Structural and Biochemical Study of the Mono-ADP-Ribosyltransferase Domain of SdeA, a Ubiquitylating/Deubiquitylating Enzyme from Legionella pneumophila. Issue 17 (17th August 2018)

• Record Type: Journal Article
• Authors: Kim, Leehyeon; Kwon, Do Hoon; Kim, Bong Heon; Kim, Jiyeon; Park, Mi Rae; Park, Zee-Yong; Song, Hyun Kyu
• Is Part Of: Journal of molecular biology. Volume 430:Issue 17(2018)
• Page Start: 2843
• Page End: 2856
• Publication Date: 2018-08-17
• Subjects: ART ADP-ribosyltransferase -- ARTT ADP-ribosylation turn–turn -- DUB deubiquitylase -- CC coiled-coil -- ESI electrospray ionization -- FL full-length -- MBP maltose-binding protein -- mART mono-ADP-ribosyltransferase -- NAD+ nicotinamide adenine dinucleotide -- NCA nicotinamide -- PDE phosphodiesterase -- SAD single-wavelength anomalous diffraction -- SAXS small-angle X-ray scattering -- SEC-MALS size-exclusion chromatography with a multi-angle light scattering -- SPR surface plasmon resonance -- TCEP tris(2-carboxyethyl)phosphine hydrochloride -- tNCS translational non-crystallographic symmetry -- Ub ubiquitin -- WT wild-type; Legionella pneumophila -- mART -- NAD+ -- SdeA -- ubiquitin; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2018.05.043
• Languages: English
• ISSNs: 0022-2836

Abstract

Abstract: Conventional ubiquitylation occurs through an ATP-dependent three-enzyme cascade (E1, E2, and E3) that mediates the covalent conjugation of the C-terminus of ubiquitin to a lysine on the substrate. SdeA, which belongs to the SidE effector family of Legionella pneumophila, can transfer ubiquitin to endoplasmic reticulum-associated Rab-family GTPases in a manner independent of E1 and E2 enzymes. The novel ubiquitin-modifying enzyme SdeA utilizes NAD + as a cofactor to attach ubiquitin to a serine residue of the substrate. Here, to elucidate the coupled enzymatic reaction of NAD + hydrolysis and ADP-ribosylation of ubiquitin in SdeA, we characterized the mono-ADP-ribosyltransferase domain of SdeA and show that it consists of two sub-domains termed mART-N and mART-C. The crystal structure of the mART-C domain of SdeA was also determined in free form and in complex with NAD + at high resolution. Furthermore, the spatial orientations of the N-terminal deubiquitylase, phosphodiesterase, mono-ADP-ribosyltransferase, and C-terminal coiled-coil domains within the 180-kDa full-length SdeA were determined. These results provide insight into the unusual ubiquitylation mechanism of SdeA and expand our knowledge on the structure–function of mono-ADP-ribosyltransferases. Graphical Abstract: Unlabelled Image Highlights: The mono-ADP-ribosyltransferase domain of SdeA consists of two sub-domains termed mART-N and mART-C. Both mART-N and mART-C domains are necessary for efficient transfer of the ADP-ribose moiety of NAD + to the substrate ubiquitin. The crystal structure of mART-C of SdeA was determined in free form and in complex with NAD + at high resolution. The overall shape and spatial orientation of the modular domains of SdeA were determined.