NMR structural studies of the first catalytic half-domain of ubiquitin activating enzyme

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• 作者：Mariusz Jaremko ; 艁ukasz Jaremko ; Micha艂 Nowakowski ; Marek Wojciechowski ; Roman H. Szczepanowski ; Renata Panecka ; Igor Zhukov ; Matthias Bochtler ; Andrzej Ejchart
• 关键词：AMP ; adenosine-5&prime ; -monophosphate ; ATP ; adenosine-5&prime ; -triphosphate ; CPMG ; Carr&ndash ; Purcell&ndash ; Meiboom&ndash ; Gill pulse train ; E1 ; ubiquitin activating enzyme ; E2 ; ubiquitin conjugating enzyme ; E3 ; ubiquitin ligase ; FCCH ; first cysteine catalytic half-domain ; HSQC ; heteronuclear single quantum correlation ; NOESY ; nuclear Overhauser effect spectroscopy ; RMSD ; root mean square deviation ; SCCH ; second cysteine catalytic half-domain ; TRIS-d11 ; perdeuterated 2-amino-2(hydroxymethyl)1 ; 3-p
• 刊名：Journal of Structural Biology
• 出版年：January, 2014
• 年：2014
• 卷：185
• 期：1
• 页码：69-78
• 全文大小：3127 K

文摘

We report a high resolution NMR structure and ¹⁵N relaxation studies of the first catalytic cysteine half-domain (FCCH) of the mouse ubiquitin-activating enzyme E1, together with interaction studies of FCCH and the other catalytic E1 subdomain - SCCH (second catalytic cysteine half-domain). In solution, mouse FCCH forms a well-defined six-stranded antiparallel 尾-barrel structure, a common fold for many proteins with a variety of cellular functions. ¹⁵N relaxation data reveal FCCH complex backbone dynamics and indicate which residues experience slow intramolecular motions. Some of these residues make contacts with the polar face of ubiquitin in the co-crystal structure of yeast E1 and ubiquitin. However, the titration of FCCH with ubiquitin does not show any visible chemical shift changes in the 2D ¹H/¹⁵N HSQC spectra of the FCCH. The 2D ¹H/¹⁵N HSQC experiments performed both for each catalytic half-domain individually and for their equimolar mixture in the milimolar concentration range display no detectable chemical shift perturbation, suggesting a lack of interaction between the two subdomains unless they are covalently linked via the adenylation domain.