Insights into catalysis and function of phosphoribosyl-linked serine ubiquitination

Sissy Kalayil, Sagar Bhogaraju, Florian Bonn, Donghyuk Shin, Yaobin Liu, Ninghai Gan, Jérôme Basquin, Paolo Grumati, Zhao Qing Luo, Ivan Dikic

Research output: Contribution to journalArticlepeer-review

33 Citations (Scopus)

Abstract

Conventional ubiquitination regulates key cellular processes by catalysing the ATP-dependent formation of an isopeptide bond between ubiquitin (Ub) and primary amines in substrate proteins 1 . Recently, the SidE family of bacterial effector proteins (SdeA, SdeB, SdeC and SidE) from pathogenic Legionella pneumophila were shown to use NAD + to mediate phosphoribosyl-linked ubiquitination of serine residues in host proteins 2, 3 . However, the molecular architecture of the catalytic platform that enables this complex multistep process remains unknown. Here we describe the structure of the catalytic core of SdeA, comprising mono-ADP-ribosyltransferase (mART) and phosphodiesterase (PDE) domains, and shed light on the activity of two distinct catalytic sites for serine ubiquitination. The mART catalytic site is composed of an α-helical lobe (AHL) that, together with the mART core, creates a chamber for NAD + binding and ADP-ribosylation of ubiquitin. The catalytic site in the PDE domain cleaves ADP-ribosylated ubiquitin to phosphoribosyl ubiquitin (PR-Ub) and mediates a two-step PR-Ub transfer reaction: first to a catalytic histidine 277 (forming a transient SdeA H277-PR-Ub intermediate) and subsequently to a serine residue in host proteins. Structural analysis revealed a substrate binding cleft in the PDE domain, juxtaposed with the catalytic site, that is essential for positioning serines for ubiquitination. Using degenerate substrate peptides and newly identified ubiquitination sites in RTN4B, we show that disordered polypeptides with hydrophobic residues surrounding the target serine residues are preferred substrates for SdeA ubiquitination. Infection studies with L. pneumophila expressing substrate-binding mutants of SdeA revealed that substrate ubiquitination, rather than modification of the cellular ubiquitin pool, determines the pathophysiological effect of SdeA during acute bacterial infection.

Original languageEnglish
Pages (from-to)734-738
Number of pages5
JournalNature
Volume557
Issue number7707
DOIs
Publication statusPublished - 2018 May 31

Bibliographical note

Funding Information:
Acknowledgements We thank J. Vogel for the gift of anti-SidC serum, R. Prabu for help with total mass analysis of proteolysed SdeA, Ö. Yildiz for sharing synchrotron time, A. Chaikuad for initial construct design of SdeA, T. Hunter and C. Lima for advice on histidine intermediate protocols, T. Hanke for the PDE mechanism scheme and discussion, T. Colby and I. Matic for their help with identifying ubiquitination sites of RTN4B by LC-MS/MS and B. Schulman and D. Scott for crystallography advice. Swiss Light Source beamtime was part of proposal 20161958. We thank the staff of SLS for their assistance in data collection as well as E. Veshkova, S. Rodriguez Gomez, S. Jelenic and F. Miljkovic for technical assistance; K. Koch, D. Höller, V. Dötsch and S. Knapp for comments on the paper; and D. Svergun’s group at beamline P12, PETRA III, EMBL-DESY for SAXS data collection. This work was supported by iNEXT (PID:3515), the DFG-funded Collaborative Research Centre on Selective Autophagy (SFB 1177), the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No 742720), the DFG-funded Cluster of Excellence ‘‘Macromolecular Complexes’’ (EXC115), the DFG-funded SPP 1580 program ‘‘Intracellular Compartments as Places of Pathogen-Host-Interactions’’ (I.D.) and the LOEWE program Ubiquitin Networks (Ub-Net) and the LOEWE Center for Gene and Cell Therapy Frankfurt (CGT), both funded by the State of Hesse/ Germany. NIH-NIAID grant R01AI127465 supported Z.-Q.L. The work of S.B. is also funded by a Goethe University Nachwuchsforscher grant.

Publisher Copyright:
© 2018 Macmillan Publishers Ltd., part of Springer Nature.

All Science Journal Classification (ASJC) codes

  • General

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