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  • Structural and biochemical characterization of NarE, an iron-containing ADP-ribosyltransferase from Neisseria meningitidis.

Structural and biochemical characterization of NarE, an iron-containing ADP-ribosyltransferase from Neisseria meningitidis.

The Journal of biological chemistry (2011-03-04)
Christian Koehler, Ludovic Carlier, Daniele Veggi, Enrico Balducci, Federica Di Marcello, Mario Ferrer-Navarro, Mariagrazia Pizza, Xavier Daura, Marco Soriani, Rolf Boelens, Alexandre M J J Bonvin
ABSTRACT

NarE is a 16 kDa protein identified from Neisseria meningitidis, one of the bacterial pathogens responsible for meningitis. NarE belongs to the family of ADP-ribosyltransferases (ADPRT) and catalyzes the transfer of ADP-ribose moieties to arginine residues in target protein acceptors. Many pathogenic bacteria utilize ADP-ribosylating toxins to modify and alter essential functions of eukaryotic cells. NarE is further the first ADPRT which could be shown to bind iron through a Fe-S center, which is crucial for the catalytic activity. Here we present the NMR solution structure of NarE, which shows structural homology to other ADPRTs. Using NMR titration experiments we could identify from Chemical Shift Perturbation data both the NAD binding site, which is in perfect agreement with a consensus sequence analysis between different ADPRTs, as well as the iron coordination site, which consists of 2 cysteines and 2 histidines. This atypical iron coordination is also capable to bind zinc. These results could be fortified by site-directed mutagenesis of the catalytic region, which identified two functionally crucial residues. We could further identify a main interaction region of NarE with antibodies using two complementary methods based on antibody immobilization, proteolytic digestion, and mass spectrometry. This study combines structural and functional features of NarE providing for the first time a characterization of an iron-dependent ADPRT.

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