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. W...

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Main Authors:	Borgnia, Mario J., Banerjee, Soojay, Merk, Alan, Matthies, Doreen, Bartesaghi, Alberto, Rao, Prashant, Pierson, Jason, Earl, Lesley A., Falconieri, Veronica, Subramaniam, Sriram, Milne, Jacqueline L. S.
Format:	Online
Language:	English
Published:	The American Society for Pharmacology and Experimental Therapeutics 2016
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4885502/

id	pubmed-4885502
recordtype	oai_dc
spelling	pubmed-48855022016-06-08 Using Cryo-EM to Map Small Ligands on Dynamic Metabolic Enzymes: Studies with Glutamate Dehydrogenase Borgnia, Mario J. Banerjee, Soojay Merk, Alan Matthies, Doreen Bartesaghi, Alberto Rao, Prashant Pierson, Jason Earl, Lesley A. Falconieri, Veronica Subramaniam, Sriram Milne, Jacqueline L. S. Accelerated Communication 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. The American Society for Pharmacology and Experimental Therapeutics 2016-06 2016-06 /pmc/articles/PMC4885502/ /pubmed/27036132 http://dx.doi.org/10.1124/mol.116.103382 Text en U.S. Government work not protected by U.S. copyright
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Borgnia, Mario J. Banerjee, Soojay Merk, Alan Matthies, Doreen Bartesaghi, Alberto Rao, Prashant Pierson, Jason Earl, Lesley A. Falconieri, Veronica Subramaniam, Sriram Milne, Jacqueline L. S.
spellingShingle	Borgnia, Mario J. Banerjee, Soojay Merk, Alan Matthies, Doreen Bartesaghi, Alberto Rao, Prashant Pierson, Jason Earl, Lesley A. Falconieri, Veronica Subramaniam, Sriram Milne, Jacqueline L. S. Using Cryo-EM to Map Small Ligands on Dynamic Metabolic Enzymes: Studies with Glutamate Dehydrogenase
author_facet	Borgnia, Mario J. Banerjee, Soojay Merk, Alan Matthies, Doreen Bartesaghi, Alberto Rao, Prashant Pierson, Jason Earl, Lesley A. Falconieri, Veronica Subramaniam, Sriram Milne, Jacqueline L. S.
author_sort	Borgnia, Mario J.
title	Using Cryo-EM to Map Small Ligands on Dynamic Metabolic Enzymes: Studies with Glutamate Dehydrogenase
title_short	Using Cryo-EM to Map Small Ligands on Dynamic Metabolic Enzymes: Studies with Glutamate Dehydrogenase
title_full	Using Cryo-EM to Map Small Ligands on Dynamic Metabolic Enzymes: Studies with Glutamate Dehydrogenase
title_fullStr	Using Cryo-EM to Map Small Ligands on Dynamic Metabolic Enzymes: Studies with Glutamate Dehydrogenase
title_full_unstemmed	Using Cryo-EM to Map Small Ligands on Dynamic Metabolic Enzymes: Studies with Glutamate Dehydrogenase
title_sort	using cryo-em to map small ligands on dynamic metabolic enzymes: studies with glutamate dehydrogenase
description	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.
publisher	The American Society for Pharmacology and Experimental Therapeutics
publishDate	2016
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4885502/
_version_	1613586150997360640

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

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