Identification of residues in DmsD for twin-arginine leader peptide binding, defined through random and bioinformatics-directed mutagenesis.
The twin-arginine translocase (Tat) system is used for the targeting and translocation of folded proteins across the cell membrane of most bacteria. Substrates of this system contain a conserved "twin-arginine" (RR) motif within their signal/leader peptide sequence. Many Tat substrates have their own system-specific chaperone called redox enzyme maturation proteins (REMPs). Here, we study the binding of DmsD, the REMP for dimethyl sulfoxide reductase in Escherichia coli, toward the RR-containing leader peptide of the catalytic subunit DmsA. We have used a multipronged approach targeted at the amino acid sequence of DmsD to define residues and regions important for recognition of the DmsA leader sequence. Residues identified through bioinformatics and THEMATICS analysis were mutated using site-directed mutagenesis. These DmsD residue variants were purified and screened with an in vitro dot-blot far-Western assay to analyze the binding to the DmsA leader sequence. Degenerative polymerase chain reaction was also used to produce a bank of random DmsD amino acid mutants, which were then screened by an in vivo bacterial two-hybrid assay. Using this hybrid method, each DmsD variant was classified into one of three groups based on their degree of interaction with the DmsA leader (none, weak, and moderate). The data from both the in vitro and in vivo analyses were then applied to a model structure of DmsD based on the crystal structure of the Salmonella typhimurium homologue. Our results illustrate the positions of important DmsD residues involved in binding the DmsA leader peptide and identify a "hot pocket" of residues important for leader binding on the structure of DmsD.
Chan
Catherine S
CS
Department of Biological Sciences, 2500 University Drive Northwest, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
Winstone
Tara M L
TM
Chang
Limei
L
Stevens
Charles M
CM
Workentine
Matthew L
ML
Li
Haiming
H
Wei
Ying
Y
Ondrechen
Mary J
MJ
Paetzel
Mark
M
Turner
Raymond J
RJ
D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019295:Computational Biology / Q000379:methods; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D011485:Protein Binding; D021382:Protein Sorting Signals; D017386:Sequence Homology, Amino Acid
D002352:Carrier Proteins; C434268:DmsD protein, E coli; D029968:Escherichia coli Proteins; D047908:Intracellular Signaling Peptides and Proteins; D021382:Protein Sorting Signals; D001120:Arginine