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

Oide, Mao; Kato, Takayuki; Oroguchi, Tomotaka; Nakasako, Masayoshi

doi:10.1111/febs.15224

Cited by 36 publications

(49 citation statements)

References 72 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Nonetheless, the conformational energy landscape proposed for T. profundus GDH is unbiased by crystal contacts that might favor some conformations more than others. On the other hand, flash-cooling may introduce other types of bias, as the authors acknowledge (Oide et al, 2020). It is also important to note that the conformational dynamic of T. profundus GDH was observed in the absence of NAD(P) or substrate/product.…”

Section: The Dynamics Of the Coenzyme Binding Domain IImentioning

confidence: 92%

See 1 more Smart Citation

Structural Studies of Glutamate Dehydrogenase (Isoform 1) From Arabidopsis thaliana, an Important Enzyme at the Branch-Point Between Carbon and Nitrogen Metabolism

et al. 2020

View full text Add to dashboard Cite

Glutamate dehydrogenase (GDH) releases ammonia in a reversible NAD(P) +-dependent oxidative deamination of glutamate that yields 2-oxoglutarate (2OG). In current perception, GDH contributes to Glu homeostasis and plays a significant role at the junction of carbon and nitrogen assimilation pathways. GDHs are members of a superfamily of ELFV (Glu/Leu/Phe/Val) amino acid dehydrogenases and are subdivided into three subclasses, based on coenzyme specificity: NAD +-specific, NAD + /NADP + dual-specific, and NADP +-specific. We determined in this work that the mitochondrial AtGDH1 isozyme from A. thaliana is NAD +-specific. Altogether, A. thaliana expresses three GDH isozymes (AtGDH1-3) targeted to mitochondria, of which AtGDH2 has an extra EF-hand motif and is stimulated by calcium. Our enzymatic assays of AtGDH1 established that its sensitivity to calcium is negligible. In vivo the AtGDH1-3 enzymes form homo-and heterohexamers of varied composition. We solved the crystal structure of recombinant AtGDH1 in the apo-form and in complex with NAD + at 2.59 and 2.03 Å resolution, respectively. We demonstrate also that both in the apo form and in 1:1 complex with NAD + , it forms D 3-symmetric homohexamers. A subunit of AtGDH1 consists of domain I, which is involved in hexamer formation and substrate binding, and of domain II which binds coenzyme. Most of the subunits in our crystal structures, including those in NAD + complex, are in open conformation, with domain II forming a large (albeit variable) angle with domain I. One of the subunits of the AtGDH1-NAD + hexamer contains a serendipitous 2OG molecule in the active site, causing a dramatic (∼25 •) closure of the domains. We provide convincing evidence that the N-terminal peptide preceding domain I is a mitochondrial targeting signal, with a predicted cleavage site for mitochondrial processing peptidase (MPP) at Leu17-Leu18 that is followed by an unexpected potassium coordination site (Ser27, Ile30). We also identified several MPD [(+/-)-2-methyl-2,4-pentanediol] binding sites with conserved sequence. Although AtGDH1 is insensitive to MPD in our assays, the observation of druggable sites opens a potential for non-competitive herbicide design.

show abstract

Section: The Dynamics Of the Coenzyme Binding Domain IImentioning

confidence: 92%

“…A very recent cryo electron microscopy (cryo-EM) study of Thermococcus profundus GDH further highlighted the dynamics of the GDH machine (Oide et al, 2020). The authors found a broad range of domain II conformations.…”

Section: The Dynamics Of the Coenzyme Binding Domain IImentioning

confidence: 99%

Structural Studies of Glutamate Dehydrogenase (Isoform 1) From Arabidopsis thaliana, an Important Enzyme at the Branch-Point Between Carbon and Nitrogen Metabolism

et al. 2020

View full text Add to dashboard Cite

show abstract

“…Recently, we began cryogenic TEM observations of phot2 [ 43 ]. Cryogenic TEM can classify images of heterogeneous structures of proteins caused by chemical modification, intrinsic molecular motions, and/or BL-induced conformational changes [ 78 ]. However, the stabilization buffer of phot2 used in SAXS experiments is unsuitable for cryogenic TEM observation of frozen-hydrated phot2 because of the high concentrations of NaCl and glycerol.…”

