Structural and Biochemical Study of the Mono-ADP-Ribosyltransferase Domain of SdeA,a Ubiquitylating/Deubiquitylating Enzyme from Legionella pneumophila |
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Authors: | Leehyeon Kim Do Hoon Kwon Bong Heon Kim Jiyeon Kim Mi Rae Park Zee-Yong Park Hyun Kyu Song |
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Institution: | 1. Department of Life Sciences, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul 02841, Korea;2. School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 61005, Korea |
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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. |
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Keywords: | 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 + 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 mART + ubiquitin |
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