The structure of human ADP-ribosylhydrolase 3 (ARH3) provides insights into the reversibility of protein ADP-ribosylation

doi:10.1073/pnas.0606762103

The structure of human ADP-ribosylhydrolase 3 (ARH3) provides insights into the reversibility of protein ADP-ribosylation

*European Molecular Biology Laboratory Hamburg Outstation, c/o Deutsches Elektronen-Synchrotron, Notkestrasse 85, D-22603 Hamburg, Germany;
^†Institute of Immunology, University Hospital, Martinistrasse 52, D-20246 Hamburg, Germany; and
^‡Leibniz-Institut für Molekulare Pharmakologie, FMP, Robert-Roessle-Strasse 10, Campus Berlin–Buch, D-13125 Berlin, Germany

Communicated by David S. Eisenberg, University of California, Los Angeles, CA, August 12, 2006 (received for review January 27, 2006)

Abstract

Posttranslational modifications are used by cells from all kingdoms of life to control enzymatic activity and to regulate protein function. For many cellular processes, including DNA repair, spindle function, and apoptosis, reversible mono- and polyADP-ribosylation constitutes a very important regulatory mechanism. Moreover, many pathogenic bacteria secrete toxins which ADP-ribosylate human proteins, causing diseases such as whooping cough, cholera, and diphtheria. Whereas the 3D structures of numerous ADP-ribosylating toxins and related mammalian enzymes have been elucidated, virtually nothing is known about the structure of protein de-ADP-ribosylating enzymes. Here, we report the 3Dstructure of human ADP-ribosylhydrolase 3 (hARH3). The molecular architecture of hARH3 constitutes the archetype of an all-α-helical protein fold and provides insights into the reversibility of protein ADP-ribosylation. Two magnesium ions flanked by highly conserved amino acids pinpoint the active-site crevice. Recombinant hARH3 binds free ADP-ribose with micromolar affinity and efficiently de-ADP-ribosylates poly- but not monoADP-ribosylated proteins. Docking experiments indicate a possible binding mode for ADP-ribose polymers and suggest a reaction mechanism. Our results underscore the importance of endogenous ADP-ribosylation cycles and provide a basis for structure-based design of ADP-ribosylhydrolase inhibitors.

Footnotes

^§To whom correspondence may be addressed. E-mail: msweiss{at}embl-hamburg.de or nolte{at}uke-hamburg.de
Author contributions: C.M.-D. and S.K. contributed equally to this work; F.H., M.S.W., and F.K.-N. designed research; C.M.-D., S.K., M.L., and J.P.v.K. performed research; C.M.-D., S.K., M.L., F.H., F.K.-N., M.S.W., and J.P.v.K. analyzed data; and M.S.W. and F.K.-N. wrote the paper.
The authors declare no conflict of interest.
Data deposition: The atomic coordinates and structure factor amplitudes of the crystal forms of hARH3 have been deposited in the Protein Data Bank [PDB ID codes: 2FOZ (orthorhombic) and 2FP0 (monoclinic)].
Abbreviations:

ADPR,

ADP-ribose;

ARH,

ADP-ribosylhydrolase;

ART,

monoADP-ribosyltransferase;

DRAG,

dinitrogenase-activating glycohydrolase;

PARG,

poly(ADP-ribose)glycohydrolase;

PARP,

polyADP-ribosylpolymerase