title: Characterization of the self-cleaving effector protein NopE1 of Bradyrhizobium japonicum.
abstract: NopE1 is a type III-secreted protein of the symbiont Bradyrhizobium japonicum which is expressed in nodules. In vitro it exhibits self-cleavage in a duplicated domain of unknown function (DUF1521) but only in the presence of calcium. Here we show that either domain is self-sufficient for cleavage. An exchange of the aspartic acid residue at the cleavage site with asparagine prevented cleavage; however, cleavage was still observed with glutamic acid at the same position, indicating that a negative charge at the cleavage site is sufficient. Close to each cleavage site, an EF-hand-like motif is present. A replacement of one of the conserved aspartic acid residues with alanine prevented cleavage at the neighboring site. Except for EDTA, none of several protease inhibitors blocked cleavage, suggesting that a known protease-like mechanism is not involved in the reaction. In line with this, the reaction takes place within a broad pH and temperature range. Interestingly, magnesium, manganese, and several other divalent cations did not induce cleavage, indicating a highly specific calcium-binding site. Based on results obtained by blue-native gel electrophoresis, it is likely that the uncleaved protein forms a dimer and that the fragments of the cleaved protein oligomerize. A database search reveals that the DUF1521 domain is present in proteins encoded by Burkholderia phytofirmans PsNJ (a plant growth-promoting betaproteobacterium) and Vibrio coralliilyticus ATCC BAA450 (a pathogenic gammaproteobacterium). Obviously, this domain is more widespread in proteobacteria, and it might contribute to the interaction with hosts.
authors.affiliation: Institute of Genetics, Dresden University of Technology, 01062 Dresden, Germany.
authors.forename: Jana
authors.initials: J
authors.lastname: Schirrmeister
authors.forename: Lars
authors.initials: L
authors.lastname: Friedrich
authors.forename: Mandy
authors.initials: M
authors.lastname: Wenzel
authors.forename: Markus
authors.initials: M
authors.lastname: Hoppe
authors.forename: Christine
authors.initials: C
authors.lastname: Wolf
authors.forename: Michael
authors.initials: M
authors.lastname: Göttfert
authors.forename: Susanne
authors.initials: S
authors.lastname: Zehner
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020369:Bradyrhizobium / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary
chemical_list: D001426:Bacterial Proteins