: Mario J
authors.initials: MJ
authors.lastname: Borgnia
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland (M.J.B., S.B., A.M., D.M., A.B., P.R., L.A.E., V.F., S.S., J.L.S.M.) and FEI Company, Hillsboro, Oregon (J.P.).
authors.forename: Soojay
authors.initials: S
authors.lastname: Banerjee
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland (M.J.B., S.B., A.M., D.M., A.B., P.R., L.A.E., V.F., S.S., J.L.S.M.) and FEI Company, Hillsboro, Oregon (J.P.).
authors.forename: Alan
authors.initials: A
authors.lastname: Merk
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland (M.J.B., S.B., A.M., D.M., A.B., P.R., L.A.E., V.F., S.S., J.L.S.M.) and FEI Company, Hillsboro, Oregon (J.P.).
authors.forename: Doreen
authors.initials: D
authors.lastname: Matthies
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland (M.J.B., S.B., A.M., D.M., A.B., P.R., L.A.E., V.F., S.S., J.L.S.M.) and FEI Company, Hillsboro, Oregon (J.P.).
authors.forename: Alberto
authors.initials: A
authors.lastname: Bartesaghi
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland (M.J.B., S.B., A.M., D.M., A.B., P.R., L.A.E., V.F., S.S., J.L.S.M.) and FEI Company, Hillsboro, Oregon (J.P.).
authors.forename: Prashant
authors.initials: P
authors.lastname: Rao
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland (M.J.B., S.B., A.M., D.M., A.B., P.R., L.A.E., V.F., S.S., J.L.S.M.) and FEI Company, Hillsboro, Oregon (J.P.).
authors.forename: Jason
authors.initials: J
authors.lastname: Pierson
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland (M.J.B., S.B., A.M., D.M., A.B., P.R., L.A.E., V.F., S.S., J.L.S.M.) and FEI Company, Hillsboro, Oregon (J.P.).
authors.forename: Lesley A
authors.initials: LA
authors.lastname: Earl
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland (M.J.B., S.B., A.M., D.M., A.B., P.R., L.A.E., V.F., S.S., J.L.S.M.) and FEI Company, Hillsboro, Oregon (J.P.).
authors.forename: Veronica
authors.initials: V
authors.lastname: Falconieri
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland (M.J.B., S.B., A.M., D.M., A.B., P.R., L.A.E., V.F., S.S., J.L.S.M.) and FEI Company, Hillsboro, Oregon (J.P.) ss1@nih.gov jmilne@mail.nih.gov.
authors.forename: Sriram
authors.initials: S
authors.lastname: Subramaniam
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland (M.J.B., S.B., A.M., D.M., A.B., P.R., L.A.E., V.F., S.S., J.L.S.M.) and FEI Company, Hillsboro, Oregon (J.P.) ss1@nih.gov jmilne@mail.nih.gov.
authors.forename: Jacqueline L S
authors.initials: JL
authors.lastname: Milne
mesh_terms: D000818:Animals; D002417:Cattle; D020285:Cryoelectron Microscopy / Q000379:methods*; D018360:Crystallography, X-Ray; D005969:Glutamate Dehydrogenase / Q000737:chemistry / Q000648:ultrastructure*; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D008322:Mammals; D008958:Models, Molecular; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D020836:Protein Structure, Quaternary
chemical_list: D008024:Ligands; D009243:NAD; D006160:Guanosine Triphosphate; D005969:Glutamate Dehydrogenase