Structural and Biochemical Characterization of NarE, an Iron-containing ADP-ribosyltransferase from Neisseria meningitidis

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.

Auto ADP‐ribosylation of NarE, aNeisseria meningitidisADP‐ribosyltransferase, regulates its catalytic activities

“…To get further insights on the molecular basis of automodification, we carried out experiments with different NarE mutants. We have previously identified E 120 and R 7 as the crucial residues for NarE catalytic activities (8). Indeed, in the NarE mutant in which E 120 was replaced with aspartic acid (E120D), no auto‐ADP‐ribosylation occurred after 1 or 3 h of incubation, while the NarE mutants E111D and E109D, residues that are not involved in catalysis (8), were ADP‐ribosylated as WT NarE ( Fig.…”

“…The R33K, R97K and R124K mutants were generated using the site‐directed mutagenesis kit by Invitrogen according to the manufacturer's instructions. The C and E mutants were obtained as described previously (7, 8). All the generated mutants were checked by DNA sequence analysis and were expressed as soluble proteins and purified as described previously (7).…”

“…A recently discovered feature of NarE is the presence of an iron‐sulfur (Fe‐S) center that we identified for the first time in a member of the ART family (7). The iron coordination site has been recently resolved, and the amino acids, 2 cysteines (C 6 and C 128 ) and 2 histidines (H 46 and H) that bind iron and zinc have been identified (8). Interestingly, the lack of assembly of the iron‐binding site with iron strongly reduced the transferase activity of NarE, while it affected the hydrolase activity only marginally (7).…”

EsiB, a Novel Pathogenic Escherichia coli Secretory Immunoglobulin A-Binding Protein Impairing Neutrophil Activation

“…To this aim, we adopted and extended an immunocapturing approach successfully used previously with monoclonal antibodies (25). The approach involves proteolytic digestion (using diverse proteases) of the target antigen prior to immunocapturing and subsequent analysis of antibody-bound peptides (containing the epitope) by mass spectrometry.…”

Understanding the Molecular Determinants Driving the Immunological Specificity of the Protective Pilus 2a Backbone Protein of Group B Streptococcus

“…The epitope-mapping approach was based on the method described by Peter and Tomer [24], which we adapted to the following two protocols [25], [26]:…”