title: The hydrophobic core of twin-arginine signal sequences orchestrates specific binding to Tat-pathway related chaperones.
abstract: Redox enzyme maturation proteins (REMPs) bind pre-proteins destined for translocation across the bacterial cytoplasmic membrane via the twin-arginine translocation system and enable the enzymatic incorporation of complex cofactors. Most REMPs recognize one specific pre-protein. The recognition site usually resides in the N-terminal signal sequence. REMP binding protects signal peptides against degradation by proteases. REMPs are also believed to prevent binding of immature pre-proteins to the translocon. The main aim of this work was to better understand the interaction between REMPs and substrate signal sequences. Two REMPs were investigated: DmsD (specific for dimethylsulfoxide reductase, DmsA) and TorD (specific for trimethylamine N-oxide reductase, TorA). Green fluorescent protein (GFP) was genetically fused behind the signal sequences of TorA and DmsA. This ensures native behavior of the respective signal sequence and excludes any effects mediated by the mature domain of the pre-protein. Surface plasmon resonance analysis revealed that these chimeric pre-proteins specifically bind to the cognate REMP. Furthermore, the region of the signal sequence that is responsible for specific binding to the corresponding REMP was identified by creating region-swapped chimeric signal sequences, containing parts of both the TorA and DmsA signal sequences. Surprisingly, specificity is not encoded in the highly variable positively charged N-terminal region of the signal sequence, but in the more similar hydrophobic C-terminal parts. Interestingly, binding of DmsD to its model substrate reduced membrane binding of the pre-protein. This property could link REMP-signal peptide binding to its reported proofreading function.
authors.affiliation: Department of Molecular Cell Biology, VU University Amsterdam, Amsterdam, The Netherlands.
authors.forename: Anitha
authors.initials: A
authors.lastname: Shanmugham
authors.forename: Adil
authors.initials: A
authors.lastname: Bakayan
authors.forename: Petra
authors.initials: P
authors.lastname: Völler
authors.forename: Joost
authors.initials: J
authors.lastname: Grosveld
authors.forename: Holger
authors.initials: H
authors.lastname: Lill
authors.forename: Yves J M
authors.initials: YJ
authors.lastname: Bollen
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry*; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D007506:Iron-Sulfur Proteins / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance
chemical_list: D002352:Carrier Proteins; C434268:DmsD protein, E coli; D029968:Escherichia coli Proteins; D047908:Intracellular Signaling Peptides and Proteins; D007506:Iron-Sulfur Proteins; D026901:Membrane Transport Proteins; D018832:Molecular Chaperones; D021382:Protein Sorting Signals; C088083:TorD protein, E coli; D049452:Green Fluorescent Proteins; D001120:Arginine; D010088:Oxidoreductases; C045664:dimethyl sulfoxide reductase