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Expression, purification, crystallization and preliminary structural characterization of the GTPase domain of human Rheb.
Acta Crystallogr D Biol Crystallogr. 2004 Oct; 60(Pt 10):1883-7.AC

Abstract

Ras homologue enriched in brain (Rheb) represents a unique group of small GTPases and shares moderate sequence identity with the Ras/Rap subfamily. It acts downstream of nutrient signalling as the direct target of the tuberous sclerosis complex (TSC) and upstream of mTOR/S6K1/4EBP in the insulin-signalling pathway. The GTPase domain of human Rheb (hRheb) has been recombinantly expressed in Escherichia coli, purified and cocrystallized in complexes with GDP, GTP and GppNHp using the hanging-drop vapour-diffusion method. Crystals of the hRheb-GDP complex belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 44.5, b = 52.3, c = 70.6 A. The hRheb-GppNHp complex crystallized in two crystal forms: one has the same space group and unit-cell parameters as the hRheb-GDP complex and the other belongs to space group C222(1), with unit-cell parameters a = 102.9, b = 99.2, c = 48.0 A. The hRheb-GTP complex also crystallized in two crystal forms: one belongs to space group C222(1), with unit-cell parameters a = 102.4, b = 98.3, c = 47.9 A, and the other belongs to space group P2(1), with unit-cell parameters a = 77.3, b = 47.9, c = 71.9 A, beta = 89.0 degrees. All these crystals diffract X-rays to better than 2.8 A resolution and at least one diffraction data set has been collected for each crystal form using an in-house R-AXIS IV++ diffractometer. Structural studies of hRheb in complexes with various substrates may provide insights into the recognition and specificity of substrate and the catalytic mechanism of mammalian Rhebs and shed light on the biological functions of Rhebs in the mTOR signalling pathway.

Authors+Show Affiliations

Yu Y
Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, People's Republic of China.
Chang Y
No affiliation info available
Li S
No affiliation info available
Hu H
No affiliation info available
Huang Q
No affiliation info available
Ding J
No affiliation info available

MeSH

BrainCatalysisCloning, MolecularCrystallography, X-RayGTP PhosphohydrolasesHumansMonomeric GTP-Binding ProteinsNeuropeptidesPlasmidsProtein BindingProtein ConformationProtein Structure, TertiaryRas Homolog Enriched in Brain ProteinRecombinant ProteinsSignal TransductionX-Rays

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

15388940

Citation

Yu, Yadong, et al. "Expression, Purification, Crystallization and Preliminary Structural Characterization of the GTPase Domain of Human Rheb." Acta Crystallographica. Section D, Biological Crystallography, vol. 60, no. Pt 10, 2004, pp. 1883-7.
Yu Y, Chang Y, Li S, et al. Expression, purification, crystallization and preliminary structural characterization of the GTPase domain of human Rheb. Acta Crystallogr D Biol Crystallogr. 2004;60(Pt 10):1883-7.
Yu, Y., Chang, Y., Li, S., Hu, H., Huang, Q., & Ding, J. (2004). Expression, purification, crystallization and preliminary structural characterization of the GTPase domain of human Rheb. Acta Crystallographica. Section D, Biological Crystallography, 60(Pt 10), 1883-7.
Yu Y, et al. Expression, Purification, Crystallization and Preliminary Structural Characterization of the GTPase Domain of Human Rheb. Acta Crystallogr D Biol Crystallogr. 2004;60(Pt 10):1883-7. PubMed PMID: 15388940.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Expression, purification, crystallization and preliminary structural characterization of the GTPase domain of human Rheb. AU - Yu,Yadong, AU - Chang,Yonggang, AU - Li,Sheng, AU - Hu,Hongyu, AU - Huang,Qiuhua, AU - Ding,Jianping, Y1 - 2004/09/23/ PY - 2004/07/03/received PY - 2004/07/24/accepted PY - 2004/9/25/pubmed PY - 2005/3/30/medline PY - 2004/9/25/entrez SP - 1883 EP - 7 JF - Acta crystallographica. Section D, Biological crystallography JO - Acta Crystallogr D Biol Crystallogr VL - 60 IS - Pt 10 N2 - Ras homologue enriched in brain (Rheb) represents a unique group of small GTPases and shares moderate sequence identity with the Ras/Rap subfamily. It acts downstream of nutrient signalling as the direct target of the tuberous sclerosis complex (TSC) and upstream of mTOR/S6K1/4EBP in the insulin-signalling pathway. The GTPase domain of human Rheb (hRheb) has been recombinantly expressed in Escherichia coli, purified and cocrystallized in complexes with GDP, GTP and GppNHp using the hanging-drop vapour-diffusion method. Crystals of the hRheb-GDP complex belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 44.5, b = 52.3, c = 70.6 A. The hRheb-GppNHp complex crystallized in two crystal forms: one has the same space group and unit-cell parameters as the hRheb-GDP complex and the other belongs to space group C222(1), with unit-cell parameters a = 102.9, b = 99.2, c = 48.0 A. The hRheb-GTP complex also crystallized in two crystal forms: one belongs to space group C222(1), with unit-cell parameters a = 102.4, b = 98.3, c = 47.9 A, and the other belongs to space group P2(1), with unit-cell parameters a = 77.3, b = 47.9, c = 71.9 A, beta = 89.0 degrees. All these crystals diffract X-rays to better than 2.8 A resolution and at least one diffraction data set has been collected for each crystal form using an in-house R-AXIS IV++ diffractometer. Structural studies of hRheb in complexes with various substrates may provide insights into the recognition and specificity of substrate and the catalytic mechanism of mammalian Rhebs and shed light on the biological functions of Rhebs in the mTOR signalling pathway. SN - 0907-4449 UR - https://www.unboundmedicine.com/medline/citation/15388940/Expression_purification_crystallization_and_preliminary_structural_characterization_of_the_GTPase_domain_of_human_Rheb_ DB - PRIME DP - Unbound Medicine ER -