Influence of the TorD signal peptide chaperone on Tat-dependent protein translocation

Abstract

The twin-arginine translocation (Tat) pathway transports folded proteins across energetic membranes. Numerous Tat substrates contain co-factors that are inserted before transport with the assistance of redox enzyme maturation proteins (REMPs), which bind to the signal peptide of precursor proteins. How signal peptides are transferred from a REMP to a binding site on the Tat receptor complex remains unknown. Since the signal peptide mediates both interactions, possibilities include: i) a coordinated hand-off mechanism; or ii) a diffusional search after REMP dissociation. We investigated the binding interaction between substrates containing the TorA signal peptide (spTorA) and its cognate REMP, TorD, and the effect of TorD on the in vitro transport of such substrates. We found that Escherichia coli TorD is predominantly a monomer at low micromolar concentrations (dimerization KD > 50 μM), and this monomer binds reversibly to spTorA (KD ≈ 1 μM). While TorD binds to membranes (KD ≈ 100 nM), it has no apparent affinity for Tat translocons and it inhibits binding of a precursor substrate to the membrane. TorD has a minimal effect on substrate transport by the Tat system, being mildly inhibitory at high concentrations. These data are consistent with a model in which the REMP-bound signal peptide is shielded from recognition by the Tat translocon, and spontaneous dissociation of the REMP allows the substrate to engage the Tat machinery. Thus, the REMP does not assist with targeting to the Tat translocon, but rather temporarily shields the signal peptide.

History of Maturation of Prokaryotic Molybdoenzymes—A Personal View

“…Using a translational fusion between the TorA signal peptide and GFP, contrasted results were obtained. Li et al [153] reported that TorD stimulates the translocation of the fusion, in support of the reported protective role of the chaperone, while Bageshwar et al [108] showed that TorD inhibited the binding of the fusion to the membrane but only moderately impacted transport efficiency in agreement with the dynamic nature of this interaction. In parallel, a structural investigation of the TorD protein revealed that the one from Shewanella massilia forms multiple and stable oligomeric species and that both the monomeric and dimeric species bind TorA [154].…”

“…This hypothesis was based on the observation that DmsD interacts with TatB and TatC located in the inner membrane [105][106][107]. However, this issue remains controversial as the TorD-related chaperone does not bind Tat structural proteins [108]. DmsD has also been shown to interact with general chaperones and several Moco biosynthesis proteins, expanding its interactome and raising questions about the universality of this property and its feasibility [109].…”

“…If the role of nucleotide binding or hydrolysis in modulating the interaction between the signal peptide and TorD remains unclear, the monomeric form is sufficient to bind both TorA sites [152,158]. Bageshwar et al [108] not only established that EcTorD is predominantly a monomer under physiological conditions but also that its main function via signal peptide binding is to protect it from degradation during folding and Moco acquisition.…”