title: NMR structural studies of the first catalytic half-domain of ubiquitin activating enzyme.
abstract: We report a high resolution NMR structure and (15)N 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 β-barrel structure, a common fold for many proteins with a variety of cellular functions. (15)N 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 (1)H/(15)N HSQC spectra of the FCCH. The 2D (1)H/(15)N 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.
authors.affiliation: Max Planck Institute for Biophysical Chemistry, Department for NMR-based Structural Biology, Am Fassber 11, 37077 Göttingen, Germany.
authors.forename: Mariusz
authors.initials: M
authors.lastname: Jaremko
authors.affiliation: Max Planck Institute for Biophysical Chemistry, Department for NMR-based Structural Biology, Am Fassber 11, 37077 Göttingen, Germany; Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Am Fassberg 11, 37077 Göttingen, Germany.
authors.forename: Łukasz
authors.initials: Ł
authors.lastname: Jaremko
authors.affiliation: Faculty of Chemistry, Biological and Chemical Research Centre, University of Warsaw, Żwirki i Wigury 101, 02-089 Warsaw, Poland.
authors.forename: Michał
authors.initials: M
authors.lastname: Nowakowski
authors.affiliation: International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland.
authors.forename: Marek
authors.initials: M
authors.lastname: Wojciechowski
authors.affiliation: International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland.
authors.forename: Roman H
authors.initials: RH
authors.lastname: Szczepanowski
authors.affiliation: International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland.
authors.forename: Renata
authors.initials: R
authors.lastname: Panecka
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5A, 02-106 Warsaw, Poland; NanoBioMedical Centre, Adam Mickiewicz University, Umultowska 85, 61-614 Poznan, Poland.
authors.forename: Igor
authors.initials: I
authors.lastname: Zhukov
authors.affiliation: International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland; Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5A, 02-106 Warsaw, Poland.
authors.forename: Matthias
authors.initials: M
authors.lastname: Bochtler
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5A, 02-106 Warsaw, Poland. Electronic address: aejchart@ibb.waw.pl.
authors.forename: Andrzej
authors.initials: A
authors.lastname: Ejchart
mesh_terms: D000818:Animals; D002384:Catalysis; D003545:Cysteine / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D051379:Mice; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D044764:Ubiquitin-Activating Enzymes / Q000737:chemistry*
chemical_list: D044764:Ubiquitin-Activating Enzymes; D003545:Cysteine
keywords: (15)N nuclear magnetic relaxation; AMP; ATP; Backbone dynamics; CPMG; Carr–Purcell–Meiboom–Gill pulse train; E1; E2; E3; FCCH; First catalytic cysteine half-domain (FCCH); HSQC; NMR structure determination; NMR study interactions in solution; NOESY; Protein structure; RMSD; SCCH; Second catalytic cysteine half-domain (SCCH); TRIS-d(11); Ub; Ubiquitin-activating enzyme E1; adenosine-5′-monophosphate; adenosine-5′-triphosphate; first cysteine catalytic half-domain; heteronuclear single quantum correlation; nuclear Overhauser effect spectroscopy; perdeuterated 2-amino-2(hydroxymethyl)1,3-propanediol; root mean square deviation; second cysteine catalytic half-domain; ubiquitin; ubiquitin activating enzyme; ubiquitin conjugating enzyme; ubiquitin ligase