Section: Discussionmentioning

confidence: 99%

Domain Organization in Plant Blue-Light Receptor Phototropin2 of Arabidopsis thaliana Studied by Small-Angle X-ray Scattering

Nakasako

Oide

Takayama

et al. 2020

IJMS

Self Cite

View full text Add to dashboard Cite

Phototropin2 (phot2) is a blue-light (BL) receptor protein that regulates the BL-dependent activities of plants for efficient photosynthesis. Phot2 is composed of two light-oxygen-voltage sensing domains (LOV1 and LOV2) to absorb BL, and a kinase domain. Photo-activated LOV domains, especially LOV2, play a major role in photo-dependent increase in the phosphorylation activity of the kinase domain. The atomic details of the overall structure of phot2 and the intramolecular mechanism to convert BL energy to a phosphorylation signal remain unknown. We performed structural studies on the LOV fragments LOV1, LOV2, LOV2-linker, and LOV2-kinase, and full-length phot2, using small-angle X-ray scattering (SAXS). The aim of the study was to understand structural changes under BL irradiation and discuss the molecular mechanism that enhance the phosphorylation activity under BL. SAXS is a suitable technique for visualizing molecular structures of proteins in solution at low resolution and is advantageous for monitoring their structural changes in the presence of external physical and/or chemical stimuli. Structural parameters and molecular models of the recombinant specimens were obtained from SAXS profiles in the dark, under BL irradiation, and after dark reversion. LOV1, LOV2, and LOV2-linker fragments displayed minimal structural changes. However, BL-induced rearrangements of functional domains were noted for LOV2-kinase and full-length phot2. Based on the molecular model together with the absorption measurements and biochemical assays, we discuss the intramolecular interactions and domain motions necessary for BL-enhanced phosphorylation activity of phot2.

show abstract

“…While the above achievements deserve praise, the full potential of electron microscopy as a technique remains veiled. EM can image multiple states of a protein simultaneously, which can be resolved and placed in an energetic landscape [16,17]. More importantly, sample preparation allows a broad range of specimens to be imaged, allowing for the structural determination of proteins from natural sources.…”

Section: Of 14mentioning

confidence: 99%

Coming of Age: Cryo-Electron Tomography as a Versatile Tool to Generate High-Resolution Structures at Cellular/Biological Interfaces

Wang

Zhang

Mim

2021

IJMS

View full text Add to dashboard Cite

Over the last few years, cryo electron microscopy has become the most important method in structural biology. While 80% of deposited maps are from single particle analysis, electron tomography has grown to become the second most important method. In particular sub-tomogram averaging has matured as a method, delivering structures between 2 and 5 Å from complexes in cells as well as in vitro complexes. While this resolution range is not standard, novel developments point toward a promising future. Here, we provide a guide for the workflow from sample to structure to gain insight into this emerging field.

show abstract

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

Cited by 36 publications

References 72 publications

Structural Studies of Glutamate Dehydrogenase (Isoform 1) From Arabidopsis thaliana, an Important Enzyme at the Branch-Point Between Carbon and Nitrogen Metabolism

Structural Studies of Glutamate Dehydrogenase (Isoform 1) From Arabidopsis thaliana, an Important Enzyme at the Branch-Point Between Carbon and Nitrogen Metabolism

Domain Organization in Plant Blue-Light Receptor Phototropin2 of Arabidopsis thaliana Studied by Small-Angle X-ray Scattering

Coming of Age: Cryo-Electron Tomography as a Versatile Tool to Generate High-Resolution Structures at Cellular/Biological Interfaces

Contact Info

Product

Resources

About